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" lymph node staging of ductal adenocarcinoma of the pancreatic head pdac by crosssectionalimaging is limited the aim of this study was to determine the diagnostic accuracy of expanded criteria in nodalstaging in pdac patientsmethods sixtysix patients with histologically confirmed pdac that underwent primary surgery were included inthis retrospective irbapproved study crosssectional imaging studies ct andor mri were evaluated by aradiologist blinded to histopathology number and size of lymph nodes were measured shortaxis diameter andcharacterized in terms of expanded morphological criteria of border contour spiculated lobulated and indistinctand texture homogeneous or inhomogeneous sensitivities and specificities were calculated with histopathologyas a reference standardresults fortyeight of patients had histologically confirmed lymph node metastases pn sensitivityspecificity and youdens index for the criterion size were and for inhomogeneous signal intensity and and for border contour and respectively there was a significant associationbetween the number of visible lymph nodes on preoperative ct and lymph node involvement pn p lymph node staging in pdac is mainly limited due to low sensitivity for detection of metastatic diseaseusing expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivityremaining low combining specific criteria yields improved sensitivity with specificity and ppv remaining highkeywords ductal adenocarcinoma of the pancreatic head staging lymph nodes computed tomography magneticresonance imaging crosssectional imaging neoadjuvant therapy correspondence florianlochcharitede1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germanyfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cloch world of surgical oncology page of pancreatic cancer remains one of the most lethal malignancies being the fourth leading cause of cancerdeath in the usa and predicted to be the secondleading cause of cancer death by the overall5year survival after diagnosis is and at thetime of diagnosis the main proportion of patients hasadvancedstage disease leaving only qualifiedfor resective surgery pancreatic cancer is locatedin the head of the pancreas in of the cases even after successful resective surgery in patients withcancer of the pancreatic head the 5year survival remains as low as these data underline theimportance of establishing multimodaltherapeuticconcepts for patients with pancreatic cancer as perother entities of abdominal cancerapart from the potential to increase the resectabilityrate of pancreatic cancer by neoadjuvant therapy [ ]there is evidence that patients which are successfullydownstaged from nodepositive disease cn1 to nodenegative disease ypn0 prior to surgery benefit in termsof higher 5year survival rate this would qualifynodalinvolvement as a sufficient basis for indicatingneoadjuvant therapy yet even given advanced imagingtechnologies identifying lymph node metastasis remainschallenging consequently the indication of a potentiallyeffective neoadjuvant therapy cn with side effects inlymph nodepositive patients cn is mainly based onunreliable clinical stagingthe established criterion for lymph node involvement in pancreatic cancer is size using the size underlies the assumption that tumor spread to regionallymph nodes leads to an enlargement of the respective lymph node the usual cutoff value is a shortaxis diameter of mm [ ] it has been shownthough that lymph nodes of ¥ mm are not seenmore frequently in patients with histopathologicallymph node involvement pn in various othertumor entities expanded morphological criteria suchas texture and border contour oflymph nodes areused for the assessment oflymph node malignancyon both computed tomography ct and magneticresonance mr imaging this is utilized in order toimprove the accuracy of lymph node staging []by applying morphological criteria instead of brown size criterion alone the sensitivity was improvedfrom to and the specificity from to inlymph node staging of rectal cancer thus the aim of this study was to determine thelymph node staging in patients withaccuracy ofductal adenocarcinoma ofthe pancreatic head byboth computed tomography and magnetic resonanceimaging using sizeand expanded morphologicalcriteriamaterial and methodspatientsin this retrospective singlecenter study approved by thelocal ethics committee consecutive patients with histologically proven ductal adenocarcinoma of the pancreatichead that underwent primary surgery between february and november at the department of surgerycampus benjamin franklin charituniversity medicine berlin germany were included patients were retrieved from the database of our pancreatic cancercenter certified by the german cancer society n inclusion criteria were primary oncologic tumor resection and the presence of preoperative crosssectional imaging of sufficient quality see below exclusion criteriawere neoadjuvant therapy presence of a potential simultaneous cause of lymphadenopathy of the upper abdominal region eg abdominal lymphoma neuroendocrinetumor and main tumor mass located outside the pancreatic head on histopathology the process of patientselection with the respective reasons for inclusion andexclusion is shown in fig crosssectional imagingall images were retrospectively analyzed for the purposeof this study by a single abdominal radiologist with morethan years of experience in staging of tumors of the visceral ans blinded to the results of histopathologyall crosssectional imaging studies were assessed forsufficient image quality by the radiologist prior to commencement for ct imaging the minimum quality wasdefined as either thinsection ct mm reconstructedslice thickness or contrastenhanced ct with a slicethickness of mm for mri minimum quality was defined as availability of an axial t2weighted sequencewith fat suppression slice thickness mm in combination with a venous phase postcontrast 3d gradientecho sequence slice thickness mmfor lymph node assessment all visible regional lymphnodes in the field of view were recorded on a score chartand the total number of visible lymph nodes per patientwas calculated then for each patient all lymph nodeswere characterized in terms of size long and shortaxisdiameter in millimeters and the expanded morphological criteria border contour lobulated spiculated indistinct or unaltered and texture homogeneous orinhomogeneous fig based on kim regional lymph nodes of the pancreas are defined asthe following lymph node station numbers 8a8p 11p 11d 12a 12b 12p13a 13b14p14d 17a17b and in all cases in which a lymph node wasnot definitively regional correlation with postoperativecrosssectional imaging was performed to assess whetherthe lymph node was resected or not only resectedlymph nodes were analyzed in this study 0cloch world of surgical oncology page of fig flowchart of patient recruitment the process of patient selection with the respective reasons for inclusion and exclusion is showna second radiologist with more than years of experience in staging of tumors of the visceral ans alsoblinded to the results of histopathology evaluated thect examinations of a representative subgroup of patients for evaluation of interobserver agreementsurgeryall patients underwent primary oncologic pyloruspreserving pancreaticoduodenectomy or whipple procedure with complete lymphadenectomy of the regionallymph nodes mentioned aboveformalinembedded surgicalhistopathologythe original histopathological reportsfor the studyspecimens wereusingreviewed cancer of the pancreatic head was defined as amalignant tumor located within the pancreas to the rightof the superior mesenteric vein and portal vein each patient with histologically proven lymph node metastaseswas classified as nodepositive pn regardless of thenumber of metastatic lymph nodes patients without anymetastatic lymph nodes were classified as nodenegativepn the ratio of metastatic lymph nodes vs the totalnumber of retrieved lymph nodes was documented infig morphological characterization of lymph nodes based on kim the morphological criteria for lymph node assessment used inthis study are shown smooth and homogeneous lymph nodes were considered normal 0cloch world of surgical oncology page of the histopathological report eg or tumorswere classified according to their respective tnm stageusing the 8th edition of tnm classification of malignant tumors table demographic data of patients with ductal adenocarcinomaof the pancreatic head undergoing primary tumor resectionpatientsagen comparison of crosssectional imaging andhistopathologysensitivity specificity and positive predictive value ofthe nodal status using ct and mri with histopathologyas a reference standard were calculated for lymph nodeinvolvement using size and morphological criteria specifically nodal involvement criteria were based on eithersize shortaxis diameter altered border contour lobulated spiculated or indistinct and inhomogeneous signal intensity fig ct and mri examinations wereconsidered nodepositive cnif at least one lymphnode met one of the respective criteria used for involvement if no lymph node with the respective criteria wasseen on ct or mri then the examination was considered nodenegative cnstatistical analysissensitivities specificities and positive predictive valueppv for the size criterion and all morphological criteria were calculated for their respective cutoff valuesan index summarizing the sensitivity and specificity foryoudens index was calculated sensitivity specificity the number of lymph nodes visible on ctand mr images in the group with pn and withoutpn nodal metastases was compared using the mannwhitney u test when calculating the association between ct and mri criteria and lymph node positivitythe Ï2test was used interobserver agreement was calculated using cohens kappa statistic a p value of was considered to indicate a statistically significantdifferenceresultspatientssixtysix patients were included in the study fig with the characteristics of the patients presented intable sixty of these patients were staged by preoperative ct twelve of which had additional stagingby mri and six patients were staged by only mri intwo patientsthe mri examinations were excludeddue to insufficient imaging quality both patients hadsufficient staging by ct and were therefore includedin the study of the patients patients receivedpreoperative biliary drainage eight ofthem werestaged by ct only and two by mri onlycomputed tomography ctlymph nodes were detected by ct in ofthe patients the median number of visible lymph nodesmedian age yearsage range yearssexfemalemalecrosssectional imagingct onlyct and mrimri onlyhistopathological stagingpnpnpt1pt2pt3 was range the smallest visible lymph node was mm of size whereas the largest measured mmshortaxis diameter the mean time between ct andsurgery was days with a median of days range the slice thickness in of the ct examinations was mm or less in five ct examinations slice thickness was mmsize criterion for lymph node involvement onpreoperative ctfigure shows the percentage of patients with pnand without pn lymph node metastases in which alymph node of the respective size was visible mmin table sensitivity specificity and youdens indexare presented for the respective cutoff values lymphnodes of small and medium size mm were visiblein patients with pn and withoutlymph node metastases pnineven frequency large lymph nodes mm wereseen more frequently in the lymph nodepositive group pnthan in the lymph nodenegative group pn the maximumvalue of youdens index for the size criterion was j ci when a cutoff value of mmwas applied yielding a sensitivity of and specificityof additionallylymph nodesgreater than mm on preoperative ct and the histopathological confirmation of a lymph node metastasispn showed a trend towards significance p the presence of 0cloch world of surgical oncology page of fig graph showing lymph node size and morphological criteria of lymph nodepositive and negative patients frequency of regional lymphnodes of the pancreatic head in percent xaxis with different shortaxis diameters and morphological features yaxis in patients with pn redbars or without histologically proven lymph node metastases pn blue bars on preoperative ct imagingexpanded morphological criteria for lymph node involvementon preoperative ctfigure shows the percentage of patients with pnand pn without lymph node metastases in which alymph node of the respective morphological criterionwas visible and table shows the sensitivity specificityand youdens index of the respective criterionlymph nodes of lobulated border contour were visiblewith a similar frequency in patients with pn and without lymph node metastases pn lymph nodes of spiculated or indistinct bordercontour were only occasionally detected in both groups vs in the lymph nodepositivegroup pn and vs in the lymphnodenegative group pnlymph nodes of inhomogeneous signal intensity were detected in only one patient of the lymph nodenegative group pn and more frequently in patients of the lymphnodepositive group pn resulting in the maximum value of youdens index for the morphological criteriaj ci consisting of a sensitivity of and a specificity of the ppv was comparison of size with expanded morphological criteriathe maximum value of the youdens index of the sizecriterion was j ci cutoff mmwhich is not inferior to the maximum value of the morphological criteria j ci inhomogeneoussignal intensity figure displays respective ct images ofpatients with and without lymph node metastasesfig ct images of patients with and without lymph node metastases a patient with enlarged suspicious lymph node adjacent to the portalvein and hepatic artery who had no lymph node metastases on pathology b patient with enlarged suspicious lymph node adjacent to thehepatic artery who had lymph node metastases on pathology c patient with multiple suspicious lymph nodes based on size and inhomogeneitywho had lymph node metastases on pathology 0cloch world of surgical oncology page of table sensitivity specificity ppv and youdens index for cutoff values and morphological criteria by ct and mrisensitivity specificity ppvyoudens indexctsize cutoff value mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctinhomogeneous mrisize cutoff value mm mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctnot visibleinhomogeneousnot visible number of visible lymph nodesthere was a significant association between number of visible lymph nodes seen on preoperativect and histopathologicallymph node involvementpn p seven or more lymph nodes were seen on preoperative ct in of patients with lymph nodemetastases pn and in of patients without lymph node metastasis pn p this resulted in a specificity of youdens index of and ppv of combination of number size and expanded morphologiccriteria on preoperative ctcombining the size criterion and the morphological criterion with the respective highest youdens index cutoff mm and inhomogeneous signalintensity andthe criterion visible lymph nodes n ¥ was significantly associated with nodal metastases pn p for this combined criterion specificity was sensitivity ppv and youdens index ci interobserver agreementinterobserver agreement was calculated for patientspn pn vs pnfor the criteria sizemorphology and number of visible lymph nodes withthe respective highest youdens indexinterobserveragreement was substantial for size mm cutoff κ p moderate for the presence of seven ormore lymph nodes κ p and fair forthe morphological criterion inhomogeneous signal intensity κ p magnetic resonance imaging mrilymph nodes were detected in of patientson preoperative mri the median number of visiblelymph nodes was range there was no significantassociation between number of visible lymph nodes seenon preoperative mri and histopathological lymph nodeinvolvement pn p the smallest visible lymph node was mm of sizewhereas the largest measured mm shortaxis diameter the mean time between mri and surgery was days with a median of days range the cutoff values of the highest diagnostic value were mm or mm for the size criterion sensitivity specificity youdens index the presence ofa lymph node of these sizes was not associated with lymphnode metastases pn p lobulated and spiculated lymph nodes were only seen in a few patients n and n and indistinct and inhomogeneous lymphnodes were not seen at all table discussionin this retrospective singlecenter study on lymph nodestaging by ct in ductal adenocarcinoma of the pancreatic head we could show that the morphologic criteriainhomogeneous signal intensity and size are specificfor regional nodal metastatic disease replacing the sizecriterion by morphologic criteria however did not improve diagnostic accuracy due to sensitivity remaininglow combining specific criteria yields improved sensitivity with specificity remaining highby ct lymph nodes of mm in shortaxis diameterwere seen just as often in patients with and without 0cloch world of surgical oncology page of lymph node metastases resulting in poor discriminationlarger lymph nodes mm had a higher prevalence inthe lymph nodepositive group leading to high specificity however these lymph nodes mm or mmwere seen infrequently resulting in a rather low sensitivity the maximum value of the youdens index for thesize criterion of was achieved when a cutoff valueof mm was applied consisting of a specificity of and sensitivity of yielding a ppv of as for morphologic criteria lymph nodes of lobulatedborder contour were seen in about half of the patients ofboth groups pn and pn and therefore isa criterion that is not suitable to differentiate betweenthe groups lymph nodes of spiculated and indistinctborder contour were seen in few cases in both patientgroups only pn and versus pn and making them poor diagnostic criteria however lymphnodes of inhomogeneous signal intensity were visible in of patients with lymph node metastases pn andonly in of the patients without lymph node metastases pn resulting in a youdens index of whichwas the maximum value for the morphological criteriaand a ppv of ideally a good discriminator for nodal metastases isnegative in patients without nodal involvement and positive for tumors with lymph node metastases in ourstudy each criterion ie size as well as different morphological features only met one of these prerequisitesthe size criterion mm as well as the presence of alymph node of inhomogeneous signal intensity as morphological criterion turned out to be negative in patientswithout nodal involvement pn and therefore highlyspecific yet lymph nodes of the respective characteristicwere not positive in a sufficiently high number of tumorswith lymph node metastases pn to reach high levelsof sensitivity and consequently did not have a significantdiagnostic valuethe maximum value of the youdens index for the sizecriterion was when a cutoff value of mm wasapplied and for the morphological criteria whenthe criterion inhomogeneous signal intensity was usedshowing that morphologic criteria do not yield in higherdiagnostic value than lymph node size in adenocarcinoma of the pancreatic head pdac patients this iscontrary to the findings of brown in rectal cancer one reason might be that brown used mri toassess morphologic criteria which has a higher soft tissuecontrast compared to ct which was used in most patients in our studyinterestingly we could show that with preoperativect the presence of seven or more lymph nodes wasseen more often in patients with lymph node metastasispn than in those without metastasis pn p when applying this as a sole criterion cn for lymphnode metastasis pn this led to a sensitivity of aspecificity of ppv of and youdens indexof in diagnostic test analysis criteria can be combinedin mainly two ways sensitive criteria can be taken together to improve specificity or specific criteria canbe accumulated to improve sensitivity when combining the highly specific criteria size cutoff value mm inhomogeneous signal intensity and number ofvisible lymph nodes n ¥ a highly significant association with nodal metastases pn p wasfound consequently the ct examination was considered nodepositive cn when at least one of thesecriteria was met the application of this criterion improved the sensitivity to with a remaining specificity of and ppv resulting in an alsoimproved youdens index of the results of the mri examinations must be viewedin a rather descriptive manner since the sample size waslimited n lymph nodes were detected in the majority of examinations generally allowing theevaluation of lymph nodes by mri as well upper abdominal mri generally has a lower spatial resolutionbut a higher soft tissue contrast compared to ct forthe size criterion a cutoff value of mm or mm ledto the best diagnostic results sensitivity specificity youdens index lymph nodes of abnormal morphological criteria were seen in only veryfew patients table the main limitation of this study is the retrospectivestudy design in which a nodebynode comparison ofcrosssectionalimaging with histopathology was notpossible this was of minor importance though sincelow sensitivity was the main factor that led to compromised diagnostic performance in our study we werealso able to correlate with postoperative crosssectionalimaging in all cases in which it was unclear whether alymph node had been resected during surgery or notalso in our cohort only patients who had subsequentsurgery were included presuming lower tumor stage ascompared to the average patient who undergoes imagingfor presurgical workupthe strength of our singlecenter study is reinforcedby a defined number of surgeons a high standardizationof the ct technique and an experienced radiologist whoperformed the analysisthe results of our study are consistent with recent andinitial data demonstrating that clinical staging by lowsensitivity underestimates histopathological lymph nodeinvolvement pn [ ] however by adding thecriterion inhomogeneous signal intensity and numberof visible lymph nodes to the size criterion we wereable to increase the sensitivity to in comparison toprevious findings roche nanashima 0cloch world of surgical oncology page of and cao with specificity remainingsufficientan additional imaging modality that has shown the potential to improve the sensitivity of detecting metastaticdisease is positron emission tomographycomputed tomography petct however a beneficial role ofpetct in locoregional nodal staging could not be established to date the majority of initial as well as recentstudies show very limited sensitivities for nodal status between and [] the petpanc study evaluated the incremental diagnostic accuracy and impact ofpetct in addition to multidetector ct in patients withsuspected pancreatic cancer in a prospective multicenterstudy that included patients in this study significantlymore patients with stage iib disease pn were correctlystaged by petct than by multidetector ct p but this only led to a moderate sensitivity of for petct versus for multidetector ct endoscopic ultrasonography eus is a wellestablisheddiagnostic procedure in pancreatic cancer with the benefitof a dynamic diagnostic examination that allows fineneedle aspiration for cytologic diagnosis two metaanalyses evaluating diagnostic accuracy of eus for locoregional nodal staging the pooled sensitivities and specificities were and li studies n patients and and nawaz studiesn patients advanced techniques such ascontrastenhanced eus cheus and eus elastographyare currently in evaluation to date ct remains the standard staging imaging modality recommended by nccn guidelines for locoregional staging of pancreatic cancer neither petctnor eus yields reliable diagnostic accuracy for nodalstagingan advantageous effect on resectability and overallsurvival os in unresectable cases including both borderline resectable and unresectable of pdac by multimodality therapy including neoadjuvanttherapy hasalready been described in several studies the benefit of neoadjuvant therapy in cases of primarily resectable disease at diagnosis is yet less revealedseveral phase ii trials showed that patients who completed neoadjuvant chemoradiation without progressivedisease at restaging had a higher chance of achieving r0resection and consequently higher median and oswhen compared to historical data as seen in othertumor entities a potential benefit of neoadjuvant therapyon the basis of positive nodal status cn is stronglyimplied cao found that the of patients thatwere successfully downstaged from nodepositive diseasecn1 to nodenegative disease ypn0 by neoadjuvanttherapy benefit in terms of higher rates of 5year survivalypn0 vs ypn1 p this is consistent with the findings of portuondo 5yearsurvival ypn0 vs ypn1 p the nccn guidelines for pancreatic adenocarcinomaappreciates these results by stating that considerationcan be given to neoadjuvant therapy for selected patientswith resectable tumor but poor prognostic features suchas large regional lymph nodes markedly elevated ca large primary tumors extreme pain and excessiveweight loss further clarification on this matter isexpected to come from the ongoing neonax trialnct02047513 a phase ii study comparing neoadjuvant plus adjuvant with only adjuvant nabpaclitaxelplus gemcitabine therapy forresectable pancreaticcancer theiii neopa trialphasenct01900327 compares neoadjuvant chemoradiotherapy with upfront surgery of resectable pancreatichead cancer a subgroup analysis in terms of nodalstatus would present reliable dataongoinggiven the suggested benefit of neoadjuvant therapybased on lymph node staging there is an urgent need tofind criteria and modalities to further improve the diagnostic value of lymph node staging by pretherapeuticcrosssectional imaging in patients with ductal adenocarcinoma of the pancreatic head to date none of theexisting modalities and criteria accomplishes reliablenodal staging larger prospective studies are ongoingand necessary to get a more precise idea of the prognostic advantage of neoadjuvant therapy in patients with regionalcn of pdac inpretherapeutic staginglymph node metastasisslymph node staging in pdac patients when using ctmorphological criteria such as border contour or homogeneity compared to diameter cutoff values does notlead to reliable diagnostic value diagnostic accuracy islimited due to low sensitivity for detection of metastaticdisease combining specific criteria yields improved sensitivity with specificity and ppv remaining high theseresults suggest an attentive interpretation of the resultsof pretherapeutic lymph node staging particularly incases in which lymph node metastases are absentabbreviationspdac adenocarcinoma of the pancreatic head ct computed tomographymri magnetic resonance imaging pn histopathologically involved lymphnodes pn histopathologically no involved lymph nodes cn clinicallyinvolved lymph nodes cn clinically no involved lymph nodes ppv positivepredictive value petct positron emission tomographycomputed tomography eus endoscopic ultrasonography cheus contrastenhancedendoscopic ultrasonography os overall survivalacknowledgementswe acknowledge support from the german research foundation dfg andthe open access publication funds of charit universittsmedizin berlinauthors contributionsall authors were involved in data acquisition and manuscript revision theconception of the design of the study and drafting of the manuscript was 0cloch world of surgical oncology page of done by fn loch c kamphues and p asbach image analysis was performedby p asbach and assisted by fn loch image analysis of the subgroup foranalysis of interobserver agreement was performed by m haas all authorshave approved the submitted version of the manuscript and account fortheir own contribution and the accuracy as well as the integrity of the workpresentedfundingthe study was not fundedavailability of data and materialsthe datasets used during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study was approved by the local ethics committee of the charituniversity medicine berlin informed consent of participation was waived bythe irb given the retrospective study design irb no ea411418consent for publicationconsent for publication is not applicable for this studycompeting intereststhe authors declare that they have no competing interestsauthor details1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germany 2charit universittsmedizin berlin corporatemember of freie universitt berlin humboldtuniversitt zu berlin and berlininstitute of health department of radiology campus benjamin franklinhindenburgdamm berlin germany 3the johns hopkins universityschool of medicine department of surgery n wolfe street blalock baltimore md usareceived december accepted july zeman rk cooper c zeiberg as kladakis a silverman pm marshall jl tnm staging of pancreatic carcinoma using helical ct am jroentgenol m¼ller mf meyenberger c bertschinger p schaer r marincek b pancreatictumors evaluation with endoscopic us ct and mr imaging radiology midwinter mj beveridge cj wilsdon jb bennett mk baudouin cj charnleyrm correlation between spiral computed tomography endoscopicultrasonography and findings at operation in pancreatic and ampullarytumours br j surg r¶sch t braig c gain t feuerbach s siewert jr schusdziarra v staging of pancreatic and ampullary carcinoma by endoscopicultrasonography comparison with conventional sonography computedtomography and angiography gastroenterology prenzel kl h¶lscher ah vallb¶hmer d drebber u gutschow ca m¶nig sp lymph node size and metastatic infiltration in adenocarcinoma of thepancreatic head eur j surg oncol rollvn e blomqvist l ¶istm¶ e hjern f csanaky g abrahamnordling mmorphological predictors for lymph node metastases on computedtomography in colon cancer abdom radiol brown g richards cj bourne mw newcombe rg radcliffe ag dallimorens morphologic predictors of lymph node status in rectal cancer withuse of highspatialresolution mr imaging with histopathologic comparisonradiology kim jh beets gl kim mj kessels agh beetstan rgh highresolution mrimaging for nodal staging in rectal cancer are there any criteria in additionto the size eur j radiol isaji s murata y kishiwada m new japanese classification of pancreaticcancer in neoptolemos j urrutia r abbruzzese j b¼chler mw editorspancreatic cancer new york springer p brierley jd gospo | 0 |
" colorectal cancer crc is the third leading cause of cancerassociated mortality the present study aimed to investigate novel biomarkers to predict prognosis and provide a theoretical basis for studies of the pathogenesis and the development of therapies for crc the present study compared mrna expression levels of patients with crc with short and longterm prognosis and of individuals with and without tumors in the cancer genome atlas tcga database differentially expressed genes degs were identified via volcano plot and venn diagram analysis gene ontology go analysis and gene set enrichment analysis gsea were performed to identify the functions of the degs and the degs were further verified using clinical crc samples a total of degs were identified as candidate genes using the tcga database and four degs [defensin 4a defb4a hyaluronan binding protein habp2 oleoylacp hydrolase and tbc1 domain family member 3g] were associated with poor prognosis of patients with crc two degs defb4a and habp2 were upregulated in tumor tissues of patients with crc in the tcga database go and gsea analyses revealed that defb4a was highly associated with immunosuppression participates in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity and was positively correlated with cd11b cd14 cd45 cd163 and il17a furthermore defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis survival analysis revealed that defb4a upregulation was associated with poor prognosis defb4a correspondence to dr yi zhang biotherapy center the first affiliated hospital of zhengzhou university jianshe east road zhengzhou henan pr chinaemail yizhangzzueducncontributed equallykey words defensin 4a colorectal cancer prognosis immunity biomarkergene knockdown experiments demonstrated that def4ba promotes cell migration these results indicated that defb4a potentially promotes tumor growth by regulating immunosuppressive activity and provided novel insights into the diagnosis and treatment of crcintroductionaccording to the global cancer statistic of colorectal cancer crc is the third most common cancer and the second leading cause of cancerassociated mortality worldwide crc was reported as the fourth most common and fatal cancer in china in patients with crc usually have a low survival rate and poor therapeutic responses and are susceptible to progression and recurrence early diagnosis and effective treatment are critical to improve the survival of patients with crc crc studies have focused on innovative ideas to identify molecular markers used to develop highprecision noninvasive screening tests for crc to increase population compliance and reduce the potentially harmful side effects associated with more invasive techniques diagnostic markers will give an indication of the likely progression of the disease targeting specific molecules in certain patients has facilitated more personalized treatments that help prevent or decelerate cancer progression the present study aimed to determine prognostic factors and novel therapeutic targets to improve the survival of patients with crcprevious studies have focused on the identification of molecules associated with tumor progression through genetic or mrna profiling and screening of patients with colon cancer for example the expression profiles of long noncoding rnas lncrnas were compared at specific tumor stages t0 t1 t2 and t3 in an azoxymethanedextran sodium sulfateinduced primary colon cancer model and upregulation of the lncrna h19 predicted a poor prognosis other studies analyzed microrna mir expression profiles between tumor tissues and matched nontumor tissues obtained from patients with crc for example mir is significantly downregulated in tumor tissues and associated with poor survival of patients with crc and may thus be considered to be a poor prognostic marker of crc furthermore high expression levels of mir 0cwu prognostic role of defb4a in colorectal cancerand mir in serum are associated with poor survival of patients with crc analysis of the cancer genome atlas tcga database revealed that mmp19 is upregulated in patients with crc and is associated with tumor progression however to the best of our knowledge no study has directly screened mrna profiles based on prognosis the present study divided patients with crc into different groups based on prognosis and screened the mrna profiles of the respective groups defensin 4a defb4a also known as bd sap1 defb2 defb4 hbd defb and defb102 belongs to the defensin family comprising cytotoxic peptides secreted by neutrophils which serve important roles in innate immune defense against microbial infections defb4a is upregulated in cutaneous squamous cell carcinoma and basal cell carcinoma it serves an important role in esophageal carcinogenesis both in vivo and in vitro the genomic copy number of defb4a has been analyzed in patients with crohn's disease and controls and an elevated defb4a copy number has been identified as a risk factor for crohn's disease regardless of disease origin however it remains unclear whether defb4a expression is associated with the prognosis of crc furthermore the role of defb4a in the immune system remains unclear the tumor microenvironment serves a significant role in tumor progression various immune elements comprise the tumor microenvironment including bone marrowderived cells such as macrophages cd4 t cells cd8 t cells b cells natural killer cells and dendritic cells myeloid cells can differentiate into macrophages or myeloidderived suppressor cells mdscs which serve a tumorigenic role in the tumor microenvironment mdscs contribute to tumor vascular development by promoting angiogenesis and tumor growth tumorassociated macrophages tams are important regulators of tumorigenesis by inhibiting the antitumor effects of other cells thus promoting tumor growth however it remains unclear whether defb4a has a regulatory effect on the tumor microenvironment or whether it promotes crc progressionto identify candidate target genes that potentially prolong patient survival mrna expression profiles of tissues from crc samples were compared in the tcga database venn analysis was performed to determine candidate genes upregulated in tumor tissues among patients with poor prognosis subsequently immuneassociated pathway enrichment was analyzed using gene ontology go and gene set enrichment analysis gsea and the correlations between candidate target genes and certain immune cells were determined finally clinical samples and crc cell lines were obtained to verify the clinical significance of the identified genes the present results may provide insights into targeted therapy for crcmaterials and methodsacquisition of microarray data microarray data were obtained from tcga httpcancergenomenihgov rnaseq data for samples were included in the dataset project id tcgacoadread including tumor samples from patients with crc and normal tissues from healthy donorsidentification of differentially expressed genes degs tcga data were divided into two groups based on different categories patient prognosis and gene expression in tumor and normal tissues venn analysis of the two groups was performed and genes associated with crc prognosis were identifiedvenn analysis to identify candidate genes associated with patient survival the gene expression profiles in the two groups were analyzed using the venn diagram web tool httpbioinformaticspsbugentbewebtoolsvenngo analysis functional analysis of the degs was performed using go httpwwwgeneontology based on biological processes gsea gsea was conducted using gsea v403 software wwwgseamsigdbgseaindexjsp and the gene used in the present study was downloaded from the molecular signatures database msigdb oftware broadinstitutegseamsigdbindexjsp v40 msigdb curates various gene sets including canonical signaling pathways from biocarta cgapncinihgovcgap_mitelman_retire_noticehtml kyoto encyclopedia of genes and genomes wwwkeggjp pid httppidncinihgov reactome reactome and other pathway databases tcga data were analyzed via gsea and pathways with a false discovery rate fdr were considered significantpatient characteristics tissue samples were obtained from patients with crc at the first affiliated hospital of zhengzhou university zhengzhou china between april and april patients underwent surgical resection or colonoscopy and the samples were verified via pathological analysis the clinical characteristics of the patients are shown in table i a total of men and women were included in the present study the median age was years age range years crc was diagnosed by two pathologists on the basis of pathological assessment the collection of specimens was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university zhengzhou china approval no sciencelw and informed consent was obtained from each patient with available followup informationreverse transcriptionquantitative pcr total rna was extracted from pairs of tumor and normal tissue samples from patients with crc using trizol reagent invitrogen thermo fisher scientific inc total rna samples µg were incubated at Ëc for min followed by incubation at Ëc for min and Ëc for sec according to the reverse transcription reaction protocol takara biotechnology co ltd the conditions of pcr were as follows Ëc10 min Ëc10 sec Ëc10 sec Ëc10 sec cycles premix ex taq ii roche target gene expression was simultaneously assessed relative to that of gapdh a housekeeping gene and internal control the following primers were used defb4a forward 'ctc ctc ttc tcg ttc ctc ttc a' and reverse 'gca ggt aac agg atc gcc tat' and gapdh forward 'gga gcg aga tcc ctc caa aat' and reverse 'ggc tgtt 0concology letters no of cases table i characteristics of patients with colorectal carcinomacharacteristic percentagesex male female age years ¥ treatment surgery others tumor size mm ¥ pathological type adenocarcinoma others lymph node metastasis yes no tnm stage i ii iii iv liver metastasis negative positive differentiation poor mediumwell others include chemotherapy and radiotherapy gtc ata ctt ctc atg g' the present study compared the expression levels of the target genes in clinical samples using the δδcq method expression levels of defb4a and gapdh were examined for each sample and the relative expression levels of defb4a were determined using the δcq value of defb4a divided by that of gapdh cell transfection sw480 and hct116 cells were seeded into a well plate sw480 and hct116 cells were purchased from chinese academy of sciences cell bank and cultivated with dmemhigh glucose containing fbs hyclone ge healthcare life sciences and penicillinstreptomycin at Ëc in co2 the growth status of the cells was closely observed until they reached a fusion rate of and then cells were transfected with ncsmall interfering rna negative control sinc sense 'uuc ucc gaa cgu guc acg utt' and antisense 'acg uga cac guu cgg aga att' and small interfering rna targeting defb4a sidefb4a sidefb4a sense 'ucc ucu uca uau ucc uga utt' and antisense 'auc agg aau aug aag agg att' purchased from shanghai genepharma co ltd with jetprime polyplus transfection reagent polyplustransfection sa after h the medium was changed to fresh medium and cells were further incubated in co2 for h subsequently cells were collected for the subsequent experimentswound healing assay for the wound healing assay sw480 and hct116 cells were cultured in µl medium with fbs hyclone and the percentage of serum was in line with previous papers subconfluent tumor cells were scraped using a sterile micropipette tip and then serumfree medium was added next cells were imaged at and h using an inverted fluorescence microscope magnification x200 olympus corporation transwell assay in the migration test the transfected cells 1x105 were inoculated into the top chamber microns with µl serumfree medium complete medium µl containing fbs was added to the lower chamber corning inc following incubation at Ëc for h the migratory cells located under the insert were fixed and stained with crystal violet staining solution beyotime institute of biotechnology at room temperature for min and observed using an inverted fluorescence microscope magnification x200 olympus corporationstatistical analysis the Ï2 test was used to compare clinicopathological factors and continuous variables were analyzed via unpaired student's ttest or oneway anova kaplanmeier analysis and the log rank test were performed for survival analysis univariate and multivariate logistic regression models confirmed the associations between defb4a expression and clinical features prism graphpad software inc was used for statistical analysis of all clinical samples anova was followed by tukey's posthoc test and performed using spss for windows spss inc r software version r foundation for statistical computing was used for bioinformatics analysis p005 was considered to indicate a statistically significant difference all experiments were performed in triplicate and data are presented as the mean ± standard deviation resultsgenes associated with poor prognosis to identify genes associated with poor survival in the crc cohort patients were divided into two groups based on os short ¤ days os patients with a survival time of days would be included in short os and long days os fig 1a in total degs fold change were identified using a volcano plot including upregulated and downregulated genes fig 1b and c hierarchical cluster analysis revealed the expression profiles of the degs fig 1d subsequently gene expression profiles in tumor and normal tissues fold change were analyzed and it was observed that genes were upregulated and were downregulated in tumor tissues compared with in normal tissues p005 fdr fold change fig 1eg the venn diagram revealed that 0cwu prognostic role of defb4a in colorectal cancerfigure screening of degs based on tcga a patients with crc were divided into two groups based on whether or not they survived for days in accordance with the rnaseq data from tcga b volcano plot of rnaseq data from tcga the red dots and blue dots represent upregulated and downregulated degs based on a fold change of the volcano plot displays different genes when comparing patients with a prolonged os and those with a short os c a total of upregulated and downregulated genes were identified d hierarchical clustering analysis of the rnaseq data of different genes in short os and long os samples e hierarchical clustering analysis of the rnaseq data of different genes in ca and n samples f using a threshold of p005 false discovery rate and fold change degs were selected using a volcano plot when comparing ca samples with normal colon mucosa samples from tcga g a total of upregulated and downregulated genes were identified h venn diagram representing the distribution of degs in different groups a total of degs were expressed in both patients with ca and patients with a prolonged os crc colorectal carcinoma degs differentially expressed genes os overall survival tcga the cancer genome atlas ca cancer n normal degs were identified in both screening methods fig 1h details are shown in table iivalidated degs are associated with poor prognosis in tcga to determine the prognostic significance of the identified degs their expression levels were determined in cases included in tcga kaplanmeier survival analysis revealed that defb4a hyaluronan binding protein habp2 oleoylacp hydrolase olah and tbc1 domain family member 3g tbc1d3g upregulation was significantly associated with poor survival in patients with crc fig 2a prognostic significance was not observed for kiss1r or5m11 chrnb3 otx2 s100a7a flj43860 and frmd7 in the patients with crc data not shown furthermore defb4a and habp2 were upregulated in tumor tissues fig 2b a previous study have reported low serum expression levels of habp2 in patients with crc therefore defb4a was selected as a candidate marker of poor prognosis in patients with crcgo and gsea to evaluate the biological role of defb4a in crc progression go enrichment and gsea analyses were 0concology letters descriptiontable ii upregulated genes n10 associated with a poor colorectal carcinoma prognosis in the cancer genome atlas databasegene symbol defb4a habp2 olah tbc1d3g kiss1r frmd7 s100a7a otx2 or5m11 chrnb3 defensin beta 4ahyaluronan binding protein oleoylacp hydrolasetbc1 domain family member 3gkiss1 receptorferm domain containing s100 calcium binding protein a7aorthodenticlehomeobox olfactory receptor family subfamily m member cholinergic receptor nicotinic beta subunitgene id performed defb4a was demonstrated to be involved in various biological processes associated functional pathways are shown in fig 3a and b and closely associated with myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity implying that defb4a potentially regulates the immune system finally the database was searched for expression profiles of defb4a and immunerelated genes and a positive correlation between defb4a expression and the expression of immune markers such as cd11b cd14 cd45 cd163 and il17a was observed fig 3c these results suggest that defb4a is associated with poor prognosis in patients with crc potentially in an immunosuppressive myeloid leukocyte and cytokinedependent mannervalidation in patient samples and clinical relevance of defb4a to further clarify the clinical significance of defb4a expression the present study analyzed tissue samples from patients with crc the associations between their mrna expression levels and clinicopathological variables were observed detailed information of the patients is provided in table iii defb4a expression was significantly upregulated in the crc tumor tissues fig 4a additionally an association between defb4a upregulation and advanced crc stage stage i cases stage ii cases stage iii cases stage iv cases and metastasis m0 cases m1 cases was observed fig 4b and c furthermore defb4a upregulation in the tumor tissues was associated with poor prognosis p00313 fig 4d additionally defb4a upregulation was significantly associated with advanced liver metastasis p0039 stage p0005 high ca72 value p0003 tumor size p0009 and lymph node metastasis p0044 table iii therefore defb4a was considered to be a prognostic marker associated with tumor progression in patients with crc logistic regression analysis was performed to determine whether defb4a can help predict the prognosis of crc univariate analyses revealed that advanced tnm stage [odds ratio or p001] liver metastasis or p003 lymph node metastasis or p004 high ca199 level or1324 p002 a high ca level or p001 and high defb4a level or p002 were associated with the survival of patients with crc furthermore multivariate analyses revealed that advanced tnm stage or p004 histological differentiation or p001 liver metastasis or p001 ca199 level or p001 high ca level or p005 and high defb4a level or p001 were independent prognostic predictors table iv overall these results suggest that defb4a serves an important role in predicting the prognosis of patients with crcdefb4a promotes proliferation and metastasis in crc to explore the biological roles of defb4a in crc defb4a expression was knocked down in hct116 and sw480 cells fig 5a and b a wound healing assay revealed that defb4a knockdown inhibited the migration of hct116 and sw480 cells fig 5c transwell assays demonstrated that the migration of cells was decreased following knockdown of defb4a in sw480 cells compared with that in the nc group fig 5d the number of migratory cells decreased following knockdown of defb4a in sw480 cells fig 5e overall these results suggested that defb4a serves an important role in crc development discussionwith the increasing availability of highthroughput technologies numerous novel biomarkers and therapeutic targets have been identified through transcriptomic analysis of various types of tumor however such studies on biomarkers in crc have not been extensively performed the identification of crc biomarkers may help predict and prolong the survival of patients with crc in the present study mrna profiling of microarray analysis data from the tcga database was performed to identify numerous novel genes associated with poor prognosis in crc a critical role of defb4a in patients with crc was identified the mrna profiles of patients were first compared between the long os and short os groups and between the tumor and normal tissue groups in the tcga database subsequently the present study investigated the association between mrna expression and prognosis defb4a habp2 olah and tbc1d3g were identified as potential predicators of poor prognosis defb4a and habp2 0cwu prognostic role of defb4a in colorectal cancerfigure defb4a is upregulated based on data from tcga and predicts poor prognosis a kaplanmeier curve of four genes defb4a habp2 olah and tbc1d3g derived from data of patients included in the tcga dataset b mrna expression levels of defb4a habp2 olah and tbc1d3g in cancer vs control samples from patients in tcga p005 ns not significant tcga the cancer genome atlas defb4a defensin 4a habp2 hyaluronan binding protein olah oleoylacp hydrolase tbc1d3g tbc1 domain family member 3g ca cancer n normalwere upregulated in crc tissues of patients in the database however habp2 has been reported to be downregulated in the sera of patients with crc p00137 therefore defb4a was considered as a candidate gene for further analysis go and gsea were used to assess the function of defb4a in promoting disease progression and to highlight the role of defb4a in the tumor microenvironment defb4a was involved in myeloid leukocyte differentiation leukocyte proliferation and leukocyte mediated immunity correlation analysis revealed that defb4a expression was positively correlated with immune markers including cd11b cd14 cd45 cd163 and il17a cd11b is expressed on the surface of a number of leukocytes including monocytes granulocytes and macrophages cd14 is expressed on both monocytes 0concology letters figure defb4a is positively correlated with inhibitory immune cells a gene ontology analysis revealed that defb4a is involved in leukocyte proliferation lymphocyte differentiation leukocyte mediated immunity myeloid cell differentiation negative regulation of type i interferon production and interleukin production b gene set enrichment analysis verified the results c correlation between defb4a and cd11b cd14 cd45 cd163 and il17a r and pvalues are indicated defb4a defensin 4a r pearson's correlation coefficientfigure defb4a predicts poor prognosis in colorectal cancer a defb4a mrna expression in groups of cancer tissues and normal tissues are displayed b defb4a mrna levels were compared with respect to tnm stage c defb4a mrna expression levels of patients with different cancer stages d effect of defb4a expression on overall survival in patients with colorectal carcinoma n52 p005 p001 p00001 defb4a defensin 4aand macrophages and cd45 is expressed on leukocytes m2 macrophages may be marked with cd163 and m2 macrophages serve a role in promoting tumor growth the os of patients with nonsmallcell lung cancer and those with esophageal cancer with high m2 macrophage infiltration rates is shorter than those with low m2 macrophage infiltration rates patients with high expression levels of il17a had a poor prognosis in a crc cohort previous studies have suggested that increased il17a promotes crc in various animal models analysis of clinical specimens of patients with crc demonstrated that defb4a expression was associated with 0cwu prognostic role of defb4a in colorectal cancer 0330a total n Ï2 pvaluedefb4a expressiontable iii association between defb4a expression and clinicopathological characteristics of patients with colorectal carcinoma characteristic sex male female age years ¥ site of lesion colon rectum differentiation poor well tumor size cm ¥ pathological type adenocarcinoma others lymph node metastasis no yes liver metastasis no yes stage iii iiiiv cea normal high ca normal high ca normal high afisher's exact test all others were assessed using a Ï2 test defb4a defensin 4ahigh n low n 0009a0003a 0575a0039a poor survival furthermore defb4a expression was upregulated in patients with crc with advanced and metastatic cancer patients with crc with high defb4a expression had poor survival in addition knockdown of defb4a affected the migration ability of crc cells tcga data of patients with crc were used to identify the degs between the long os days and short os days groups in addition mrna expression was compared between tumor tissues and normal tissues in the same database defb4a was highly expressed in tumors and associated with a poor prognosis defb4a upregulation was associated with poor prognosis and defb4a expression was significantly upregulated in patients with large tumors advanced cancer stage lymph node metastasis and liver metastasis another study used the gene expression omnibus database to screen genes that are increased in patients with recurrence hierarchical clustering and pathway analyses revealed that thrombospondin thbs2 and cartilage 0concology letters table iv logistic regression model analysis of liver metastasis predictors in patients with colorectal carcinoma characteristics sex male vs female age vs ¥ years tumor size vs ¥ mm pathological type adenocarcinoma vs others tnm stage iii vs iiiiv differentiation medium vs poor liver metastasis no vs yes lymph node metastasis no vs yes cea vs ¥ ca199 vs ¥ ca724 vs ¥ defb4a high vs low or odds ratio or pvalue univariate ci or pvalue multivariate ci figure defb4a promotes colorectal cancer cell migration a expression levels of defb4a were detected using rtqpcr following transfection of hct116 cells with sinc and sidefb4a b expression levels of defb4a were detected using rtqpcr following transfection of sw480 cells with sinc and sidefb4a c migration ability of cells was examined using a wounding healing assay d representative images were obtained for the transwell assay magnification x200 e proportions of migrated cells after h were quantified defb4a defensin 4a nc negative control rtqpcr reverse transcriptionquantitative pcr si small interfering rna p001 p0001 0cwu prognostic role of defb4a in colorectal canceroligomeric matrix protein comp are associated with the ecmreceptor interaction focal adhesion and tgf signaling pathways the hypergeometric distribution test demonstrated that the association between thbs2 and crc is stronger than that of comp pearson test results indicated that thbs2 might be considered to be a prognostic biomarker for crc to the best of our knowledge this screening method and the hypothesis that defb4a may serve a protumor role through immunosuppression have not been seen in other studies defb4a stimulates keratinocytes to release il and il proinflammatory cytokines serving as deciding factors in the pathogenesis of psoriasis furthermore defb4a induction is required for tolllike receptor tlr activation in monocytes through the convergence of il and vitamin d receptor signaling and exerts direct bactericidal effects against m tuberculosiss the antimicrobial peptides defb4a and camp are inhibited by hsamir leading to suppression of the tlr21induced vitamin d antimicrobial signaling pathway defb4a has been suggested as a biomarker for psoriasis because the clinical efficacy of targeted antibody therapy in psoriasis is associated with the inhibition of defb4a expression defb4a expression can directly be inhibited by anthralin in vitro and in vivo thus benefiting patients with psoriasis however it has remained unclear whether defb4a is involved in the immunoregulation in crc go analysis revealed that defb4a is involved in myeloid leukocyte differentiation leukocyte proliferation and positive regulation of leukocytemediated immunity therefore defb4a may be associated with immunity in crc to the best of our knowledge the present study was the first to report defb4a as a prognostic marker for crc and as an immunoregulatory factor in the tumor microenvironment in patients with crc however a limitation of the present study was that the research cohort was not large enough which may affect the statistical results in addition the specific role of defb4a and immune factors in colon cancer and the underlying molecular mechanism need to be further exploredin conclusion to the best of our knowledge defb4a is upregulated in patients with crc and is closely associated with poor prognosis defb4a regulates immune function and potentially promotes immunosuppression therefore defb4a may be considered as a prognostic marker and immunotherapeutic target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos u1804281 and and funding from state's key project of research and development plan grant no 2016yfc1303500availability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author on reasonable requestauthors' contributionsyz qw and dw participated in the design and conception of the present study yz qw dw zs jl and wty were involved in data acquisition and analysis of certain clinical data qw dw zz and yw performed the clinical experiments and analysis of the data the manuscript was written by qw and critically reviewed by yz dw zz yw wny and nrm wny ks and nrm were involved in performing and analyzing the cell experiments all authors read and approved the final manuscriptethics approval and consent to participatethe present study was approved by the institutional ethics committee of the first affiliated hospital of zhengzhou university approval no sciencelw and informed consent was obtained from each patient with available followup informationpatient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences siegel rl miller kd and jemal a cancer statistics ca cancer j clin cidon eu the challenge of metastatic colorectal cancer clin med insights oncol vreeland tj clifton gt herbert gs hale df jackson do berry js and peoples ge gaining ground on a cure through synergy combining checkpoint inhibitors with cancer vaccines expert rev clin immuno siegel rl miller kd fedewa sa ahnen dj meester rgs barzi a and jemal a colorectal cancer statistics ca cancer j clin dickinson bt kisiel j ahlquist da and grady wm molecular markers for colorectal cancer screening gut nikolouzakis tk vassilopoulou l fragkiadaki p mari | 0 |
Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigators discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA level by ¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics years years ¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had exceptional responses defined as normalisation or ¥ decline of CA patients or normalisation PET SUVmax in In Arm B had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an exceptional CA response and had an exceptional PET response as defined by either ¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection statusAAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA decrease FDG PET SUVmax decrease ¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options | 2 |
" the development of a safe effective reversible nonhormonal contraceptive method for men hasbeen an ongoing effort for the past few decades however despite significant progress on elucidating the functionof key proteins involved in reproduction understanding male reproductive physiology is limited by incompleteinformation on the genes expressed in reproductive tissues and no contraceptive targets have so far reachedclinical trials to advance product development further identification of novel reproductive tractspecific genesleading to potentially druggable protein targets is imperativeresults in this study we expand on previous single tissue single species studies by integrating analysis of publiclyavailable human and mouse rnaseq datasets whose initial published purpose was not focused on identifyingmale reproductive tractspecific targets we also incorporate analysis of additional newly acquired human andmouse testis and epididymis samples to increase the number of targets identified we detected a combined totalof genes for which no previous evidence of male reproductive tractspecific expression was annotated manyof which are potentially druggable targets through rtpcr we confirmed the reproductive tractspecific expressionof novel orthologous human and mouse genes without a reported mouse model of these we ablated fourepididymisspecific genes spint3 spint4 spint5 and ces5a and two testisspecific genes pp2d1 and saxo1 inindividual or double knockout mice generated through the crisprcas9 system our results validate a functionalrequirement for spint45 and ces5a in male mouse fertility while demonstrating that spint3 pp2d1 and saxo1 areeach individually dispensable for male mouse fertilitys our work provides a plethora of novel testis and epididymisspecific genes and elucidates thefunctional requirement of several of these genes which is essential towards understanding the etiology of maleinfertility and the development of male contraceptiveskeywords contraception drug target male reproductive tract paralog sperm maturation spermatidspermatozoa correspondence coarfabcmedu thomasgarciabcmedu1dan l duncan comprehensive cancer center baylor college of medicine baylor plaza houston tx usa3department of pathology and immunology baylor college of medicine baylor plaza houston tx usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crobertson bmc biology page of the world human population reached nearly eight billion people in august this number continues torise and is predicted to reach nearly ten billion by theyear the increasing need to promote familyplanning through the development of reliable contraceptive options available to both men and women is widelyrecognized currently there are numerous contraceptiveoptions available to women however identification of asafe nonhormonal contraceptive option for men is stillan ongoing challenge although several different fertilitycontrol alternatives for men have been investigatednone are currently clinically approved for use our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as theidentification and elucidation of the function of key reproductive proteins is still an ongoing effort identifyingdruggable protein targets expressed in the male reproductive tract has been the focus of numerous studiesdedicated to the development of male contraceptionessentialroletheitsthe mammalian epididymis is a segmented ancomprised of a single highly coiled tubule with functionally and morphologically distinct regions that can besubdivided most simplistically into a proximal centraland distal region conventionally named the caput corpus and cauda regions respectively as mammalianspermatozoa transit through the epididymis they acquire the ability to recognize and fertilize an egg properties that they did not possess upon exiting the testis epididymisinconsideringaddition to maturing germ cells of the testis and spermatozoais a prime target for the development of a malecontraceptive to advance progress towards the development of a nonhormonal male contraceptive several previous highthroughput studies have been published thatidentified a number of human mouse and rat genes astestisspecific or epididymisspecific [ ] in schultz conducted the first study to identify malereproductive tractspecific genes using microarraysthroughgeneexpression analysis of meiotic and postmeiotic spermatogenic cells together with parallel analysis of available data from the ncbi unigene database the authorsidentified mouse genes as testisspecific which included genes with both known and unknown function atthe time in the following years through two additional microarraybased studies of rat testes and purifiedrat testicular cells johnston identified and additional or overlapping genes astestisspecific in as part of the continued effort to identify novel contraceptive targets the newer rnaseqbased transcriptomics methodology was utilized identifying human genes as testisspecific together withantibodybased protein profiling many of these genesaffymetrixbasedgenomewidewere characterized in terms of the spermatogenic cellpopulations showing expression the first highthroughput transcriptomics study to identify epididymisspecific genes was a mouse epididymal transcriptome studyin which rna isolated fromeach of the epididymal segments was analyzed bymicroarray analysisidentifying epididymisspecificgenes with distinct patterns of segmental gene regulation later in additional transcriptome profiling utilizing whole genome microarrays resulted in identification of previously unreported epididymisspecific transcripts inthe mouse and epididymisspecific transcripts inthe rat a significant number of the identified mouseand rat genes in these studies were not known at the timeand only the probe identification numbers were presentedwhen evaluating potential druggability in a targetbased drug discovery process one must consider theprotein properties that are required for safe and effectiveinhibition among the most significant is tissue expression specificity to minimize potential adverse effectsprotein function and whether protein activity or interaction with other proteins is potentially druggable sequence similarity to closely related paralogs that may beubiquitously expressed and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest several noteworthyreview publications have mentioned numerous geneswhose critical functions high expression and specificityto the testes or epididymides make them viable nonhormonal male contraceptive targets [] howeveramong the identified genes a significant number either are required for fertility but are expressed in nonreproductive tissues or are reproductive tractspecific but when disrupted lead to subfertility ineither case both are ineffective and highly undesirableoutcomes for a potential male contraceptive targettherefore the identification of additional novel male reproductive tractspecific genes would allow for furtheradvances to be made in the quest to develop an effectiveand safe nonhormonal male contraceptivein this study newly acquired and previously published human and mouse rnaseq datasets [ ]were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tractenriched transcripts additional databases obtained from illuminating the druggable genome mouse genome informatics andensembl biomart were utilized to stratify the resultsinto subgroups based on protein druggability and on theavailability of a mouse model numerous reproductivetractspecific and reproductive tractenriched potentiallydruggable targets for which no published mouse modelexists congruent in expression across both mouse and human datasets were identified through our analysis and 0crobertson bmc biology page of verified through conventional polymerase chain reactionpcr we present the data in a manner that should bemost relevant and of substantialinterest to the malecontraceptive development field since identification ofnew targets worthy of consideration for further functionalanalysis in a knockout animal model and potential drugtargeting continues to be of vast importanceresults wethrough ouridentified four novelepididymisspecific genes spint3 spint4 spint5 andces5a and two novel testisspecific genes pp2d1 andsaxo1 worthy offunctional validation in an animalmodel through the crisprcas9 system we generatedfour individual gene knockouts spint3 ces5a pp2d1and saxo1 and one double knockout mouse modelspint45 revealing an essential requirement for spint4and spint5 in male mouse fertility and the potential utility of pursuing spint4 in humans as a nonhormonalcontraceptive targetresultsstudy approaches and datadespite significant advances in our understanding of thehuman and rodent testis and epididymis transcriptomemostly through microarraybased studies no prior studies have utilized purified human testis cells for the identification of human testisspecific transcripts no priorstudies have utilized the more stateoftheart rnaseqbased transcriptomics methodology for analysis of human epididymisspecific transcripts and no prior studieshave utilized rnaseq analysis of rodent reproductivetissues or cells to identify rodent reproductive tractspecific transcripts to address these gaps in knowledgeand to increase the number of identified reproductivetractspecific genes in both species using the most relevant highthroughput transcriptomics methodology weanalyzed in parallel on a custom bioinformatics pipelinea large number of published and newly acquired humanand mouse rnaseq datasets one hundred and sixtytwo previously published human and previously published mouse rnaseq datasets were retrieved from thesequence read archive sra the sra value for eachsample is listed in additional file table s1 and additional file table s2 we also generated new humanand new mouse reproductive tissue rnaseq samplesgeo accession gse150854 the final dataset is comprised of new and previously published human testisdatasets previously published purified humangerm cell datasets [ ] previously published purified human sertoli cell datasets [ ] new and previously published human epididymis segment datasets previously published mouse testis datasets new mouse epididymis datasets previouslypublished purified mouse germ cell datasets [ ]and previously published purified mouse sertoli celldatasets an additional previously publisheddatasets contributed to the nonreproductive humantissues and previously published datasets contributed to the nonreproductive mouse tissues figure 1a b summarizes all the samples acquired for thestudywe performed a principal component analysis tovisualize the variation in the samples after correcting forbatch effects human reproductive and nonreproductivetissues grouped according to sample type the reproductive tissue samples clustered by tissue type whetheror not they were newly generated or acquired from thesra fig 1c mouse data showed a similar variation inthe samples based on the tissue type fig 1d for bothhuman and mouse reproductive tissues samples separated by whether or not the rnaseq was performed onisolated cells or the whole tissue epididymal tissue wasdistinct from testis tissue in both human and mousefig 1c didentified in the mouseto identify potential male reproductive tractspecificdrug candidates we analyzed the aggregated rnaseqdata to find genes that were statistically significant in expression when compared to the nonreproductive tissuethat had the maximum expression for that gene thisgene list was then further refined by filtering for genesthat were lowly expressed in the nonreproductive tissuethat had the maximum expression for that gene tpmless than or equal to for human tpm less than orequal to for mouse finally this tpm filtered listwas then filtered for the genes that had a reproductivetissue or cell expression value greater than or equal to tpm for human or tpm for mouse fig 2aacross all the reproductive tissues candidate geneswere identified in the human and candidate geneswerefig 2badditional file fig s1 additional file table s3 andadditional file table s4 summarize the differentialfold change identity of the nonreproductive tissue withmaximal gene expression based on the differential geneanalysis fdr average and standard deviation tpm expression values and log2 cpm gene expression value forthe human and mouse samples respectively the resultsfrom the fdr and tpm expression value filtering forthe human and mouse samples are summarized inadditional file table s5 and additional file tables6file table s5 andadditional file table s6 report the log2 fold changefor the reproductive tissue or cell of interest comparedto the tissue with maximal gene expression the genesidentifiedandadditional file table s6 pass the filters in at least oneofinadditional file table s5 and additional file tables6 a value of zero for a given gene and fold expressionthe reproductive tissues or cells ofrespectively additionalinterestsamplesin additionalfile tables5 0crobertson bmc biology page of fig see legend on next page 0crobertson bmc biology page of see figure on previous pagefig summary of the human and mouse rnaseq samples used in the identification of novel male reproductive tractspecific drug targets thernaseq samples used in the human a and mouse b analyses are schematically shown principal component analysis was performed on thehuman c and mouse d nonreproductive and reproductive samples separately the colors of the circles next to the tissues listed in a and bcorrespond to the colors used in the circles for the pca in c and d sample size n values in red andor black denote the number of new redand previously published black samples included in our analysiscomparison indicates that for that comparison the genedid not pass the filters the majority of genes weredownregulated in the reproductive tissue ofinterestcompared to the maximal geneexpressing nonreproductive tissue additional file fig s2 from theanalysis the majority of the candidate genes that passedthe fdr and tpm filters were identified in the testis orspermrelated cells in both human and mouse samplesadditional file fig s2the majority of candidate genes identified in ourscreen that were testisspecific were already identified bythe human protein atlas andor our reanalysis offig identification of candidate drug male reproductive gene targets a diagrammatic representation of overall methodology used to identifyreproductive tractspecific candidate genes in humans genes and in mice genes the maximum gene expression was determinedacross all the nonreproductive tissue samples for each gene for a reproductive tissue or cell sample of interest genes were then filtered forsignificance using a false discovery rate fdr of less than or equal to based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest genes that passed the fdr filter werefiltered such that the average tpm expression value of the maximum expressing nonreproductive tissue was less than or equal to tpm andthe average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to tpm b diagrammaticrepresentation of the number of human and mouse candidate genes in terms of the number of orthologs in the opposite species thenumber of genes previously or not previously identified in a prior transcriptomicsbased drug target report the availability and phenotypicoutcome of any reported mouse models and the number of novel genes without a reported mouse model congruent across both speciesthe main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in the numbers inparentheses comprise the total number of candidate genes that are either epididymisspecific or specific to testis and epididymis but not testisandor testis cellspecific only 0crobertson bmc biology page of the hpa testis datasets additional file fig s3 andadditional file table s7 thirtysix out of the genes that were identified across all the human epididymis tissue were also identified by the human combinednewly acquired and previously published datasets testiscandidate gene list finally the majority of the candidategenes identified from the combined newly generated and previously published human testis datasetswere shared with genes identified from the various testiscell datasets we identified more candidate genes in thenewly generated human epididymis tissues compared topreviously published data out of genes wereunique to the newly generated caput samples comparedto only out of genes which was unique to the previously published samples out of genes were uniquein the newly generated corpus samples compared to out of genes which were unique to the previously published corpus samples and genes were unique to thenewly generated cauda samples compared to genes inthe previously published cauda data with no overlap between the two cauda gene lists additional file fig s3and additional file table s7 there were candidategenes that overlapped between the newly generated human testis samples and mouse testis sample gene listswhile there were candidate genes that overlapped between the previously published human testis sample andmouse testis sample gene lists additional file fig s3and additional file table s7 across all human epididymis tissue samplesincluding the newly generated andpreviously published samples there were genes in common with the combined list of candidate genes across allthe mouse epididymis tissue samples there was a smalloverlap between the human and mouse samples when thenewly generated human caput corpus and cauda tissueswere individually compared to the mouse caput corpusand cauda tissues there was an overlap of and forrespectivelyadditional file fig s3 and additional file table s7this trend was continued for the candidate gene lists derived from the previously published human caput corpusand cauda samples when compared to the candidate genelist from the mouse caput corpus and cauda with and genes in common for the caput corpus and caudacomparisons respectively additional file fig s3 andadditional file table s7 additional file table s7 details the genes that are unique and in common for each ofthe comparisonscorpuscaputtheandcaudato assess the potential usefulness of the candidategenes identified in each human reproductive tissue asdrug targets we assigned the genes to a protein familyie gpcr or ion channel the majority of identifiedgenes were not from a traditional drug target family likekinases or enzymes the testis and germ cell datasetsprovided the most potential targets while the epididymisdatasets provided the fewest additionalfile figs4a the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file table s8 the majority of thecandidate genes do not have a reported mouse modeladditional file fig s4b additional file table s9summarizes mouse model availability for each candidategene identified from human reproductive tissues or cellsfigure shows the complete list of novel human geneswithout a reported mouse model as identified in each ofthe respective cell andor tissue datasets digital pcrsheatmap and conventional pcrs demonstrating expression of a subset of the novel human reproductivetractspecific genes without a reported mouse modelthat we identified are shown in figs and respectively additional file fig s5 shows the complete listof previously identified human genes that remain without a reported mouse model as identified in each of therespective cell andor tissue datasets additional file fig s6 shows the complete list of male reproductivetractspecific human genes for which a previously generated mouse model shows male infertility phenotype asidentified in each of the respective cell andor tissuedatasetsreproductive tractspecific genes identified throughhuman datasetsthrough our bioinformatics analysis of previously published and newly acquired rnaseq datasets we identified a total of genes as reproductive tractspecific inhumans fig of these genes genes do not have amouse gene ortholog while genes have a mousegene ortholog fig of those with a mouse geneortholog have a single gene ortholog have thesame symbolin mouse while have a differentsymbol in mouse while have two or more orthologous mouse genes seventysix human genes had orthologous mouse symbols genes had orthologous mouse symbols and genes fam205a krtap106 magea10 or2ag1 pramef11 pramef2ssx2 ssx3 and ssx4b had greater than orthologous mouse symbols symbols additional file table s5 of the human genes that we identified asmale reproductive tractspecific have not been previously identified in a transcriptomicsbased male reproductive tractspecific study [ ] the sum of ourhuman data confirms the findings of out of genes from djureinovic after reidentificationof gene symbols from reported affymetrix ids and consideration of orthologous genes mouse to human andrat to human our human data confirm the findings of out of genes from johnston out of genes from schultz out of genes fromjohnston out of genes from johnston 0crobertson bmc biology page of fig two hundred and thirtythree novel human reproductive tractspecific genes that each have mouse orthologous genes but with noreported knockout mouse models the listed genes were identified in one or more datasets as indicated in the venn diagram underlined geneswere also identified in our studies as reproductive tractspecific in mouse genes genes written in blue encode either enzymes kinasesgpcrs ogpcrs transporters transcription factors or proteins involved in epigenetic regulation genes genes written in dark red wereidentified in both testis testis andor testis cell and epididymis genes out of genes from johnston and out of genes from jelinsky of the genes that have a mouse gene ortholog have notbeen previously identified as male reproductive tractspecific and of these human genes currently lackmouse phenotype information based on data obtainedfrom ensembl biomart mgi impc and ncbihuman testisspecificthree hundred and eightysix genes were identified astestisspecific through either the reanalysis of djureinovic testis datasets genes identified analysis ofour de novo testis datasets genes identified orboth additional file table s5 three hundred andthirteen genes were congruent across both datasetswhile genes were uniquely identified through our reanalysis of djureinovic s datasets and only genes[ac1363524 ankrd20a1 ankrd62fam230aggtlc2iqcm potec prnt and utf1] wereuniquely identified through our de novo datasetsadditional file table s5 interestingly of the genes we identified through djureinovic s reanalyzed datasets were not previously identified in theirreport or any of the other previous reports [ ]of these genes we randomly verified of thesegenes as testisspecific in humans through conventionalpcr fig we also verified through rtpcr an additional genessuch as allc cdkl3 cox7b2or2h1 and sppl2cthat had been identified throughprevious studies additional file fig s7 of the genes identified through either testis datasets havenot been previously identified of these genes haveone or more mouse orthologs and of these genes arelacking reported phenotype information of the novelgenes lacking a reported mouse model genes encodeenzymes adam20 cpa5 dusp21 naa11 plscr2prss38 and triml1 encode transcription factorsbhmg1znf560znf729 encode transporters slc22a14 slc25a52and encode proteins of unknown drug target typetgif2lyfoxr2prdm9 0crobertson bmc biology page of fig representative novel reproductive tractspecific human a and mouse b genes without a reported mouse model the listed genes wereidentified through our studies as reproductive tractspecific in both humans and mice the digital pcr heatmap depicts the average transcriptsper million tpm value per tissue per gene from the indicated human and mouse rnaseq datasets as processed in parallel through ourbioinformatics pipeline the data was obtained from published and newly acquired datasets white tpm black ¥ tpm the expressionprofile of the human and mouse housekeeping genes gapdh and eif3l is included as reference for data obtained from published datasetssuperscript values succeeding the sample names reference the publications as follows djureinovic fagerberg guo zhu kumar browne consortium helsel da cruz and zimmermann such as etdb smim36 bend2 btg4 cnbd1dppa2 efcab5 erich6 fthl17 iqcm mroh2bms4a5 oosp2 pnma6e ppp4r3c rbmxl3 rtl9spdye4 spem2 all of these genes are listed in fig and many of these genes are listed in figs andor to the best of our knowledge no prior studies haveutilized purified human testis cells for the identificationof human testisspecific transcripts through our analysis we identified genes as human testisspecificthrough one or more of the human germ cell datasetsbut not through either of the human testis datasetsadditionalfile table s5 seventysix genes wereidentified exclusively through one or more of the fivehuman spermatogonia datasets genes such as anp32dc13orf42 dscr4 or13g1 or2d2 or52e4 ssx2tle7 while genes were identified exclusivelythrough the human spermatocyte datasets genes suchas h2bfm mageb17 mageb18 or2b6 tcp10 andznf709 and genes were identified exclusivelythrough the human spermatid datasets genes suchas ac0132691 clec20a or7e24 pramef2 spata31a3 tmem191c znf679 thirtyfour geneswere identified through all three cell types datasetsgenes such as ccdc166 eloa2 fam47a heatr9 and spata31a1 many of these genes are listedin figs andor of the genes identified as human testisspecificthrough one or more of the human germ cell datasets genes have not been previously identified ofwhich have one or more equivalent mouse orthologswith of these genes having not been knocked out inmouse of these novel genes with no mouse model encode enzymes glt6d1 prss48 satl1 sult6b1tmprss7 tpte2 triml2 and ttll8 encodes anepigenetic protein taf1l encode gpcrs gpr156tas2r13 tas2r30 tas2r46 tas2r50 vn1r2 encodes a kinase cdkl4 encode ogpcrs such asor2d3 or3a2 or52e5 or8g5 or10j1and 0crobertson bmc biology page of adrenal glandadiposecolonbrainskeletal musclesmooth musclesmall intestinesalivary glandleukocytespancreasstomachprostatethyroidkidneyspleentestisheartlungliverskincorpusuteruscaudaovarycaputaac0221675ac1876531adam20al6720431bhmg1bsph1bx2760929c1orf105c2orf92c4orf51c16orf95ccdc196ces5acpa5dcaf8l1efcab5erich6etdbfer1l5glt6d1iqca1llcn6magea11mageb6mroh2bnaa11pp2d1ppp4r3cprr23aprr23bprr23cprss38saxo1slc22a14spag11aspag11bspata31a1spata31d1spata31d3spata31d4spint3spint4tcp10l2tex13dtex44tex48tex51tle7tpte2triml1trpc5oswfdc8wfdc9wfdc10awfdc10bwfdc13gapdhskeletal musclesmooth musclesmall intestinestomachprostatethyroidspleentestislungskinkidneyheartlivercorpusuteruscaudaovarycaputadiposebladderbraincoloneyebgm5767gm5294adam201700042g07rikgm4969bsph14933412e24rik4930558k02rik4933424g06rik1700011l22rik1700018b08rikgm6657ces5acpa5pet2efcab5erich6etdfer1l5glt6d14931409k22riklcn6magea4mageb3mroh2bnaa11pp2d14932429p05rikprr23a3prr23a3prr23a3prss38saxo1slc22a14spag11bspag11bspata31spata31d1bspata31d1bspata31d1bspint3spint4spint5tcp10ctex13c1tex44tex48gm35060tle7tptetriml1trpc5oswfdc8wfdc9wfdc10wfdc10wfdc13hprtfig rtpcr confirmation of reproductive tract specificity in both humans a and mice b the genes listed in this figure are novel as identifiedthrough our studies and without a reported mouse model humans do not have an equivalent proteincoding equivalent to mouse spint5 gapdh and hprt are included as housekeeping genes 0crobertson bmc biology page of fig three hundred and two novel mouse genes with human orthologs without a reported mouse model the listed genes were identified inone or more mouse datasets as indicated in the venn diagram underlined genes were also identified through our studies as reproductive tractspecific in human genes genes written in blue encode either enzymes kinases gpcrs ogpcrs transporters transcription factors orproteins involved in epigenetic regulation genes genes written in dark red were identified in both testis testis andor testis cell andepididymis genesnkx24or14a2 encode transcription factorsznf99 znf648 znf705b znf705g and znf709 encode transporters abcc12 slc35g3 slc35g5 and encode proteins of unknown drug target type suchas ac0080733 ac1135541 aknad1 al0496342dnlz ervw1 etda fbxw10 fer1l5 lmntd1lrrc72 nms prr23a prr23b prr23c pxt1rfpl4b and ssx4b many of these genes are listed infigs andor fam236a figs and and obp2b met candidatethreshold through our analysis of human testes datasetsbut did not meet candidate threshold from any of thegerm cell or sertoli cell datasets indicating potential expression in peritubular myoid cells leydig cells or othercell outside of the seminiferous epithelium fam36a hasnot been previously identified and neither mouse orthologs 1700011m02rik gm9112 have been knocked outobp2b was previously identified through djureinovic and johnston however of the equivalent mouse orthologs lcn4 obp2a obp2b only obp2ahas been knocked out revealing abnormal coathair pigmentation ism2 and magec2 were identified through both human sertoli cell datasets while also identified throughtestis andor germ cell datasets both genes have beenpreviously identified ism2 magec2 ism2knockout mice display nonreproductive phenotypes consistent with this finding our mouse data do notidentify ism2 as reproductive tractspecific in micemagec2 lacks a mouse ortholog for functional analysis in micehuman sertoli cellspecifickrtap23 krtap412 lhx9 and psg5 were identified through one or both human sertoli cell datasets butwere not identified through any of the testis or germ cell 0crobertson bmc biology page of datasets indicating sertoli cellspecific expression in thetestes additional file table s5 none of these geneshave been previously identified as reproductive tractspecific in humans although lhx9 and psg5 havemouse orthologs that have been knocked out []human krtap23 has mouse orthologs krtap52gm4559 gm40460 and gm45618 and human krtap412 has mouse orthologs krtap47 and gm11555none of these mouse orthologs have been knocked outfig and additional file table s5knockin mousepsg5 knockout mice display nonreproductive phenotypes [] however lhx9 knockout mice display absent testes and sterility due to an essential requirementfor lhx9 during mouse gonad formation a lhx9gfpcreerbyknockingin gfpcreer at the endogenous lhx9 locuscrossed with the rosa26tdtomato reporter mouse linerevealed cre recombinase activity in retinal amacrinecells developing limbstestis hippocampal neuronsthalamic neurons and cerebellar neurons thuslhx9 is not reproductive tractspecific in mice ourmouse data confirm this findinglinegeneratedhuman epididymisspecificto the | 0 |
Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M 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delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663 Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229 Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | 2 |
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes | 2 |
"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c" | 2 |
CASE REPORTpublished August 103389fbioe202000929SecondGeneration RTQuIC Assayfor the Diagnosis ofCreutzfeldtJakob Disease Patientsin BrazilBreno Jos Alencar Pires Barbosa1 Bruno Batitucci Castrillo1 Ricardo Pires Alvim1Marcelo Houat de Brito1 Helio R Gomes1 S´nia M D Brucki1 Jerusa Smid1Ricardo Nitrini1 Michele C Landemberger2 Vilma R Martins2 Jerson L Silva3 andTuane C R G Vieira3 Department of Neurology Hospital das Clnicas University of S£o Paulo Medical School S£o Paulo Brazil Tumor Biologyand Biomarkers Group International Research Center AC Camargo Cancer Center S£o Paulo Brazil National Centerof Nuclear Magnetic Resonance Jiri Jonas Institute of Medical Biochemistry Leopoldo de Meis National Institute of Scienceand Technology for Structural Biology and Bioimaging Federal University of Rio de JaneiroUFRJ Rio de Janeiro BrazilThe recent development of IQCSF the second generation of realtime quakinginducedconversion RTQuIC using cerebrospinal ï¬uid CSF for the diagnosis of CreutzfeldtJakob Disease CJD represents a major diagnostic advance in the ï¬eld Highly accurateresults have been reported with encouraging reproducibility among different centersHowever availability is still insufï¬cient and only a few research centers have access tothe method in developing countries In Brazil we have had suspected cases of CJDsince when surveillance started Of these were undiagnosed This lack ofdiagnosis is due among other factors to the lack of a reference center for the diagnosisof these diseases in Brazil resulting in some of these samples being sent abroad foranalysis The aim of this research study is to report the pilot use of IQCSF in a smallcohort of Brazilian patients with possible or probable CJD implementing a referencecenter in the country We stored CSF samples from patients with possible probable enetic CJD one case during the time frame of December through June AllCSF samples were processed according to standardized protocols without access tothe clinical data Eight patients presented to our team with rapidly progressive dementiaand typical neurological signs of CJD We used CSF samples from seven patientswith other neurological conditions as negative controls Five out of seven suspectedcases had positive tests two cases showed inconclusive results Among controls therewas one falsepositive a CSF sample from a 5yearold child with leukemia undertreatment The occurrence of a false positive in one of the negative control samplesraises the possibility of the presence of interfering components in the CSF sample frompatients with nonneurodegenerative pathologies Our pilot results illustrate the feasibilityof having CJD CSF samples tested in Brazilian centers and highlight the importance ofinterinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Braziland Latin AmericaKeywords CreutzfeldtJakob disease prionconversion biomarkersrapidly progressive dementiarealtime quakinginducedEdited byMaria Dos Anjos PiresUniversity of Tr¡sosMontes and AltoDouro PortugalReviewed byAssia AngelovaGerman Cancer Research CenterDKFZ GermanySumit GhoshThe Research Institute at NationwideChildrens Hospital United StatesCorrespondenceBreno Jos Alencar Pires BarbosabrenojbgmailcomTuane C R G VieiratuanebioqmedufrjbrSpecialty sectionThis was submitted toBiosafety and Biosecuritya section of the journalFrontiers in Bioengineering andBiotechnologyReceived May Accepted July Published August CitationBarbosa BJAP Castrillo BBAlvim RP de Brito MH Gomes HRBrucki SMD Smid J Nitrini RLandemberger MC Martins VRSilva JL and Vieira TCRG SecondGeneration RTQuIC Assayfor the Diagnosis of CreutzfeldtJakobDisease Patients in BrazilFront Bioeng Biotechnol 103389fbioe202000929Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alINTRODUCTIONtogroupTSEsconditionsencompasstransmissibleasecondaryspongiformPrion diseases also known asofrareencephalopathiesneurodegenerativeabnormalconversion of a constitutively expressed cellular glycoproteinthe prion protein PrPC into an abnormally folded isoformPrPsc Geschwind Zanusso SporadicCreutzfeldtJakob disease sCJD is the most common priondisease in humans and usually presents as rapidly progressivedementia in combination with variable degrees of multisystemneurologicalimpairment Zerr and Hermann Sinceits clinical and molecular manifestations are heterogeneousand nonspeciï¬c early diagnosis of prion diseases remainschallenging in clinical practice Geschwind and Murray Brain histopathological evaluation andor detection of PrPScare still the standard criteria for establishing a deï¬nitive diagnosisfor CJD CDC However invasiveness when performingthis type of analysis antemortem brings very little beneï¬t to thepatient since these diseases are still incurable Clinical signs andparaclinical tests are the most commonly used approaches duringthe course of the disease and can be used to classify it as a possibleor probable prion disease Brown CDC Among the paraclinical tests brain diï¬usion weightedMRIDWMRI and cerebrospinal ï¬uid CSF analysis have increaseddiagnostic accuracy Eisenmenger Staï¬aroni Bizzi The presence of protein Tauprotein neuronspeciï¬c enolase NSE the astroglial proteinS100B and PrPSc itself are used as biomarkers in CFS for TSEdiagnosis Schmitz Connor Overallthe only protein that is a speciï¬c biomarker for TSE is PrPScPrPSc can be detected in CSF based on its selfpropagating abilityconverting and seeding the aggregation of the nonpathogenicPrPC into PrPScThe realtime quakinginduced conversion RTQuIC assayits experimentalwas developed in and since thenconditions have been improved and tested on a large number ofsamples in several laboratories worldwide Wilham Green It ultrasensitively detects limited amounts of PrPScin CSF and other tissue samples Wilham The recentdevelopment of the secondgeneration IQCSF RTQuIC assayusing CSF for the diagnosis of CJD represents a major diagnosticadvance in the ï¬eld Wilham Orrº In humans RTQuIC analysis showed a sensitivity of and speciï¬city of with encouraging reproducibility amongdiï¬erent centers Franceschini RTQuIC started to be clinically used in and becamea criterion of the Centers for Disease Control and PreventionCDC to diagnose CJD as probable CDC Its highsensitivity and speciï¬city make it an important clinical laboratorytestits global availability is stillinsuï¬cient only a few research centers have access to the methodespecially in developing countriesfor widespread use butSurveillance of TSE cases has been compulsory in Brazil since Martins and suspected CJD cases werereported up to June Of these were undiagnosed deOliveira Cardoso Ministrio da Saºde RTQuIC for the Diagnosis of Brazillian PatientsinimagingperformingexaminationsandDiï¬cultiesneuropathological analyses are found in several medicalunits in the country making diagnosis problematic Somediagnosed patients had samples sent to centers outside thecountry for biomarker analysis allowing for greater coverageof the diagnostic criteria The implementation of a speciï¬ctest in Brazil such as RTQuIC which can provide diagnosisfor diï¬erent TSEs with ease and conï¬dence is urgent to havenotiï¬cation and diï¬erential diagnosis for these diseases Thisis also important to guide medical decisions Here we reportthe pilot use ofthe secondgeneration IQCSF RTQuICassay in a small cohort of Brazilian patients with possibleor probable CJD to implement a reference center for thisanalysis in the countryMATERIALS AND METHODSClinical InvestigationPatients with suspected CJD were admitted for investigationunder a protocol for rapidly progressive dementia RPD aspreviously reported Studart Neto Following a fullneurological examination all patients underwent complete bloodcell count serum electrolytes blood glucose acute Creactiveprotein liver renal and thyroid function tests antithyroid andantinuclear antibody testing as well as treponemal and HIVserology Patients also underwent brain magnetic resonanceimaging [T1 T2 ï¬uidattenuated inversion recovery FLAIRgradient echo and diï¬usion weighted imaging sequences] EEGand CSF cell count biochemistry and g globulin levels CSF protein was assessed in cases of suspected prion diseasesas required by Brazilian norms Selected patients underwentchest and abdomen computed tomography mammography forwomen testicular ultrasound for men and thyroid ultrasoundto rule out paraneoplastic RPD In addition we obtainedonconeural andor neuronal surface antigen antibody testingwhen paraneoplastic or autoimmune encephalitis was suspectedA ï¬owchart for patient inclusion is provided in Figure Three cases had access to molecular analysis of the PRNP genefor polymorphisms in the codon Samples were analyzedusing denaturing highperformance liquid chromatographyTechnical details about this procedure as well as ampliï¬cationreactions and DNA extraction have been previously describedCastro Smid CSF SamplesWe analyzed eight CSF samples from seven patients withpossible probable or genetic CJD referred to the Departmentof Neurology at University of S£o Paulo from December to June One patient named ABT had her CSF testedtwice at diï¬erent times We used CSF samples from sevenpatients with other neurological conditions as negative controlsCSF samples mL were collected by lumbar puncture LPfollowing a standard procedure Two milliliters of the CSFsample were then centrifuged at g for min andstored in polypropylene tubes at ¦C until blind analysis byresearcher TCRGV at UFRJFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsof CJD The patient named ABT had her CSF tested twice atdiï¬erent times ABT1 and ABT2 samples therefore renderinga total of eight CSF samples ABT patient results will be betterreported in Case descriptionWe randomly used CSF samples from seven patients withother neurological conditions as negative controls Five out ofeight suspected samples had positive RTQuIC results in ourhands Figure 2A Clinical diagnosis CSF analysis and RTQuICresults are summarized in Table Samples from patients LRC and DAS were used as positivecontrols given that they were previously analyzed using RTQuICin a reference center outside the country The NationalPrion Disease Pathology Surveillance Center Cleveland OHUnited States Our RTQuIC analyses were also positivecorroborating this result Figure 2A Patient ASM was a geneticCJD positive for the E200K mutation and in our analysis thispatient was also positive presenting a very short lag phase and ahigh ï¬uorescence signal Figure 2AAmong the negative controls there was one false positivesample CT2 Figure 2A The falsepositive case was a5yearold child with leukemia who was receiving intrathecalchemotherapy GBTLI protocol with methotrexate aracitincitrabin dexamethasone during the time frame of the studyIn the following two cases are presented in which the resultsobtained with RTQuIC show interesting aspects related toRTQuIC sensitivity and disease progressionCase ABT was a 72yearold woman with years of formal educationShe had an atypical presentation characterized by a 4monthhistory of rapidly progressive cognitive impairment associatedwith visual hallucinations and gait disturbances with repeatedfalls She had no relevant past medical history or family history ofany neurological conditions On neurological examination shescored on the MiniMental State Examination MMSEand physical tests revealed only a prominent axial syndrome asshe could not sit or stand up without bilateral support There wereno pyramidal signs parkinsonism or visuospatial impairmentA comprehensive investigation with metabolic inï¬ammatoryparaneoplastic and infectious tests was unremarkable Brainmagnetic resonance imaging MRI revealed symmetric diï¬usionweighted image DWI hyperintensities in the basal gangliaFigure a ï¬nding that raised the suspicion of CDJ TheCSF analysis was unremarkable with elevated tau protein levelsand negative The RTQuIC results were inconclusiveonce from wells only two crossed the threshold ABT1sample in Figure 2B Electroencephalogram EEG revealeddisanized electrical brain activity with no periodic wavesThree months later she developed a signiï¬cant worsening withthe need for aid for most activities of daily living severe cognitiveimpairment she could not be tested with the MMSE myoclonusand the presence of prominent frontal reï¬exes on neurologicalexamination At this point a prolonged EEG eventually showedbilateral periodic sharp waves and another lumbar puncturewas performed with a second sample sent for RTQuIC analysiswith a positive result with a high ï¬uorescence ABT2 samplein Figure 2B She eventually died of clinical complicationsFIGURE Flowchart of samples included for RTQuIC analysisRTQuICThe RTQuIC assay was performed using the improvedQuICCSF IQCSF conditions as published Orrº Brieï¬y µL of CSF was added to µL of reaction mixturein each well of a black well plate with a clear bottomNunc The ï¬nal solution contained mM phosphate buï¬er atpH mM ethylenediaminetetraacetic acid tetrasodium saltdihydrate EDTA at pH mM NaCl µM thioï¬avinTThT sodium dodecyl sulfate SDS and mgmLrecombinant Syrian hamster truncated form of prion proteinHa rPrP Samples were tested in quadruplicate threeor four times independently generating a total of or wellsevaluated for each sample The plates were sealed and incubatedin a FLUOstar OMEGA plate reader BMG Labtech Germanyat ¦C with intermittent cycles of shaking s doubleorbital rpm and rest s ThT ï¬uorescence was collected every min using ± nm excitation and ± nmemission wavelengths The threshold was deï¬ned as the averageï¬uorescence for all samples within the ï¬rst h of incubationplus standard deviations SD A sample was consideredpositive when at least two of four replicate wells crossed thisthreshold All IQCSF RTQuIC analyses were performed at theFederal University of Rio de JaneiroUnseeded reaction not shown and random CSF samplesfrom patients with other neurological conditions Table wereselected as negative controls Given the descriptive nature of thisstudy no statistical analyses were performedRESULTSIn the referred time frame seven patients presented to our teamwith rapidly progressive dementia and typical neurological signsFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsTABLE Results of diagnostic investigations in the tested patient cohortSample ageDiagnosisaCSF analysisRTQuIC statusWBCµ LProt mgdLGlu mgdLTaub pgmLCT1 CT2 CT3 HCS HBV RMC ABS LRC DAS ABT1 ABT2 ASM NAJJN GMT TN Control chronic meningitisControl leukemiaControl cranial nerves syndromeControl multiple sclerosisControl HIVControl SAHventriculitisControl chronic meningitisRPD probable CJDRDP probable CJDRPD possible CJDRPD possible CJDRPD genetic CJDRPD probable CJDRPD possible CJDRPD probable CJD1cNANANANegativePositiveNegativeNANANANegativeNegativePositiveNegativeNegativeNegativeNegativeNegativePositivedPositivedNegativePositivePositivePositiveNegativePositiveWBC white blood cells Prot protein levels Glu glucose levels RPD rapidly progressive dementia SAH subarachnoid hemorrhage NA not available aControls andtheir suspected diagnosisinvestigation bReference value for total tau levels was pgmL cThis sample had red blood cells dThese patients also had their CSFsamples sent abroad with positive results The National Prion Disease Pathology Surveillance Center Cleveland OH United States months after the ï¬rst symptoms appeared PRNP gene analysisrevealed codon heterozygous MVin hisCase JJN was a 65yearold male who presented to our clinicwith an 8month history of behavioral changes He had years of education and a medical history of hypertensionand gouty arthritis There was no family history of anyneurological conditions His wife described the ï¬rst symptomsas prominent changesfood preferences with anunusual demand for rice and chicken Two months laterhe developed visual hallucinations mostly described as thepresence of spiders in the ceiling In the ï¬rst evaluationhe was independent of instrumental activities of daily livingNeurological examination revealed an MMSE of withan unremarkable physical examination A laboratory workupincluding metabolicinï¬ammatory and serology studies wasnegative At this point we were surprised by the ï¬nding ofbrain MRI diï¬usion weighted imaging revealing marked bilateralhypersignal in the frontotemporoparietooccipital cortex basalganglia thalamus and less markedly in the hippocampusraising the suspicion of sporadic CreutzfeldtJakob DiseasesCJD Figures 4AB The patient was hospitalized for furtherinvestigation The EEG study revealed bilateral and synchronousslow waves A brain 18FDG PET showed hypometabolism in thetemporal and frontal lobes caudate nuclei and temporoparietalcortex Figure 4C The CSF study was initially unremarkableexcept for the high total tau protein levels whereas ptau and betaamyloid values were within the normal rangeA CSF sample was sentfor autoantibodies and RTQuICtesting The patient was empirically treated with intravenousmethylprednisolone for days and was discharged for outpatientfollowup Atthis point we received CSF results negativefor autoantibodies with an inconclusive RTQuIC responsefrom wells only two crossed the threshold Figure 2CIn the following months he eventually developed cognitivedeterioration and parkinsonism with the presence of morecomplex visual hallucinations He eventually died of clinicalcomplications approximately months after initial symptomsPRNP gene analysis revealed codon heterozygous MVDISCUSSIONsyndromes ofencephalopathiesThe present study reports the results of pilot secondgenerationreferred forRTQuIC testing in a small patient cohortrapidly progressive neurologicalsuspectedprion nature Our center previously reported cases ofrapidly progressive dementia among patients in a 3yearintervalrepresentedthe majority of the samples followed by CJD Studart Neto All cases from that study werediagnosed with CJD according to the University of CaliforniaSan Francisco Modiï¬ed Criteria Vitali which doesnot include CSF testingImmunemediatedAs we described here IQCSF RTQuIC proved to be anextremely important tool in the diagnosis of ABT and JJN casesCases and Both patients presented with rapidly progressiveneurological syndromes and could not initially be classiï¬ed withpossible or probable CJD according to the most recent criteriaCDC Both ABT and JJN showed slightly elevated levelsof total Tau protein but although this biomarker has sensitivity it is only speciï¬c for CJD Connor protein was not elevated in both cases despitetheir genetic subtype MV which is often associated with positive protein levels Manix Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsFIGURE Blind RTQuIC analysis of CSF samples A RTQuIC responses from reactions seeded with µl of CSF from suspected CJD cases LRC DAS ASMJJN GMT TN The RTQuIC responses from reactions seeded with CSF samples from patients with other neurological disorders CT1 CT2 CT3 HCS HBV RMCABS were used as a negative control Each curve represents the mean of four replicate readings of three or four repetitions B Singlewell analysis of CSF samplesfrom patient ABT ABT1 and ABT2 refer to the ï¬rst and second collections respectively C Singlewell analysis of CSF samples from patient JJNIn the case of ABT patient the clinical presentation ofrapid cognitive impairment with visual hallucinations and gaitimpairment was considered atypical In this case the brain MRIwas an important diagnostic clue with DWI hyperintensitiesin the basal ganglia leading to a diï¬erential diagnosis withmetabolic encephalopathies hypoxicischemic lesions or toxicFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian Patientsdisease progression To our knowledge this approach performedwith samples of a patient collected at diï¬erent times has not yetbeen tested and this is the ï¬rst report suggesting that RTQuICresults change according to disease evolution A more feasiblehypothesis would be that the ï¬rst sample had some interferencesuch as blood that could mask the positive result Cramm despite a only modest presence of red blood cells in thisparticular sample cellsµL see Table Atypical features for sCJD in JJN Case included a presentation with mild behavioral changes the initialsparing of motor systems with evidence of motor ï¬ndingsin the neurological examination only almost months afterï¬rst symptoms an unremarkable EEG with months ofevolution and the presence of a bilateral T2 hypersignalin the hippocampus The RTQuIC was inconclusive buttogether with MRIit could suggest early diagnosis ofprobable CJD Perhaps with disease progression JJNs CSFsample would obtain a positive RTQuIC result as observedfor ABT patientThe occurrence of one falsepositive case in our RTQuICtest weakens our diagnostic accuracy and underscores the needfor improvements in the protocol Despite having an extremelyhigh speciï¬city IQCSF RTQuIC falsepositive results have beenreported in the literature Hayashi reported thecase of a 61yearold man who presented with rapid cognitiveimpairment myoclonus and recurrent seizures A brain MRIrevealed cortical hyperintensities and the CSF analysis showedelevated and tau levels with a positive RTQuIC Despiteaggressive treatment with corticotherapy the patient died andpostmortem assessment revealed only pathological changes afterconvulsion with no signs of prion disease The authors concludethat convulsion may cause falsepositive RTQUIC results andthat a postmortem evaluation remains the gold standard fordiagnosing similar cases The shaking eï¬ect was analyzed in vitroby Orrº and they showed that long shaking periodsreduced scrapieseeded reaction times but continuous shakingpromoted falsepositive reactionsFIGURE Diffusionweighted images DWI from ABT case revealingenlarged ventricles and symmetric hyperintensities in the basal ganglia Ayellow arrows B shows corresponding apparent diffusion coefï¬cient ADChypointensities in the same territorylesions ie carbon monoxide intoxication Finelli and DiMario A careful clinical assessment made all possibilities lesslikely and CJD became our main hypothesis Despite aninconclusive RTQuIC result for the ï¬rst collected sample thepatient eventually evolved with a more typicalimpairmentand the second CSF sample with a 3month interval yieldedpositive RTQuICThe diï¬erent results obtained with the ABT patient sampleswere very intriguing We hypothesize that this diï¬erence could beattributed to higher loads of PrPsc following disease progressionAlthough disease duration does not seem to be related toRTQuIC results McGuire it is not yet clear whetherthere is a change in the presence of seeds in CSF according toFIGURE A MRI diffusionweighted images from JJN case revealing hyperintensities on frontal temporoparietal and posterior cingulate cortical areas Ayellow arrow B MRI apparent diffusion coefï¬cient maps with corresponding hypointensities in the same regions above B yellow arrow C 18FDG PETCTshows hypometabolism on the bilateral temporoparietal cortex posterior cingulate precuneus and caudate nucleusFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsRegarding our falsepositive case we did not ï¬nd any similarcases reporting the use of intrathecal medications as a possiblereason for a positive RTQuIC result A positive case in a5yearold child without any neurological symptoms wouldnever be expected an 18yearold woman was the youngestperson diagnosed with probable sCJD using RTQuIC Yao Therefore this sample was selected as a negative controlalthough infant CSF was never RTQuIC analyzed Despiteoptimized protocols sample processing issues can always be apossibility for such unexpected resultsThe present study is limited by its pilot nature with a modestsample size Improvements in the establishment of the protocolare necessary requiring a greater number of analyses The useof nasal brushes to collect patient samples for RTQuIC analysisis also valid to improve protocol sensitivity and speciï¬cityHowever this is the ï¬rst study to our knowledge to report speciï¬cbiomarkerbased feasible results performed in a developingcountry for prion diagnosis in addition to pointing out newpossible interferences in the protocol and the need to understandhow the diï¬erent current diagnostic approaches can revealdisease progressionCONCLUSIONThe cases reported here illustrate the importance of usingRTQuIC for patients with neurological syndromes enabling thediagnosis of probable CJD while no other method was suï¬cientto support this diagnosis even with atypical clinical presentationIn addition the identiï¬cation of a false positive in a sample froma leukemic pediatric patient undergoing intrathecal treatmentwith chemotherapy suggests new possible interferences in themethod This will require future investigation of the eï¬ect of thesechemotherapeutic agents for inclusion or not as a limitation forcarrying out the assayCSF samples are often evaluated only once due to theinvasiveness of CSF collection and the absence of curativetreatment for TSEs in addition to rapid disease progressionOne of the cases we report points out the importance ofcarrying out studies that evaluate the progression of the diseaseand that RTQuIC is a useful approach for such studies Inthis case the use of nasal brushes might be prioritized overCSF analysis since this provides greater sensitivity and enablesmore frequent sample collection given the less invasive natureof the procedureFinally our study illustrates the feasibility of having CJDCSF samples tested in Brazilian centers and highlights theimportance of interinstitutional collaboration in order to pursuea greater diagnostic accuracy for CJD in developing countriesREFERENCESBizzi A Pascuzzo R Blevinsetal Evaluation ofM E Mdetecting prion disease with diï¬usion magneticJAMA Neurol101001jamaneurol20201319print]J Grisoli M Lodi R Moscatellia new criterion forimagingofresonance[EpubaheadIt also demonstrates that RTQuIC can be clinically availablefor testing patients from Brazil and other Latin Americancountries and points to the need and feasibility of establishinga national reference center for the diagnosis of these rare butdevastating diseasesDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialETHICS STATEMENTEthical review and approval was not required for the studyon human participants in accordance with the local legislationand institutional requirements Written informed consent toparticipate in this study was provided by the participants legalguardiannext of kin Written informed consent was obtainedfrom the individuals and minors legal guardiannext of kinfor the publication of any potentially identiï¬able images or dataincluded in this AUTHOR CONTRIBUTIONSBB study design patient data collection and manuscript writingBB BC RA MB HG and SB patient data collection andinterpretation JS and RN study design data interpretation andmanuscript critical revision ML and VM genetic analysis JLSand TV RTQuIC facility implementation TV study designRTQuIC evaluations RTQuIC data analysis and manuscriptwriting All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by research grants from the CarlosChagas Filho Foundation for Research Support in the State of Riode Janeiro FAPERJ and the National Council of Technologicaland Scientiï¬c Development CNPq to JLS and TVACKNOWLEDGMENTSWe would like to acknowledge all the patients and their familiesfor their contributions to this studyBrown P Brunk C Budka H Cervenakova L Collie D Green A WHO Manual for Surveillance of Human Transmissible SpongiformEncephalopathies Including Variant CreutzfeldtJakob Disease Geneva WorldHealth anizationCastro R M R P S Landemberger M C Walz R Carlotti C GHuang N Cunha D R High capacity and low costdetection of prion protein gene variant alleles by denaturing HPLCFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cBarbosa et alRTQuIC for the Diagnosis of Brazillian PatientsJ Neurosci Methods101016jjneumeth2004CDC CDCs Diagnostic Criteria for CreutzfeldtJakob Disease CJD[Internet] Atlanta Centers for Disease Control and PreventionConnor A Wang H Appleby B S and Rhoads D D Clinical laboratorytests used to aid in diagnosis of human prion disease J Clin Microbiol57e0076919Cramm M Schmitz M Karch A Mitrova E Kuhn F Schroeder B Stability and reproducibility underscore utility of RTQuICfor diagnosis of CreutzfeldtJakob disease Mol Neurobiol 101007s1203501591332de Oliveira Cardoso C A de Albuquerque Navarro M B M Soares B E Cand Oliveira Cardoso T A Avalia§£o epidemiol³gica dos ³bitos pordoen§as pri´nicas no Brasil sob o enfoque da biosseguran§a Cadernos SaºdeColetiva 1015901414462x201500010002Eisenmenger L Porter MC Carswell C J Thompson A Mead S RudgeP Evolution of diï¬usionweighted magnetic resonance imagingsignal abnormality in sporadic creutzfeldtjakob disease with histopathologicalcorrelation JAMA Neurol Finelli P F and DiMario F J Diagnostic approach in patients withsymmetric imaging lesions of the deep gray nuclei Neurologist 10109701nrl0000087718555976aFranceschini A Baiardi S Hughson A G McKenzie N Moda F Rossi M High diagnostic value of second generation CSF RTQuIC acrossthe wide spectrum of CJD prions Sci Rep Geschwind M D Rapidly progressive dementia Continuum 101212con0000000000000319Geschwind M D and Murray K Diï¬erential diagnosis with other rapidprogressive dementias in human prion diseases Handb Clin Neurol 101016b9780444639455000209Green A J E RTQuIC a new test for sporadic CJD Pract Neurol 101136practneurol2018001935Hayashi Y Iwasaki Y Yoshikura N Asano T Mimuro M Kimura A An autopsyveriï¬ed case of steroidresponsive encephalopathywith convulsion and a falsepositive result from the realtime quakinginduced conversion assay Prion Manix M Kalakoti P Henry M Thakur J Menger R Guthikonda B CreutzfeldtJakob disease updated diagnostic criteria treatmentalgorithm and the utility of brain biopsy Neurosurg Focus 39E2Martins V R Gomes H R Chimelli L Rosemberg S and Landemberger M C Prion diseases are under compulsory notiï¬cation in Brazil surveillanceof cases evaluated by biochemical andor genetic markers from to Dement Neuropsychol 101590s198057642008dn10400004McGuire L I Peden A H Orrº C D Wilham J M Appleford N EMallinson G Real time quakinginduced conversion analysisof cerebrospinal ï¬uid in sporadic CreutzfeldtJakob disease Ann Neurol 101002ana23589Ministrio da Saºde Protocolfor Notiï¬cation and Investigation ofCreutzfeldtJakob Disease with a Focus on the Identiï¬cation of the New Variant[Internet] Brazil Ministrio da SaºdeOrrº C D Bongianni M Tonoli G Ferrari S Hughson A GGroveman B R A test for CreutzfeldtJakob disease usingnasal brushings N Engl J Med 101056nejmoa131Orrº C D Groveman B R Hughson A G Zanusso G Coulthart M Band Caughey B Rapid and sensitive RTQuIC detection of humancreutzfeldtjakob disease using cerebrospinal ï¬uid mBio 6e0245114Orrº C D Hughson A G Groveman B R Campbell K J Anson K J MancaM Factors that improve RTQuIC detection of prion seedingactivity Viruses 103390v8050140Schmitz M Ebert E Stoeck K Karch A Collins S Calero M Validation of protein as a marker in sporadic creutzfeldtjakob diseasediagnostic Mol Neurobiol Smid J Landemberger M C Bahia V S Martins V R Nitrini R SmidJ Codon polymorphism of prion protein gene in is nota risk factor for Alzheimers disease Arquivos Neuro Psiquiatr 1015900004282x20130055Staï¬aroni A M Kramer A O Casey M Kang H Rojas J C Orrº C D Association of blood and cerebrospinal ï¬uid tau level and otherbiomarkers with survival time in sporadic creutzfeldtjakob disease JAMANeurol Studart Neto A Soares Neto H R Simabukuro M M Solla D J F Gon§ | 2 |
"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Deï¬ning advance care planning for adults Aconsensus deï¬nition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneï¬ciaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conï¬ict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" | 2 |
collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identiï¬ed in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the antiammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe ï¬nal protein what is more its only amino acid posttranslational modiï¬cation is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains ï¬ve exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by ï¬broblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myoï¬broblasts and locates in the sites ofcollagen matrix deposition in micethe ï¬rst exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ ] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the ï¬rst enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ ]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossiï¬cation and other 0cmediators of ammationcthrc1tgfð½tð½r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1ð½ð¼ð¼ð½integrincxrc4tð½r1p ppsmad23smad4cthrc1dvlpdvlaxinapcð½cateninck1ð¼gsk3dpagt1stabilizationð½cateninpkcð¿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhifð¼vegfecminvasionpsmad23smad4ð½catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespeciï¬c tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury uencing ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver ï¬brosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespeciï¬c the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied []current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the destructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasiondestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 []the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic ï¬broblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ ]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to ï¬bronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation []erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and ï¬bronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac ï¬broblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it brieï¬y cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was ï¬rst discovered more than a decad | 0 |
recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junÃn virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ ] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavirritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm | 0 |
Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelialmesenchymal transition EMT and apoptosis in BC are not fully understood We ï¬nd that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4 EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8 Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspeciï¬c transmembrane protein foundin a variety of peripheral cells11 is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored at °C for western blotting and PCR analysis The othergroup of specimens was ï¬xed in formalin and parafï¬nizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and veriï¬ed for their authenticities by STR proï¬ling Epirubicin was purchased fromPï¬zer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The ï¬nal protein staining score was the percentage score multiplied by the intensity score ï¬nalprotein staining scores were divided into three categoriesas follows negative low expression and high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturers instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments à cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were ï¬xed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantiï¬edby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturers protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magniï¬cation à b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplanMeier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magniï¬cation à f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell ï¬uorescence was measured with a FACScan ï¬owcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefï¬ciency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of à stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacriï¬ced by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups à stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacriï¬ced by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were ï¬xed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were ï¬xed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunoï¬uorescenceImmunoï¬uorescence staining was performed as previously described19 In brief after corresponding treatments the cells ï¬xed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfï¬cial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by KaplanMeier analysis and thelogrank test was used to compare survival differencesbetween groups Pearsons correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically signiï¬cantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe ï¬rst measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfï¬cial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassigniï¬cantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and KaplanMeier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was signiï¬cantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot signiï¬cant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown signiï¬cantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further conï¬rm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efï¬ciency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was conï¬rmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1BE Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown signiï¬cantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunoï¬uorescence showed the same results as thoseOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magniï¬cation à f Correlation analyses of proteinexpression levels between DNER and catenin g KaplanMeier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further conï¬rm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were signiï¬cantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunoï¬uorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally KaplanMeier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfï¬cial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a speciï¬c Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were signiï¬cantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These ï¬ndingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown signiï¬cantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to conï¬rm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efï¬ciency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efï¬ciency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantiï¬cation of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see ï¬gure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightï¬eldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t | 2 |
Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young at diagnosis patients who received curative resection for breastcancer The signiï¬cance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identiï¬ed variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI P and OS HR CI P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identiï¬ed as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI and CI respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identiï¬ed for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to beneï¬t prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and Chinascontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkins lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classiï¬cation system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassiï¬ed as luminal Alike ER PR¥ HER2 andKi6715luminal Blike ER andor PR HER2HER2enriched ER PR HER2 or triplenegative ERPR HER2 subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have deï¬niteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insuï¬cient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was deï¬ned as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was deï¬ned asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the KaplanMeier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asigniï¬cance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the rms package of R The discriminationofthe nomogram models was estimated by the Harrellsconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank à statistics with the Xtileprogram Pearsons chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the ï¬ndings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsuï¬cientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patientsFIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical signiï¬cancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The models explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI and CI respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS patients intermediateriskgroup TPS patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS patientsintermediaterisk group TPS patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the ï¬rsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentiï¬ed as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE KaplanMeier curves for A diseasefree survival and B overallsurvival stratiï¬ed by HBsAg status in very young patients with breast canceridentiï¬ed as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may beneï¬t patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insuï¬cientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population conï¬nes the universality of our resultsFurthermore the information was insuï¬cient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the ï¬rst evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to conï¬rm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the ï¬nal versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer 101093annoncmdf039at presentation Ann Oncolfeaturesof 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HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell 107314APJCP201516135285lung cancer Asian Paciï¬c J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in Vickers AJ Elkin EB Decision curve analysis a novel method for evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res 10115810780432CCR040713Conï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in 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" Replication of these findings supports the robustness of these markers for CMH and suggests that they are useful in defining a subset of subjects with CMH who could benefit from computed tomography (CT) screening for lung cancer [46]. Indeed low cost gene-specific methylation screening assays could be incorporated into clinical practices for patients suspected to be at risk for lung cancer. Conclusions Especially male former smokers with persistent chronic mucous hypersecretion have markedly increased promoter methylation of lung cancer risk genes in cell obtained by sputum collection. These smokers may be at increased risk of lung cancer and may benefit from further tests for lung cancer such as CT screening. Competing interests The authors declare that they have no competing interests. Authors contributions YT SB and HP made substantial contributions to conception and design; SB JW JS HP SB and MP made substantial contributions to acquisition of data or analysis and interpretation of data. All authors made substantial contributions to drafting the or revising it critically for important intellectual content and final approval of the version to be published. Supplementary Material Additional file 1: Table S1 Select variables by CMH status in the combined cohorts. Table S2. Select variables by high and low methylation tertile in combined cohorts. Table S3. Select variables by CMH in males from the LSC and PLuSS. Table S4. Select variables by CMH in females from the LSC and PLuSS. Figure S1. ROC curves comparing the sensitivity and specificity of the 3-gene methylation panels for classifying CMH. ROC curves were generated by applying logistic regression models to male former smokers independently in the PLuSS (n?=?52) and LSC (n?=?87). The covariates included age pack years education and COPD. AUC is indicated in parentheses. " | 1 |
" distribution and reproduction in any medium provided the original author and source are credited. The epithelial mesenchymal transition (EMT) is an important process in tumor development. Despite previous investigations it remains unclear how p120-catenin (p120ctn) isoforms 1A and 3A affect the EMT of tumor cells. Here we investigated expression of p120ctn E-cadherin and vimentin in 78 human non-small cell lung cancer (NSCLC) samples by immunohistochemistry and found that p120ctn membrane expression positively correlated with E-cadherin expression (P<0.001) and negatively correlated with vimentin expression and lymph node metastasis (P<0.05). Meanwhile p120ctn cytoplasmic expression negatively correlated with E-cadherin expression (P<0.001) and positively correlated with vimentin expression and lymph node metastasis (P<0.05). Cells expressing high (H460 and SPC) and low (H1299 and LK2) levels of p120ctn were screen to investigate its impact on EMT. E-cadherin was restricted to the cell membrane in H460 and H1299 cells whereas it was expressed in the cytoplasm of SPC and LK2 cells. Ablation of endogenous p120ctn isoform 1A in cells expressing high levels of the protein resulted in decreased E-cadherin expression increased N-cadherin vimentin and snail expression and enhanced invasiveness in H460 cells. Meanwhile completely opposite results were observed in SPC cells. Furthermore transfection of in H1299 cells expressing low p120ctn levels with the p120ctn isoform 1A plasmid resulted in increased E-cadherin expression decreased N-cadherin vimentin and snail expression and weakened invasiveness while LK2 cells showed completely opposite results. Both cell lines expressing low p120ctn levels and transfected with the p120ctn isoform 3A plasmid appeared to have increased E-cadherin expression decreased N-cadherin vimentin and snail expression and weakened invasiveness. In in cells with membrane E-cadherin both p120ctn isoforms 1A and 3A inhibited EMT and decreased cell invasiveness. In cells with cytoplasmic E-cadherin p120ctn isoform 1A promoted EMT and increased cell invasiveness while p120ctn isoform 3A inhibited the EMT and decreased cell invasiveness. This work was supported by grants from the National Natural Science Foundation of China (grants 81071905 and 81272606 to E.-H. W. grants 81101780 to Y.Z.). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction The epithelial mesenchymal transition (EMT) is a rapid and often reversible change of cell phenotype and plays a particularly important role in tumor development. In the process of EMT epithelial cells undergo a phenotypic switch to form mesenchymal cells that are similar in appearance to fibroblasts [1] [2]. Such phenotypic changes cause epithelial cells to lose their characteristic cell-cell adhesion structures alter their polarity modulate the anization of their cytoskeletal systems switch expression from keratin- to vimentin-type intermediate filaments as well as become isolated motile and resistant to anoikis [3] [4]. Typically cells undergoing EMT show decreased E-cadherin expression [5] [6] and decreased expression of mesenchymal biomarkers such as N-cadherin vimentin snail slug and twist [7] [8]. Previous studies on the relationship between p120-catenin (p120ctn) and EMT have been confined to the switch from short to long p120ctn isoforms during the EMT induced by expression of SIP1/ZEB2 [9] twist [10] or Zeppo1 [11]. However the mechanism by which p120-catenin isoforms 1A and 3A affect EMT of tumor cells remains unknown. The p120ctn protein has four isoforms (1 to 4) resulting from four transcriptional start sites and each isoform has a full central Armadillo repeat domain that can interact with the juxtamembrane domain of E-cadherin in order to participate in the formation of an adhesion complex on the cell membrane [12]. These observations suggest that the subcellular localization and function of p120ctn can be affected by the localization of E-cadherin. Previous studies have shown that p120ctn may play opposing roles depending on whether it is located on the membrane or in the cytoplasm of cells [13] [14]. Others have also found that p120ctn isoforms 1A and 3A have different regulatory functions on tumor cell proliferation invasion and metastasis [15] [16] . These studies indicate that if p120ctn has an impact on the EMT it is likely to be different between p120ctn isoforms 1A and 3A. Some studies have shown that p120ctn may promote or inhibit tumor growth and invasiveness depending on whether E-cadherin expressed or not [18] [19]. Yu and colleagues also found different effects of p120ctn isoforms 1A and 3A on proliferation and invasion in tumor cells exhibiting different localizations of E-cadherin [20]. Thus whether p120ctn isoforms 1A and 3A also play different roles in regulating EMT in tumor cells with E-cadherin at different locations remains unknown. The aim of this study was to determine the potential effects and regulatory mechanisms of p120ctn isoforms 1A and 3A on EMT in lung cancer cells. We first revealed that the membrane or cytoplasmic expression of p120ctn correlated with expression of E-cadherin and vimentin or lymph node metastasis by immunohistochemistry. We further detected the expression levels of p120ctn E-cadherin and vimentin in lung cancer cells by Western blot and screened cell lines expressing both low and high levels of p120ctn and with E-cadherin in the membrane or cytoplasm. Changes in expression of EMT-related molecules and cell invasion were also investigated by knockdown of endogenous p120ctn-1A or overexpression by transfection of p120ctn-1A and 3A plasmids into cells. Materials and Methods Materials This study was conducted with the approval of the institutional review board at China Medical University. Written consent was given by the participants for their information to be stored in the hospital database and for their specimens to be used in this study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Samples were collected from 78 cases of squamous cell lung cancer and lung adenocarcinoma diagnosed at the First Affiliated Hospital of China Medical University (Sheny-ang China). The samples were from 46 male and 32 female patients with an average age of 57 years. The samples were classified according to lung tumor histological criteria (2004) of the World Health anization (WHO) [21] as squamous cell lung carcinoma (32 cases) or lung adeno-carcinoma (46 cases). Thirty cases were highly differentiated and forty-eight were moderately or poorly differentiated. Lymph node metastases were present in 43 cases but not in the other 35. We selected cases with lymph node metastases to compare the metastatic nodules with the primary tumor. Tumor staging was performed according to the tumor-node-metastasis (TNM) staging system of the International Union against Cancer (UICC) [22]. There were 39 cases at stage III and 39 cases at stage IIIaIIIb. None of the patients had received radiotherapy or chemotherapy before the operation and were given the standard treatment following the surgery. All samples were fixed in formalin embedded in paraffin and stained with hematoxylin and eosin for pathological analysis and diagnosis. Cell culture Normal human bronchial epithelial (HBE) cells and A549 H1299 H460 and H157 cell lines were obtained from the American Type Culture Collection (Manassas VA USA). The SPC-A-1 LTEP-A-2 and LK2 cell lines were purchased from the Shanghai Cell Bank of Chinese Academy of Science. The human lung ADC Anip973 and AGZY83a cell lines were purchased from Shanghai Bioleaf Biotech Co. Ltd (http://www.bioleaf.com) and stored in the Department of Pathology Harbin Medical University. Cells were cultured in RPMI 1640 (Invitrogen Carlsbad CA USA) containing 10% fetal calf serum (Invitrogen) 100 IU/ml penicillin (Sigma St. Louis MO USA) and 100 mg/ml streptomycin (Sigma). Plasmid construction and transfection Expression plasmids for p120ctn isoforms 1A and 3A (donated by Dr. Albert B. Reynolds Department of Cancer Biology Vanderbilt University School of Medicine TN USA) have been described previously [16]. Sequences of p120ctn-1A-siRNA (Guangzhou Ruibo Co. Ltd Guangzhou China) used in the experiments were as follows: si-h-CTNND1: 5?-CACAAGAUGCCAACCCACU dTdT-3? 3?-dTdT GUGUUCUACGGUUGGGUGA-5?. The cells were transiently transfected with p120ctn-1A-siRNA and plasmids expressing p120ctn isoforms 1A and 3A using Lipofectamine 2000 (Invitrogen Carlsbad CA) or Attractene Transfection Reagent (QIAGEN GmbH Hilden Germany) according to the manufacturer's instructions. Immunohistochemistry The paraffin embedded samples were cut serially into 4-?m thick sections. Normal bronchial epithelium present in the tumor slides was used as an internal positive control. Immunostaining was performed by the streptavidin-peroxidase (S-P) method. The tissue sections were incubated with a p120ctn mouse monoclonal antibody (1?100 cat. 610134 BD Transduction Laboratories Lexington KY USA) E-cadherin rabbit monoclonal antibody (1?100 cat. SC-7870; Santa Cruz Biotechnology Santa Cruz CA USA) or vimentin rabbit monoclonal antibody (ready-to-use cat. RMA-0547 MaiXin Bio Fuzhou China) at 4°C overnight. PBS was used as a negative control. Biotinylated goat anti-mouse serum IgG or biotinylated goat anti-rabbit serum IgG (ready-to-use cat. KIT-9922 MaiXin Bio) was used as the secondary antibody. After washing the sections were incubated with streptavidinbiotin conjugated with horseradish peroxidase (Ultrasensitive MaiXin Bio) and then the peroxidase reaction was developed with 33-diaminobenzidine tetrahydrochloride (MaiXin Bio). Light counterstaining was performed with hematoxylin and then the sections were dehydrated in alcohol before being mounted. Two investigators independently examined all the tumor slides. Five random fields were examined per slide and 100 cells were observed per high magnification field (400Ã). The percentage of positive cells was scored as follows: 0?=?no staining; 1+?=?025%; 2+?=?2650%; 3+?=?5175%; and 4+?=?76100%. The staining intensity was scored as follows: 0?=?no staining; 1?=?light yellow granules; 2?=?dark yellow or brown granules. The labeling score defined by multiplying the percentage of positive cells by the staining intensity was the final score for the section. When the total score was ?3 the case was defined as positive. When the total score was <3 the case was defined as negative. For scores greater than 3 points when more than 30% of the tumor cells stained strongly and continuously for p120ctn signal on the cell membrane the sample was defined as membrane positive. When fewer than 30% of the tumor cells displayed membrane expression but stained strongly and continuously for p120ctn in the cytoplasm the sample was defined as cytoplasm positive. Western blot analysis Fifty micrograms of proteins were separated by SDS-PAGE (10%). After transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore Billerica MA USA) the proteins were incubated overnight at 4°C with antibodies to the following: p120ctn (1?500 cat. 610134) E-cadherin (1?300 cat. 610181) N-cadherin (1?1000 cat. 610920) (BD Transduction Laboratories Lexington KY USA) vimentin (1?1000 cat. 5741) snail (1?500 cat. 3879) (Cell Signaling Technology Boston MA USA) and twist (1?200 cat. sc-15193 Santa Cruz Biotechnology). After incubation with anti-mouse (1?2000 E030110-01) or anti-rabbit (1?2000 E030120-01) IgG (EarthOx LLC San Francisco CA USA) at 37°C for 2 h the protein bands were visualized using enhanced chemiluminescence (ECL Thermo Fisher Scientific Waltham MA USA) and quantified using BioImaging Systems (UVP Upland CA USA). Relative protein levels were calculated in reference to GAPDH as the loading control. Immunofluorescent staining Cells grown on glass coverslips were fixed with ice-cold 4% paraformaldehyde for 15 min followed by permeabilization with 0.2% Triton X-100 and incubation with normal goat serum for 30 min at 37°C. Cells were then incubated overnight with p120ctn mouse monoclonal antibody (1?200 cat. 610134; BD Transduction Laboratories Lexington KY USA) and E-cadherin rabbit polyclonal antibody (1?100 SC-7870; Santa Cruz Biotechnology). Primary antibodies were applied overnight at 4°C followed by incubation with a rhodamine/fluorescein-5-isothiocyanate (FITC)-labeled secondary antibody goat anti-mouse or TRITC-labeled goat anti-rabbit IgG (1?100 cat. E031210-01 and E031320-01 EarthOx San Francisco CA USA). The nuclei were counterstained with propidium iodide/4 6 diamidino-2-phenylin-dole. Epifluorescent microscopy was performed using an inverted Nikon TE300 microscope (Melville NY USA) and confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope (Carl Zeiss Thornwood NY USA). Matrigel cell invasion assay Matrigel cell invasion assays were performed according to the manufacturer's instructions (Corning Acton MA USA). A 100-?l cell suspension (5Ã105 cells) was added to the upper chamber while the lower chamber was filled with RPMI 1640 medium containing 10% fetal calf serum. Each upper and lower chamber was separated by a 8-?m porous polycarbonate membrane. The cells were incubated for 24 h at 37°C in a humid atmosphere with 5% CO2. After the medium was discarded the cells were fixed with methanol for 30 min and stained with hematoxylin (Sigma). For each filter the numbers of cells that invaded to the lower surface of the porous membrane in five different fields of 400Ã magnification were counted randomly using a Nikon E200 microscope. The mean was calculated from data obtained from each experiment repeated three times. Statistical analysis All statistical analyses were performed using SPSS 17.0 (SPSS Inc. Chicago IL USA) for Windows software. The chi-square test was used to analyze immunohistochemistry data. The independent samples T test was used to examine transwell experimental data. P values<0.05 were considered statistically significant. Results Membrane expression of p120ctn positively correlates with E-cadherin expression and negatively correlates with vimentin expression and lymph node metastasis Normal bronchial epithelial tissues showed p120ctn in the membrane (Figure 1A) while the proportion of lung cancer tissues expressing p120ctn in the membrane was significantly lower (35% 27/78) than that with p120ctn cytoplasmic expression (65% 51/78). E-cadherin was expressed in the membrane in normal bronchial epithelium tissues (Figure 1A) while the rate of positive expression was decreased (28% 22/78) and that of negative expression was significantly increased (72% 56/78) for E-cadherin in lung cancer tissues. Vimentin was negatively expressed in normal bronchial epithelial tissues (Figure 1A) while the rate of positive expression was increased to 32% (25/78) in the lung cancer tissue. It appears lung cancer tissues with cytoplasmic/nuclear localization of p120ctn tended to express vimentin in comparison with those with the membranous localization (41.2% [21/51] versus 14.8 [4/27]).. Cytoplasmic/nuclear localization of p120ctn showed increased lymph node metastasis (29/51) in comparison with the membranous localization (8/27). Statistical analysis showed that the localization of p120ctn was closely related with E-cadherin expression vimentin expression and lymph node metastasis (P<0.05) (Table 1). In other words p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis (Figure 1B); meanwhile p120ctn cytoplasmic expression was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis (Figure 1C). 10.1371/journal.pone.0088064.g001 Figure 1 Immunohistochemical analysis of p120ctn E-cadherin and vimentin localization in NSCLC. (A) E-cadherin and p120ctn were membrane positive and vimentin was negative in normal bronchial epithelial cells. (B) E-cadherin was membrane positive and vimentin was negative in p120ctn membrane-positive lung cancer cells. (C) E-cadherin was negative and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells. 10.1371/journal.pone.0088064.t001 Table 1 Correlation between E-cadherin vimentin and lymph node metastasis and p120ctn. p120ctn N membrane cytolymph/nucleolus X2 p E-cadherin negative 56 9 47 30.166 <0.01 positive 22 18 4 Vimentin negative 53 23 30 5.633 0.022 positive 25 4 21 Lymph node metastasis No 41 19 22 5.251 0.032 Yes 37 8 29 Localization of p120ctn is consistent with E-cadherin in lung cancer cells We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn (Figure 2A). Although the protein expression levels of p120ctn were not related to E-cadherin the localization (membrane or cytoplasm) of p120ctn was always consistent with that of E-cadherin. We then screened cells expressing high levels of p120ctn and E-cadherin in the membrane (H460 cells) or cytoplasm (SPC cells) as well as those expressing low levels of p120ctn and E-cadherin in the membrane (H4299 cells) or cytoplasm (LK2 cells) for further study (Figure 2B). 10.1371/journal.pone.0088064.g002 Figure 2 Expression and localization of p120ctn and E-cadherin in H460 SPC H1299 and LK2 cells. (A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells whereas they both were confined to the cytoplasm in SPC and LK2 cells. Different functions of p120ctn isoform 1A in EMT are dependent on E-cadherin subcellular localization Knockdown of endogenous p120ctn isoform 1A by siRNA-p120ctn-1A resulted in decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells (Figure 3A). However knockdown of endogenous p120ctn-1A by siRNA-p120ctn-1A showed opposite results in SPC cells where we found increased E-cadherin expression and decreased N-cadherin snail and vimentin expression (Figure 3B). In comparison with the control the ablation of p120ctn isoform 1A also enhanced the H460 cells invasiveness (17.33±1.25 vs. 36.33±1.70 P<0.01) (Figure 3C) whereas reduced the SPC cells invasiveness (23.0±0.82 vs. 13.0±0.82 P<0.01) (Figure 3D). These results revealed that the p120ctn isoform 1A plays a different role in EMT and cell invasiveness in different E-cadherin subcellular locations. 10.1371/journal.pone.0088064.g003 Figure 3 p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells. (A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01). Inhibitory function of p120ctn isoform 3A on EMT is not affected by differences in E-cadherin subcellular localization To verify whether p120ctn isoforms 1A and 3A play different roles in regulating EMT their expression plasmids were transiently transfected into lung cancer cells with low expression of p120ctn (H1299 with membrane E-cadherin expression and LK2 with cytoplasmic E-cadherin expression). The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin vimentin and snail expression (Figure 4A); on the contrary the decreased E-cadherin expression and increased N-cadherin vimentin and snail expression were observed in LK2 cells (Figure 4B). Overexpression of the p120ctn isoform 1A also reduced the H1299 cell invasiveness (52.0±2.65 vs. 33.33±2.64 P<0.01) (Figure 4C) while enhanced the LK2 cell invasiveness (18.0±0.82 vs. 39.66±2.05 P<0.01) (Figure 4D).. Overexpression of p120ctn isoform 3A led to increased E-cadherin expression decreased N-cadherin vimentin and snail expression (Figure 4A 4B) and reduced cell invasiveness (52.0±2.65 vs. 29.66±1.53 P<0.01; 18.0±0.82 vs. 8.33±expression 0.47 P<0.01) (Figure 4C 4D) in both of these cell lines. These results further confirmed that the p120ctn isoform 1A had a different effect on EMT depending on the subcellular localization of E-cadherin. They also revealed that the p120ctn isoform 3A maintained an inhibitory role in the EMT of lung cancer cells whether E-cadherin was localized to the membrane or the cytoplasm. 10.1371/journal.pone.0088064.g004 Figure 4 p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization. (A B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin vimentin and snail expression. (C D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid. Discussion The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin vimentin and other mesenchymal biomarkers [3] [4] [5] [6] [7]. As an important factor for stabilizing E-cadherin p120ctn plays a role in inhibiting or promoting tumor cell proliferation and invasion that is dependent on whether E-cadherin is expressed or not [16] [17]. Furthermore p120ctn isoforms 1A and 3A have shown different effects on E-cadherin expression and tumor cell invasiveness which are based on differences in the localization of E-cadherin [18]. These results strongly suggest that p120ctn most likely regulates the EMT of tumor cells by affecting E-cadherin expression and that p120ctn isoforms 1A and 3A play different roles in EMT expressing E-cadherin in different subcellular locations. We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies [13] [14] they further suggested that p120ctn likely affects the EMT by influencing the expression of E-cadherin and vimentin and thereby the cell invasion and metastasis in non-small cell lung cancer (NSCLC). To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A [24] [25] we could not design an interference sequence specifically for p120ctn isoform 3A. Therefore we had to further study the impact of the two isoforms on EMT and cell invasiveness in lung cancer cells with different E-cadherin locations specifically by knocking down p120ctn isoform 1A in H460 and SPC cells with high p120ctn expression and transfecting cDNA plasmids for exogenous p120ctn isoforms 1A and 3A into H1299 and LK2 cells with low expression of p120ctn. Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor p120ctn isoform 1A which was consistent with previous studies [20] [26]. Decreased E-cadherin expression and disrupted cell-cell adhesion may induce EMT [27] [28] [43] [44] which results in increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. On the other hand overexpressed p120ctn isoforms 1A and 3A was shown to bind E-cadherin located on the membrane proactively in tumor cells [29] and then inhibit the degradation of E-cadherin and stabilize its expression contributing to the formation of effective epithelial cell adhesion complexes [30] [31] [32]. As these series of processes maintained the normal cell-cell adhesion connection and inhibited EMT there was increased E-cadherin expression and decreased N-cadherin vimentin and snail expression as well as inhibited cell invasiveness in H1299 cells. Previous studies have shown that although p120ctn isoform 1A could bind E-cadherin in the cytoplasm they could not form effective adhesion complexes on the membrane between epithelial cells [33]. Furthermore the cytoplasmic E-cadherin is likely not to be the full-length E-cadherin but instead cleaved E-cadherin fragments such as E-cad/sE-cad (80 kDa) and E-cad/CTF2 (33 kDa) [34]. The E-cad/CTF2 fragment can bind to p120 in the cytoplasm and then translocate into the nucleus and bind the transcriptional repressor of Kaiso to activate the Wnt/b-catenin pathway [35] [36] finally promoting the EMT of tumor cells and enhancing cell invasion and metastasis [37]. Moreover others have shown that p120ctn-1A is related to abnormal expression of E-cadherin and poor prognosis [38]. These studies illustrated that the cytoplasmic p120ctn isoform 1A can play a role in promoting tumor cell EMT invasion and metastasis. Based on the above we observed on the one hand that the effect of p120ctn isoform 1A to promote tumor cell EMT invasion and metastasis would be lifted by its ablation resulting in increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited invasiveness in SPC cells. On the other hand transfection of the p120ctn isoform 1A plasmid into LK2 cells expressing cytoplasmic E-cadherin resulted in decreased E-cadherin expression increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. Although the precise role of p120ctn during EMT induction is still unclarified previous studies suggested that knockdown of all isoforms of p120ctn could induce EMT indirectly [27] [28] [43] [44]. All inductions were based on decreased E-cadherin expression and intercellular adhesion in previous studies which were also confirmed by our study in H460 cells with E-cadherin membrane localization. Unlike the H460 cells knockdown of the p120ctn isoform 1A in SPC cells with E-cadherin cytoplasmic expression could not decrease E-cadherin expression and intercellular adhesion. Instead we found increased E-cadherin expression and decreased cell invasiveness indicating that the EMT could not be induced by this pathway in SPC cells. It was worth noting that the same result was observed in LK2 and H1299 cells transfected with the p120ctn isoform 3A plasmid both showing increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited cell invasiveness. These results suggested that p120ctn isoform 3A has the function of inhibiting EMT of lung cancer cells and this function is independent of the cellular E-cadherin localization. Past research had also confirmed a shift from p120ctn isoform 3A to p120ctn isoform 1A expression after the induction of EMT [9] [10] [11] which indirectly indicates that p120ctn isoform 3A" | 1 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l 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" while the impact of family caregiving has been welldocumented many of such studies center oninvestigating external factors such as socioeconomic status accessibility to resources and availability of socialsupport as the primary causation of caregiver wellbeing outcomes this paper explores the motivations that drivefamily caregivers in supporting their family members at the endoflife and critically examines how internalappraisal processes of such motivations can both positively and negatively impact their wellbeingmethods this study adopted an interpretative phenomenological analysis ipa to investigate the motivations andinternal appraisal processes of asian family caregivers in singapore who were tending to a dying family memberqualitative dyadic interview data n was drawn from a larger randomized controlled trial for a novel familydignity intervention fdi for palliative care patients and their families the sampling population consisted ofparticipants aged and above who were identified to be the primary caregivers of older palliative care patientswith a prognosis of less than months data collection was conducted in the homes of patients and familycaregiverscontinued on next page correspondence andyhyhontuedusg1psychology programme school of social sciences nanyang technologicaluniversity singapore singapore2centre for population health sciences lee kong chian school of medicinenanyang technological university singapore singaporefull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctanho bmc palliative care page of continued from previous pageresults findings revealed six themes that could either nurture or diminish caregiver wellbeing honoringfidelity caregivers were motivated to commit to their caregiving roles in order to avoid regret alleviatingsuffering caregivers were motivated to relieve their family members pain enduring attachment caregiverswere motivated to spend time together with their family member preserving gratitude caregivers weremotivated to express their appreciation to their family member by caregiving navigating change caregiverswere motivated to adapt accordingly to changes in the illness trajectory and reconciling with mortalitycaregivers were motivated to respond accordingly to their family members prognosis the final theme of thewellbeing determinant is posited as an indication of selfdetermination and is conjectured to influence howcaregiving motivations are appraised by the caregiver fulfilling and enhancing ones sense of selfdetermination appears central to infusing ones caregivingmotivations with positive meaning and consequently nurturing ones wellbeing in the endoflife caregivingjourney these findings are discussed with recommendations for healthcare professionals working with familycaregivers of palliative care patientskeywords palliative care caregiver motivations wellbeing meaning burnout resilience selfdetermination endoflife qualitative research the experience of an endoflife eol family caregivercan be likened to a paradox what could evoke a senseof pleasure appreciation and gratitude could also bringabout feelings of anxiety distress and pain while somehave related the caregiving journey to the metaphor ofascending a mountain the expedition of an eol family caregiver usually spans beyond merely a couple ofdays weeks or months they must navigate the peaks ofdiagnosis to prognosis and eventually death and bereavement in what often unfolds into a lifelong climbthe role of the eol family caregiver is often multifaceted and interminable daily duties involve managingmedical regimes traversing the healthcare system andtaking charge of other dependents alongside providingphysical mental and emotional support throughout theillness trajectory [ ] many family caregivers are rarelyequipped with formal or adequate training and nor dothey possess sufficient resources and skills before theyfind themselves embroiled in eol caregiving responsibilities family caregivers must also process and manage amultitude of thoughts and emotions as they come toterms with the changes and sometimes losses in theirpersonal lives this complex experience is not limited to a minorityof people despite strong global advancements in medical technology and healthcare systems older populations remain highly susceptible to chronic and terminalmorbidities that are incapacitating in the unitedstates alone over million caregivers tend to their ailing family members annually while an estimated of patients in europe requiring longterm care areattended to by informal caregivers with the anticipated number of older adults in the world soaring to billion by the year there will certainly be a surging demand for family caregivers to relieve the ensuingresource strains on healthcare settingsthe impact of family caregiving stressors has beenwelldocumented [] with various studies exploringcaregiver burnout and its effects on the community andsociety complementing these studies are literature thatreveal characteristics of resilience and transformationalgrowth displayed by family caregivers in adversity [] the common thread that impacts both caregiverburnout and resilience appears to be a lack of or adequate coping a process that requires the individual toconstantly change efforts in both thoughts and behaviorsin order to manage internal or external demands thatare considered stressful despite this indication thatones psychological resources are key to maintainingones wellbeing many studies often consider externalfactors such as socioeconomic status accessibility toresources and availability of social support as the primary causation for the degree of caregiver wellbeingthus these studies often recommend pragmatic interventions that focus on improving external circumstancesaccordinglyperception emotion and motivation of the familycaregiverwhile it is undoubtedly beneficial to mitigate tangiblestressors one must not lose sight of the magnitude of apersonsthedemands of caregiving this perception and appraisalembody ones subjective caregiver burden observed inreviews of over caregiver studies to pose deepseatedimplications on caregivers quality oflevels ofdepression and anxiety and stress coping [ ]internal perception and appraisal oflife 0ctanho bmc palliative care page of campbell later confirmed this observation in astudy that evaluated multiple variations in the caregiverexperience subjective caregiver burden was repeatedlyidentified as the primary indicator for caregiver stressit was around the same time that folkman andmoskowitz discovered that caregivers experiencepositive emotions alongside negative emotions duringstressful events they put forth the tenet of meaningfocused coping in which caregivers derive mental andemotional sustenance thus effecting positive emotionsin challenging circumstances by deferring to their beliefsvalues and existential goals folkman and moskowitzfound that meaningfocused coping is an intrapsychicprocess of discovering benefits in caregiving reminding oneself of such benefits setting goals thatinspire a sense of mastery and competence realigningpriorities in view of changes and infusing ordinaryevents with positive meaning folkman furtherattested that meaningfocused coping exists alongsidenegative appraisals in a caregivers stress process inorderand psychologicalresources during stressful eventsto reinstate physiologicalgiven the importance of psychological appraisal it isreasonable to postulate that gaining insight into a caregivers beliefs values and goals that stimulate them incaregiving defined as motivations in this paper wouldyield valuable information that could be used to enhancemeaningfocused coping such interventions would target the essence of a persons selfconcept that is theindividuals perception of who they are and what theybelieve in and transcend current cursory social mentaland emotionalfor caregiverstresssymptom management[ ] a studybridging the research gap in asian caregivingwhile the multidimensional nature of caregiving burdenand coping is not confined to any specific culture thereare distinctive elements that bear influence on the asiancaregiving experience family takes precedence in asiansocieties with strongly inculcated values and expectations of filial piety and filial responsibility placed uponfamily membersconducted insingapore a multiethnic society that is predominantlychinese found that family caregivers who internalizedand prioritized societal expectations as motivations overtheir personal wellbeing most often faced internalconflict and were highly likely to experience difficultiesin maintaining their mental health familial ties and caregiving duties this is corroborated by the evolvingattitudes towards such confucian rules younger asiangenerations no longer perceive absolute submission orcomplete obedience to the family as instrumental valuesin a modern and globalized society and it would bevaluable to further understand how these complexitiesmight manifest in a family caregivers motivationsthis paper aims to contribute to and grow the currentbody of knowledge for asian eol family caregivers byanswering the following research questions what arethe internalized motivations defined here as uncononessciously assimilated beliefs and valuesattitudes or behaviors of asian family caregivers how might these motivations affect the way they respond to caregiving and impact their wellbeing howcan an understanding of these motivations be integratedinto psychosocial interventions to enhance and sustaincaregiver wellbeingintomethodsresearch design and proceduresthe current study draws qualitative dyadic interviewdata n from a larger randomized controlled trialfor a novel family dignity intervention fdi for asianpalliative care patients and their families n thesampling methods inclusion criteria interview procedure and study protocol for the fdi are comprehensivelydescribed by ho briefly the fdi is developedbased on an integrative body of empirical investigationthat focuses on dignified endoflife care in both western and asian contexts [ ] it integrates elements oflogotherapy and narrative life review to provide psychosociospiritual support to patients and families facingmortality and has been piloted for acceptability andfeasibility before being fully adapted into the intervention study in practice fdi comprises a recorded dyadicsemistructured interview with a patient and a familycaregiver conducted in their homes the fdi therapistuses a guided question framework to facilitate joint conversation on shared memories and living wisdoms thatlead to meaningmaking and the expression of appreciation and reconciliation this is done with the ultimategoal of creating a legacy document that tells the life storyof the patient and is bestowed to the rest of the familythrough an open reading each interview lasted between and min and was conducted in english malaymandarin or a chinese dialect hokkien teochew orcantonese these recorded interviews were transcribedverbatim translated into english by a native languagespeaker where applicable and edited into legacy documents transcripts and legacy documents were reviewedand finalized by patients and caregivers to ensure accuracy and authenticitysamplingthe sample drawn for this study consisted of primaryfamily caregivers of older palliative care patients aged and above with mainly a cancer prognosis of lessthan months eleven were spousal caregivers seven 0ctanho bmc palliative care page of were adultchildren caregivers and two were siblingcaregivers the majority were female aged between to with a mean age of years see table for caregiver demographics they were recruited through theinpatient daycare and homecare hospice service unitsof hca hospice care dover park hospice tan tockseng hospital singapore cancer society and methodistwelfare services the inclusion criteria required familycaregivers to be above years old and identified by thepatient to be their primary carer patients and familycaregivers came from varioussocioeconomic s and were predominantly of chinese ethnicityas the fdi focused on patient narratives transcriptsbearing a moderate to sizeable amount of input from thefamily caregiver were selected for data analysisdata analysisstudy adopted an interpretative phenomenothislogical analysis ipato investigate the internalizedmotivations of family caregivers in tending to a dyingfamily member the ipa is an approach in qualitativeresearch that aims to provide insights into how an individual makes meaning out of a phenomenon itis important to note that as the current study drawsqualitative data from the larger fdi study no explicitinternalized caregiver motivationsquestionsaboutwere asked reflections on motivations towards caregiving occurred anically throughout the interviewtranscripts and were identified by the researcher usingipa through a process of data reduction and datareconstructionfirst authors and screened all transcripts andselected those that had adequate inputfrom familycaregivers to be used for analysis authors and thenconducted linebyline coding to develop descriptivethemes and analytical categories conceptualizing newinterpretation of the data this was followed by regularmeetings among all authors for the further refinement ofthemes and categories to encapsulate the meaning andcontent within the cluster of similar codes with theemergent themes and subthemes created via a summarychart all authors reviewed and defined the emergentthemes once consensus was reached operational definitions were created finally relationships between categoriesthemes and subthemes were proposed andmapped with supporting quotes from transcripts to address issues of trustworthiness and credibility emergentthemes were constantly compared and contrasted withinand across groups during regular meetings the finaltheme categorization and definitions were agreed uponby the entire research team and data saturation and investigator triangulation were achieved table demographics of family caregiversidentifierdph14caregiver relationshipchildcaregiver ethnicitychinesepatients diagnosislung cadph19dph34dph42dph53dph59dph68hca12hca68hca75hca81hca87hca109hca114hca116hca117mws004scs18ttsh61ttsh65spousespousesiblingspousechildspousespousechildchildchildspousespousespousespousespousesiblingspousechildchildchinesechinesechinesechinesechinesemalayeurasianchinesemalaychinesemalaychinesechinesechinesechinesechinesechinesemalaychineseprostate calung casigmoid calung cagynaecological malignancylung caprostate cacolon cabreast caendometrial carenal caendometrial cabrain canasopharyngeal capancreas cacopdliver calung cagynaecological capatients prognosis months 0ctanho bmc palliative care page of didnt i do this why didnt i do that dph19spousein some instances family caregivers displayed poignant emotion and dedication to their family membersconveying the great extent to which they would goto give theirthe best care andcomfortfamily membersi wish to care for him till the very end¦ i want thebest for him and i will do whats best for him i amwilling to sacrifice my soul to make that happen ortake his place if i could dph68 childalleviating suffering n family caregivers displayed awareness and empathytowards their family members physical and emotionalsuffering tied in with a desire to relieve their painthis morning she was upset with me for forcing herto drink the bitter medicine i told her i love you iwouldn't do this if i had a choice i want you todrink this for your own benefit not mine i'm just encouraging you from thedph53spousesidelinesunderlining this motivation to alleviate suffering was thefamily caregiversinnate compassion for their familymember an empathic bond so strong that witnessingtheir family members suffering caused them deep emotional distress¦ it hurts a lot to drain the fluids im heartbrokenwhen i see how much pain she is in especially wheni see the tubing being inserted it must hurt somuch hca109 spousefindingsfigure presents the six caregiving motivations and onewellbeing determinant generated from data to form theblessings or burdens of endoflife caregiving bobecmodelthe six caregiving motivations honoring fidelityalleviating suffering enduring attachment preservinggratitude navigating change and reconciling withmortality represent the beliefs values and goals that areassimilated into the eol family caregivers daily life eachmotivation is posited to affect the way a caregiver makesmeaning of their role hence leading to a nurturance ofcaregiver wellbeing termed in this paper as blessings ora diminishment in caregiver wellbeing termed in thispaper as burdens the wellbeing determinant which ischaracterized by the caregivers sense of control selfempowerment and kinship derived from their experiences serves as an indication of selfdetermination andis theorized to have a positive influence on how the sixcaregiving motivations are appraised by the caregiverthese themes are described in greater detail and demfrom study participantsonstrated by direct quotesbelowhonoring fidelity number of transcripts theme hasoccurred in n family caregivers expressed their faithfulness and commitment to attend to the needs and wishes of their family members for the remainder of their lives fearingregret to see things through till the end this motivatedthem in fulfilling their sense of duty to the utmost oftheir abilitiesi shouldnt regret anything whatever i can do forhim i will do my best and [instead of waiting till]hes in the coffin you know [and then say] oh whyfig the blessings or burdens of endoflife caregiving bobec model 0ctanho bmc palliative care page of enduring attachment n family caregivers experienced a prevailing attachment totheir family member that motivated them in spendingcherished time together and doing all they could toensure that their family member was well taken care ofi think i try to make him as comfortable as he canbe every medical checkup every appointment wewill keep to it and i will always be there for him[there will never be] any appointment that i am notgoing with him hca117 spousesome family caregivers found that the motivation tosustain this attachment was also driven by feelings ofanxiety about their family members wellbeing thesecaregivers felt the need to be within their family members physical proximity as much as possiblei get worried when shes lying there and sleepingbecause im not sure if anything has happened toher im much happier when shes sitting here withme when shes just lying there i would think ohno what if something has happened to her and idbe worried dph59 childpreserving gratitude n family caregivers described past circumstances and beliefs that motivated present feelings of gratitude to theirfamily members this consequently influenced their efforts and responses in caregivingshe was constipated for as long as a week and shedidnt tell me when she eventually relieved herselfshe made a mess on the bathroom floor as i wascleaning the mess i thought about how she hadcleaned me up when i was little so i didnt mindhca81 childwhile many family caregivers reported feelings of gratitude stemming from how their family member hadtreated them in the past some indicated that religiousand cultural beliefs had indoctrinated a sense of indebtedness to their family membersmy mother says i was indebted to my brother in mypast life this is why i have to settle my debt in thislifetime [by caregiving] because he is here to get hiscompensation mws004 siblingnavigating change n family caregivers reflected on their perceptions of thechanges that had taken place in their lives and that oftheir family members throughout the illness trajectorysome caregivers found motivation in helping their familymembers adaptenergy to liftsupportto changes by dedicating time andtheirspirits and provide emotionali would bring my father food when i visit while myhusband would share words of encouragement andtalk to him to cheer him up we just want him to behappy so that he wouldn't spend the whole day innegativity ttsh65 childothers saw the changes as a temporary setback andfound motivation in steering their family member backto their previous condition if possiblesometimes i will move his legs a little to give himthat exercise i hope that he can walk again but itdepends on how strong his willhca116spouseisreconciling with mortality n the knowledge of their family members prognosis motivated some family caregivers to make the most of thetime left with their family members creating treasuredmemories and remembering their legaciesall of us just want to cherish the time that we haveleft with her and we want her to help us spend moregood times together we [want to] learn about mygrandmother learn about my mother so that wecan pass on to the next generation share with themthe traits and the role models to look up tottsh61 childother family caregivers perceived their family membersprognosis to be unacceptable choosing to push for further treatment in order to stall deaths journey to theirdoorsmy grandmother lived past years old so ithought my mother would live till atleast without any problems i felt really shocked becausei always thought she still has more than years istill have time ¦ so we felt that if it was possibleshe should extend her life dph14 childthe wellbeing determinant n family caregivers reflected on the discovery of positivetheir family memberschanges amidstillness one such change was found within strengthenedkinshipthe trials ofas we grow up its a bit harder [to have familygatherings] because we are all working so when thedisease came even though its not a good thing not 0ctanho bmc palliative care page of something you will ask for it united us again maybewithout it [we] would have been a bit more separated ttsh61 childi feel like we are more united now maybe in thepast we didn't really chat with each other¦ theamount of communication we had increased i feelthat our unity has become stronger dph14 childthey expressed pride in overcoming initial fears by taking charge of and learning to perform unfamiliar taskswith a newfound confidence in their abilities to faceboth practical and emotional difficulties family caregivers also demonstrated a sense of selfempowermentand strengthbased reflection in their sharingi know nothing about going to visit the government¦ or to do this do that but somehow i findmy way there [i am a] much stronger person so ifanything happens to me i think i know i can faceup to it dph19 spousethis is how you grow i learnt to grow because of[my husband] you have to face the insurmountablechallenges that come your way i learnt how toshoulder my responsibilities on my own scs18spousediscussionthis is the first known study that investigates and bringsattention to the internalized motivations of eol familycaregivers while the family dignity intervention studyquestions did not specifically query family caregivers ontheir motives these motivationcentred responses occurred spontaneously and abundantly throughout the interviews an indication that internalized motivationsare profoundly espoused within eol caregiving attitudesand behaviors the bobec model fig illustrates theduality ofthese caregiving motivations in regard tomeaningfocused coping and intrapsychic strains as well as identifies an important influence on caregiver wellbeingmotivations with cultural influencessome themes revealed cultural undertones that reflectedthe internalization of asian values into family caregivermotivations in their motivation for alleviating sufferingfamily caregivers displayed the desire to do so by practical means such as administering medication to theirfamily member and experiencing distress when suchmethods were not feasible this is in line with the asianculture of preferring to show concern for their familymembers through pragmatic ways in their motivation for preserving gratitude family caregivers demonstrated the significance of filial piety as well as culturalbeliefs about karma and pastlife within theirattitudes towards caregiving finally in their motivationfor reconciling with mortality family caregivers indicated the importance placed on close intergenerationalconnections as well as longevity for their elders motivations as blessings meaningfocused copinghonoring fidelityall eol caregiving motivationsalleviating suffering enduring attachment preservinggratitude navigating change and reconciling with mortality were found to embody the tenets of meaningfocused coping fuelled by these motivationsfamilycaregivers displayed the propensity for benefitfindingand benefitreminding even in witnessing their familymembers suffering and imminent mortality adaptivegoal processes in adjusting their expectations and aspirations in accordance with their family members physicalcondition and prognosis reordering priorities in hopesof making the most of the time left with their familymember and infusing ordinary events both past andpresent with positive meaning that allowed them to feelaffirmed encouraged and grateful in their daily caregiving as such the capacity for imbuing stressful caregiving events with positive meaning and responseswould make these motivations advocates of perceivedblessings in the eol caregiving journeymotivations as burdens intrapsychic strainsparadoxically the authors found that these eol caregiving motivations also ran parallel to the intrapsychicstrains as postulated by pearlin and colleagues intheir seminal stress process model intrapsychic strainsoccur when the caregivers selfconcept is diminisheddue to the chronicity of providing care intrapsychicstrains unique to caregivers were defined as role captivity in which the caregiver feels entrapped within hisor her role whether or not by personal choice the lossof self in which the caregiver experiences a loss of identity and sense of personhood as enmeshment with thepatient ensues perceived low competencein whichthe caregiver does not see the value and skill of the workthey do leading to a sense of helplessness and perceived lack of gain in which the caregiver does not findpersonal growth orcaregivingprocessenrichmentin theshould their eol caregiving motivations personifythese strains it is only a matter of time before familycaregivers experience outcomes of mental and emotionaldistress such as depression anxiety and irritability aswell as a decline in physical health and a disengagementfrom their caregiving roles in short these motivations would most certainly bludgeon the family caregiverwith great burdens within the eol caregiving journey 0ctanho bmc palliative care page of the crucial factor selfdeterminationselfdetermination theory suggests that people needto feel a sense of competence gaining mastery of tasksand having selfefficacy relatedness feeling like theybelong and mutually relating to others and autonomycontrol over their own choices behaviors and goals inorder to fuel high quality motivation that helps one tothrive the theme of the wellbeing determinant alignswith this concept in a caregivingcentric phenomenonfamily caregivers contributing to this theme displayedconfidence in carrying out previously unfamiliar caregiving tasks competence affirmed a stronger sense of kinship relatedness with the patient and their families andtook ownership of their caregiving responsibilities andchallenges autonomy encouragingly numerous studieshave proposed that high quality motivations stemmingfrom selfdetermination can elicit outcomes of greaterfortitude higher commitment and more positive emotions and selfconcepts []building on said studies the authors propose that family caregivers who feel a sense of competence relatedness and autonomy within their caregiving motivationswould be further inclined to meaningfocused copingsuch as deriving perceived benefits from their caregiving even in difficult events reminding themselves ofthese benefits when faced with similar circumstances setting their own goals in caregiving having the competence and confidence to be flexible and adaptive and finding positivity in normal everyday situations possessing a sense of selfdetermination in the caregivingrole would in essence safeguard ones caregiving motivations from the intrapsychic strains of perceived entrapment a sense of disempowermentineptitude andfruitlessnessas such the bobec modelidentifies the wellbeingdeterminant as an indicative element of caregiver selfdetermination and a crucial factor as to whether the eolfamily caregiver perceives their journey as one lined withblessings or laden with burdens competencetargeted mediators apart fromgeneral psychoeducation on symptom managementmedical care and selfcare interventions could incorporate mediums to develop and improve selfefficacy these can take the form of personalstrengths journaling facilitating peer support between new and experienced caregivers rolemodelling and goalsetting such interventions canbe created in the form of structured support groupsonline platforms or mobile applications autonomytargeted mediators in addition toproviding adequate and appropriate eol caregivereducation autonomytargeted interventions suchas those involving mindfulness practice and thearts can serve to give caregivers a sense of control as well as help them make meaning out oftheir thoughts emotions and circumstances todate the mindfulcompassion art therapymcat for eol care professionals showsgreat potential to be converted into a programfor eol family caregivers relatednesstargeted mediators dyadic or familyprojects that recall shared memories expressappreciation seek fiveness impart wisdom andcreate generativity such as the fdi are valuable incrafting the bond of relatedness among familycaregivers and their family members such projectsshould be implemented as a foundation ofpsychosocial interventions at the endoflifeinterventions should be offered to family caregiversin a culturallyrelatable manner this can be donethrough emphasizing how these mediators will helpfamily caregivers enhance their practical caregivingwith increased competencesense of control andmeaningmaking as well as enrich their intergenerational familial bonds with conversations that focus onlegacy creationimplications and recommendationsfindings from a number of studies show that fulfillingones sense of selfdetermination appears central to sustaining ones motivation and innate satisfaction in caregiving [] the findings from this paper indicatethat caregivers are driven by motivations that couldequally contribute to wellbeing nurturance or diminishment at the same time our findings also indicate thatcaregivers possess a caregivercentric sense of selfdetermination the wellbeing determinant that is theorized to have positive affect on their motivations thusthis paper recommends that caregiver support interventions should comprise all of the following mediators inorder to fulfil the need for selfdeterminationlimitations and future directionswhile the anically occurring responses emphasize thesignificance of motivations within eol caregiving theseresponses were not examined further during the fdi interviews due to other pri | 0 |
"Evidence on the association between exposure to perfluoroalkyl and polyfluoroalkyl substancesPFASs and blood glucose concentrations in pregnant women is inconsistent This study aimed to examine theassociation between PFAS exposure and the concentrations of fasting plasma glucose FPG and onehour plasmaglucose hPG after a 50g oral glucose tolerance test in pregnant womenMethods The study was based on the ShanghaiMinhang Birth Cohort in which pregnant women were recruitedAmong them women provided blood samples at gestational weeks for PFAS measurement FPG data collectedfrom women at GW and hPG data collected from women at GW were obtained through medicalrecords from the routine prenatal care system High FPG or hPG was defined as ¥90th percentile of FPG or hPG Theanalysis of eight PFASs was conducted in this study perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOSperfluorooctanoic acid PFOA perfluorononanoic acid PFNA perfluorodecanoic acid PFDA perfluoroundecanoic acidPFUdA perfluorododecanoic acid PFDoA and perfluorotridecanoic acid PFTrDA The odds ratios ORs and associated confidence intervals CIs were estimated to determine the associations of each PFAS compound with high FPG and hPG from a logistic regression modelResults After adjustment for potential confounders most PFASs were positively associated with high hPG concentrationsThe OR for high hPG concentrations was CI with a one log unit increase of PFOS similar associationswere observed for PFNA OR CI PFDA OR CI PFUdA OR CI and PFDoA OR CI When the PFAS concentrations were categorized into three groups by tertiles thehighest tertiles of PFOS PFOA PFNA PFDA PFDoA and PFTrDA had a statistically significant increase in the risk of high hPG concentrations compared with the lowest tertiles No statistically significant association was observed between PFASexposure and high FPGConclusion PFAS exposure was associated with an increased risk of high hPG among pregnant women but no suchassociation was observed for FPGKeywords Perfluoroalkyl and polyfluoroalkyl substances Plasma glucose Cohort study Pregnancy Correspondence miaomaohua163com Yanfeng Ren and Longmei Jin contributed equally to this work4NHC Key Lab of Reproduction Regulation Shanghai Institute of PlannedParenthood Research Fudan University Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cRen Environmental Health Page of IntroductionPerfluoroalkyl and polyfluoroalkyl substances PFASs agroup of manmade chemicals with water stain andgreaseresistant properties are used in a wide range ofconsumer products including fast food packaging stainresistant carpets windshield washing fluid firefightingfoam insecticides and paints [] Humans are widely exposed to PFASs through the ingestion of contaminateddrinking water and food as well as the inhalation ofcontaminated indoor air and dust [] Some PFASs havebeen shown to bioaccumulate in anisms [] Themean halflives of PFASs in adult humans vary from to years [ ] The most commonly studied PFASsincluding perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOS perfluorooctanoate PFOAand perfluorononanoate PFNA are detected in the majority of human serum samples []Animal studies have shown that PFAS exposure is associated with a wide range of adverse health effects including the disruption of endocrine hormones such astestosterone estrogen and thyroid hormones [ ] alterations in serum lipid levels [] impaired glucose metabolism and insulin hypersensitivity [] and immune systemdisturbance [ ] Human studies have also suggested theadverse effects of PFASs on the immune system [] carcinogenesis [] pregnancyinduced hypertension arterialatherosclerosis [ ] and glucose metabolism []Although epidemiological studies have suggested thatPFASs are associated with impaired glucose toleranceand homeostasisinsulin resistance betacell dysfunction and a higher risk of diabetes [] the associations observed in the general population cannot begeneralized to metabolically vulnerable pregnant womenowing to their specialinsulinresistant state duringpregnancy The current evidence on the effects of PFASson glucose metabolism in pregnant women is limitedand inconclusive In the Odense Child Cohort studyPFHxS and PFNA concentrations were associated withimpaired glycemic status in pregnant women and maytherefore enhance the risk of developing gestational diabetes mellitus GDM [] In another prospective studyof women higher prepregnancy PFOA concentrations were associated with an increased risk of GDMbut the associations for six other PFASs were not statistically significant [] In contrast Valvi found noassociations between PFOA PFOS PFHxS PFNA orperfluorodecanoic acid PFDA concentrations and therisk of GDM in pregnant women []In the present study we sought to evaluate the associations between PFAS exposure and fasting plasma glucose FPG and 1h plasma glucose concentrations hPG measured after a 50g oral glucose tolerance testOGTT in pregnant women by using data from theShanghaiMinhang Birth Cohort Study SMBCSMethodsStudy participantsAll study participants were recruited from the SMBCSbetween April and December Pregnantwomen attending their first routine antenatal care at theMaternal and Child Health Hospital of Minhang districtin Shanghai were consecutively recruited if they wereat gestational weeks GW of pregnancy theywere registered residents of Shanghai they had nohistory of chronic disease of the liver kidney or otherans they planned to give birth in the study hospital and they were willing to participate in specifiedinterviews during pregnancy and after delivery Among pregnant women who were invited pregnantwomen were recruited corresponding to a response rateof Exposure assessment and quality controlBlood samples for PFASs measure were collected at recruitment and plasma samples were separated andstored at °C before they were transported to theCenter for Disease Control and Prevention in HubeiProvince for the assay of PFASHighperformanceliquid chromatographycoupledwith tandem mass spectrometry Agilent TechnologiesInc USA was used for the quantitative measurement ofPFASs The information on sample collection separation reservation transportation quantification limit ofdetection LOD and quality control has been detailedpreviously [] Among the PFASs measured in ourstudy eight PFASs with detection rates above including PFHxS PFOS PFOA PFNA PFDA perfluorododecanoic acid PFDoA perfluoroundecanoic acidPFUdA and perfluorotridecanoic acid PFTrDA wereincluded in the final analysesAn internal standard approach was used to aidquantification MilliQ water was used to performprocedural blank analysis for each batch of samplesThe concentrations of each detected congener shouldbe more than three times of that in the proceduralblank and were corrected by subtracting the procedural blank concentration in the present study LODwas defined as the concentration with a signaltonoise ratio equal to or greater than All the recoveries ranged from A five point calibration curve was drawn and each precursor rangedfrom ngmL Calibration curves presenteda linear pattern over the concentration range of theprecurslucose and covariate measurementThe information on plasma glucose concentrations inpregnant women was collected from the medical recordsof the prenatal care system and included results for FPG 0cRen Environmental Health Page of and hPG In the study hospital within the studyperiod pregnant women were asked to provide bloodafter overnight fasting for FPG testing at their earliestconveniences generally within week after their firstantenatal care It was suggested that pregnant womenunderwent a h 50g OGTT at GW in order toscreen for gestational diabetes if they were consideredto have a high risk of GDMn ie FPG ¥ mmolL mgdL [] or overweight and obeseieBMI ¥ kgm2 [] otherwise it was suggested thatthe examination of hPG was performed between and GW The 50g OGTT was performed after anovernight fasting also The distribution of gestationalweeks in which the FPG and hPG examination wasperformed is shown in Supplemental Table S1 Information on whether the women had been diagnosed withGDM was extracted through medical records at birthA structured questionnaire was used by trained interviewers to collect information on the covariates Thewomen were asked about age per capita household income education level passive smoking height prepregnancy weight parity history of abortion and stillbirth pregnancy complications etc Prepregnancy BMIkgm2 was calculated as body weight divided by bodyheight squaredStatistical analysisAmong the pregnant women recruited womendelivered singleton live births and women providedblood samples at enrollment for PFAS measurement FPGconcentrations measured at GWs were obtainedfor women and hPG concentrations measured at GWs were obtained for pregnant womenPregnant women who had data on PFASs and FPG concentrations were included in this study Fig We firstdescribed and compared the demographic characteristicsofthe included and excluded pregnant women Themeans and standard deviations SD were used to describethe distributions of FPG and hPG according to thedemographic characteristics ofthe included pregnantwomen A logistic regression model was used to examinethe association between PFAS exposure and plasma glucose with the 90th percentiles of FPG mmolL ie mgdL and hPG mmolL ie mgdL usedlogarithm lntransformedas the cutoff value NaturalPFAS concentrations were firstincluded in logisticFig Study population of the present study from SMBCS FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance test SMBCS ShanghaiMinhang Birth Cohort Study 0cRen Environmental Health Page of regression models and those with concentrations belowthe LOD were assigned a value of LOD PFAS concentrations were also categorized into three groups by tertilesT1 lowest tertile T2 middle tertile and T3 highest tertile and included in the logistic regression models withthe lowest tertile as the reference group Odds ratiosORs and associated confidence intervals CIs wereestimated for the association between each PFAS and highFPG1 hPG ie ¥90th of FPG concentration or ¥ 90th of hPG concentration Based on tertiles the concentrations were transformed to ordinal data and assigned to allpersons to calculate ptrend values In addition multiplelinear regressions were used to analyze the associationbetweenglucoseconcentrationscontinuousplasmaPFASsandPotential confounders were identified a prior according to the previous literature Age of pregnant womeneducation economic income prepregnancy BMI passive smoking parity history of abortion and stillbirthand pregnancy complicationsincluding bleeding thyroid disease and pregnancyinduced hypertension [ ] were identified and a directed acyclic graphsSupplemental Figure S1 was used to evaluate the appropriation of covariates We did not adjust for alcoholconsumptionn in the final models because of thelow prevalence The statistical assumptions of logistic regressions were evaluated and met including linear relationship of independent variables with logitp outliersand colinearity of independent variablesSeveral sensitivity analyses were performed to test therobustness of the primary results1 Considering the potential effect of prepregnancy BMI on GDM [] andthe variation in PFAS concentrations across BMI we repeated the analysis in women with a prepregnancy BMIof kgm2 to eliminate the confounding effect ofBMI To test the generalizability of the results we repeated the analyses in pregnant women without GDM To examine whether the associations of PFASs withFPG1 hPG were timedependent we performed subgroup analyses for different spans of GW at glucosemeasurement for FPG at GWs and GWsfor hPG at GWs and GWs StatisticalAnalysis System SAS software version SAS Institute Inc Cary NC USA was used for statistical analysis P values of were considered statisticallysignificantResultsTable presents the characteristics of the included pregnant women are compared with those of the excludedwomen in the study The majority of women included inthe present analyses were nulliparous years of age with a BMI between kgm2 with a household income per capita of Table Characteristics of the included and excluded pregnantwomenCharacteristicsPvalue of Studentsttest or Chisquare testIncludedN N Mean ± SDExcludedN N Mean ± SDMaternal age at enrollment yearsMean ± SD ± ¥ Prepregnancy BMI kgm2Mean ± SD ± ¥ Maternal education ± ± Below highschool High School College orabove Per capita household income CNY Passive smokingYesNo Pregnancy complicationYesNo History of abortion and stillbirthYesNoParity¥ CNYmonth well educated collegeleveleducation or above without pregnancy complication without history of abortion and stillbirth Approximately of women were exposed topassive smoking during pregnancy The distributions ofthese demographic characteristics were not significantlydifferent between the included and excluded womenexcept parityTable presents PFHxS PFOS PFOA PFNA andPFDA were detected in all maternal plasma sampleswhile PFUdA PFDoA and PFTrDA were detected in 0cRen Environmental Health Page of Table PFASs concentrations ngmL of the includedpregnant women N PFASLODLOD NGMGSDPercentiles5th25th 50th 75th 95thPFHxS PFOSPFOAPFNAPFDAPFUdA PFDoA LOD PFTrDA Note LOD limit of detection GM geometric mean GSD geometricstandard deviationLOD about samples PFOA and PFOS had the highestconcentrations PFOA GM ngmL PFOS GM followed by PFHxS GM ngmL ngmLPFDA ngmL PFNA ngmL and PFUdA ngmL while PFDoA and PFTrDA had the lowestconcentrationsTable presents the concentrations of FPG and hPGaccording to the demographic characteristics of the subjects The mean SD FPG and hPG concentrationswere mmolL ie mgdL and mmolL ie mgdL respectively Theconcentrations of FPG and hPG were comparableacross pregnant women with different BMI household income passive smoking status pregnancy complicationand history of abortion and stillbirth The concentrationof hPG was higher in pregnant women who were olderor had higher education levels but not in those with FPGThe concentration of FPG was lower in nulliparous pregnant women but not in those with hPGTable presents that higher concentrations of PFOSPFOA PFNA PFDA PFDoA and PFTrDA were associatedwith an increased risk of high FPG however the associationswere not statistically significant AORPFOS CI AORPFOA CI AORPFNA CI AORPFDA CI AORPFDoA 95CI AORPFTrDA CI Higher concentrations of PFASs were associated with an increased risk of high hPG except for PFHxSand the associations with PFOS PFNA PFDA PFUdA andPFDoA were statistically significant after adjustment for potential confounders AORPFOS CI AORPFNA CI AORPFDA CI AORPFUdA CI AORPFDoA CI In addition multiple linearregressions were also used to analyze the association betweenPFASs and plasma glucose Similar results were found inmultiple linear regression as in logistic regression modelalthough the association of PFDoA with hPG is not statistically significant Supplemental Table S2We further examined the associations between the categorized PFAS concentrations and FPG1 hPG Weak associations between the highest tertiles of PFASs and anincreased risks of high FPG were observed but the associations were not statistically significant Fig Comparedwith pregnant women with the lowest tertiles of PFASsthe risk of high hPG was increased in women with thehighest tertiles of PFASs with statistically significant associations observed for PFOS PFNA PFDA PFUdA andPFDoA AORPFOS CI AORPFNA CI AORPFDA CI AORPFUdA CI AORPFDoA CI Fig Linear trends were observed between the tertiles of PFOS PFNA PFDAPFUdA and PFDoA and high hPG P for trend and respectivelyWe repeated the analysis after excluding women withGDM The pattern of associations between PFASs andhigh FPG and hPG did not change substantially except that the association between PFDoA and high hPG was no longer statistically significant SupplementalTable S3 In addition the analysis among pregnantwomen with a BMI of kgm2 produced similar results Supplemental Table S4thatIn the subgroup analysis for different GW spans theassociations between PFASs and high FPG remainednonsignificant disregard of the timing of FPG measurements exceptthe increased concentrations ofPFNA were associated with an increased risk of highFPG at GWs AORPFNA CI The pattern of association between PFASs andhigh hPG did not substantially change disregard ofmeasurement time of hPG with the exception thatthe association with high hPG became nonsignificantfor PFOS PFUdA PFDoA at GWs and PFOSPFNA PFDA and PFUdA at GWs largely owingto the reduced sample size Supplemental Table S5DiscussionIn this prospective cohort study PFAS exposures inpregnant women were found to be associated with high hPG but not FPG and the association persisted forpregnant women without GDM or with BMI kgm2Many studies have demonstrated that PFASs wereassociated with impaired glucose homeostasis and anincreased risk of diabetes in the general population [] However in pregnant women the associations between PFASs and glucose homeostasis have not beenwell investigated Wang et als study showed that severalPFAS compounds were associated with increased postpartum FPGincluding perfluoro1metylheptylsulfonat mPFOS perfluoro34metylheptylsulfonat m 0cRen Environmental Health Page of Table The distribution of FPG and hPG mmolL according to participants demographic characteristicsCharacteristicsFPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueTotalMaternal age at enrollment years ¥ Prepregnancy BMI kgm2 ¥ Maternal educationBelow high schoolHigh SchoolCollege or abovePer capita household income CNY Passive smokingYesNoPregnancy complicationYesNoHistory of abortion and stillbirthYesNoParity¥ hPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvalue FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucose tolerance test p compared with the first groupmPFOS perfluoro5metylheptylsulfonat mPFOSand PFHxS [] The Longitudinal Investigation of Fertility and the Environment LIFE study reported thateach SD increment in PFOA concentrations was associated with a 187fold increase in GDM risk [] In theOdense Child Cohort study in metabolically vulnerablepregnant women ie BMI ¥ kgm2 family history ofdiabetes mellitus previous GDM multiple pregnancy ordelivery of a macrosomic child PFHxS and PFNA concentrations were associated with impaired glycemic status however no associations were found in women withlow GDM risk [] Its a pity that the absence of information on history of family diabetes and subjects withprevious GDM limited our ability of examining the[]association in subjects with high risk Higher concentrations of PFASs in our study may partially contributeto the differences with other studies In our studyconcentrations of most PFASs were much higher thanthose in the Odense Child Cohortthe LIFEStudy [] and Wang et al study [] except thatPFOS is higher in the LIFE Study compared to thecurrent study The differences in concentrations aswell as outcome indices of impaired glucose homeostasistiming of measurement and population included make the comparison between these studiesdifficult nevertheless the potential for PFAS exposureto disturb glucose homeostasis has been supported inmost studies 0cRen Environmental Health Page of Table Association between PFAS concentrations lntransformed and high FPG and hPG in pregnant womenInPFASngmlPFHxS hPGN COR CIFPGN COR CI AOR CIPFOSPFOAPFNAPFDAPFUdAPFDoA AOR CI PFTrDACOR crude odds ratio AOR adjusted odds ratio CI confidence interval FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance testModels were adjusted for maternal age at enrollment years prepregnancy BMI kgm2 per capita household income education level passive smokingpregnancy complication history of abortion and stillbirth and parity Although the underlying mechanism linking PFASs toglucose homeostasis is not yet clear it has been suggestedthat inhibition of phosphorylation of protein kinase BAkt and the activation of peroxisome proliferator activated receptors PPARs may play a role [ ] Studies using animal models and HepG2 cells have indicatedthat PFAS compounds reduce the expression of the phosphatase and tensin homolog protein and affect the Aktsignaling pathway [ ] The inhibition of Akt a keymediator of cellular insulin sensitivity may stimulate gluconeogenesis and hepatic insulin resistance [] BothPFOA and PFOS have been certified to affect glucose metabolism by AKT signaling pathway However the otherPFASs were not investigated in these studies [ ] Inaddition studies have demonstrated that PFASs can bindto and activate the PPAR α and γ receptors [] PPAR anuclear transcription receptor is known to play essentialroles in the regulation of gene expression glucose homeostasisfatty acid metabolism and inflammation []Therefore PFASactivated PPAR could disturb glucosehomeostasis by influencing insulin resistance [] and insulin secretion [] PFOA have the highest potential ofPPARα activation than the other PFASs with a shortercarbon chain length including PFHxS PFNA PFDA andPFDoA [] Moreover PFAS exposure may interferewith secretion and function of glucocorticoids andthyroid hormones via hypothalamicpituitaryadrenalaxis and hypothalamicpituitarythyroid axis whichmay further disturb glucose metabolism [ ]The physiologicaleffects of PFASs on glycemichomeostasis may depend on the potency and concentration of individual PFASs [ ]Fig Association between PFAS concentrations divided by tertiles and high FPG Notes All the ptrend values for PFASs with FPG were insignificant 0cRen Environmental Health Page of Fig Association between PFAS concentrations divided by tertiles and high hPG Notes p for trend The strengths of the present study were the prospective nature of the study design the large sample size andthe measurement of a wide range of PFAS compoundsHowever the potential limitations of the study shouldbe considered First a considerable proportion of subjects was lost to followup which may have led to selection bias However the characteristics of the includedsubjects were similar to those excluded in terms of ageeducation prepregnancy BMI and household incomeand thus a substantial selection bias was not expectedSecond the followup period from the measurement ofPFAS exposure to the endpoints FPG and hPG wasshort but the singlepoint measurement of PFAS concentration may reflect PFAS exposure long before thedate of blood collection owing to their long halflifeThirdthe relationships between PFASs and bloodglucose measures may have been confounded by unmeasured confounders such as family diabetes historyand dietary habits this should be examined in futurestudiesinformation on maternal activesmoking was not collected since the proportion of activesmoking was quite low in Chinese women [] For example only of pregnant women have been exposedto active smoking during pregnancy in a Shanghai BirthCohort [] Thus the current result is not expected tobe severely biased by the unadjustment of active smoking Fourth not all the subjects had information on hPG after the 50g OGTT which may have led to missedcases of GDM and affect the association between PFASsand outcome indicesin the sensitivity analysis ofpregnant women with GDM However the absence ofGDM cases if any would have attenuated the observedassociation Fifth data for FPG GWs or hPG GWs were collected over a long time span andIn additionthus the associations between PFASs and FPG and hPG may have been confounded by the gestational weekHowever we performed subgroup analyses using different GWs spans at glucose measurement and found thatthe results did not change significantlyConclusionExposure to certain PFASs ie PFOS PFNA PFDAPFUdA and PFDoA was associated with an increasedrisk of high hPG among pregnant women Furtherstudies are needed to clarify the effect of PFASs ongestational glycemic homeostasis and the underlyingmechanismSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12940020006408Additional file Table S1 The distribution of gestational week atglucose measurement for the included pregnant women Table S2Association between PFAS concentrations lntransformed and high FPGand hPG using multiple linear regression Table S3 Associationbetween PFAS concentrations lntransformed and high FPG and hPGin pregnant women without GDM Table S4 Association between PFASconcentrations lntransformed and high FPG and hPG in pregnantwomen with BMI kgm2 Table S5 Subgroup analysis of theassociation between PFAS concentrations lntransformed and high FPGand hPG in pregnant women by gestational age Figure S1 Assumeddirected acyclic graph for PFASs and plasma glucoseAbbreviationsPFASs Perfluoroalkyl and polyfluoroalkyl substances FPG Fasting plasmaglucose hPG Onehour plasma glucose GWs Gestational weeksPFHxS Perfluorohexane sulfonate PFOS Perfluorooctane sulfonatePFOA Perfluorooctanoic acid PFNA Perfluorononanoic acidPFDA Perfluorodecanoic acid PFUdA Perfluoroundecanoic acidPFDoA Perfluorododecanoic acid PFTrDA Perfluorotridecanoic acidORs Odds ratios CIs Confidence intervals GDM Gestational diabetesmellitus OGTT Oral glucose tolerance test SMBCS ShanghaiMinhang Birth 0cRen Environmental Health Page of Cohort Study LOD Limit of detection SD Standard deviations HOMAIR Homeostasis model of assessment for insulin resistance Akt Proteinkinase B PPARs Peroxisome proliferator activated receptorsAcknowledgementsThe authors thank fieldworkers involved in the survey for their efforts in datacollection and quality control and all the pregnant women investigatedAuthors contributionsWY HL and MM conceived and designed the study YR LJ and MMperformed data analysis and drafted the WY MM YR FY HL XS ZZand JD revised the manuscript and critically discussed the results All authorswere involved in interpreting the data and approved the final FundingThis work was supported by grants from the National key research anddevelopment program [grant numbers 2016YFC1000505 2018YFC1002801]Shanghai Municipal Commission of Health and Family Planning [grantnumber ] Innovationoriented Science and Technology Grantfrom NHC Key Laboratory of Reproduction Regulation [grant numbersCX2017] and Shandong Medical and Health Science and Technology Development Project [grant numbers 2018WS060]Availability of data and materialsThe datasets used during the current study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the ethical review committee of ShanghaiInstitute of Planned Parenthood Research SIPPR Written informed consentwas obtained before the data collection and analysis and the survey wasconducted in accordance with the Declaration of Helsinki PrinciplesConsent for publicationNot applicableCompeting interestsThe authors declare they have no actual or potential competing financialinterestsAuthor details1Department of Health Statistics School of Public Health Weifang MedicalUniversity Weifang Shandong China 2Minhang District Maternal and ChildHealth Hospital Shanghai China 3Department of Global Public HealthKarolinska Institute Stockholm Sweden 4NHC Key Lab of ReproductionRegulation Shanghai Institute of Planned Parenthood Research FudanUniversity Shanghai ChinaReceived May Accepted August ReferencesLau C Anitole K Hodes C Lai D PfahlesHutchens A Seed J Perfluoroalkylacids a review of monitoring and toxicological findings Toxicol Sci Tittlemier SA Pepper K Seymour C Moisey J Bronson R Cao XL DabekaRW Dietary exposure of Canadians to perfluorinated carboxylates andperfluorooctane sulfonate via consumption of meat fish fast foods andfood items prepared in their packaging J Agric Food Chem Conder JM Hoke RA De Wolf W Russell MH Buck RC Are PFCAsbioaccumulative A critical review and comparison with regulatory criteria andpersistent lipophilic compounds Environ Sci Technol Olsen GW Burris JM Ehresman DJ Froehlich JW Seacat AM Butenhoff JLZobel LR Halflife of serum elimination of perfluorooctanesulfonateperfluorohexanesulfonate and perfluorooctanoate in retired fluorochemicalproduction workers Environ Health Perspect Bartell SM Calafat AM Lyu C Kato K Ryan PB Steenland K Rate of declinein serum PFOA concentrations after granular activated carbon filtration attwo public water systems in Ohio and West Virginia Environ HealthPerspect Tian Y Zhou Y Miao M Wang Z Yuan W Liu X Wang X Wang Z Wen SLiang H Determinants of plasma concentrations of perfluoroalkyl andpolyfluoroalkyl substances in pregnant women from a birth cohort inShanghai China Environ Int Biegel LB Liu RC Hurtt ME Cook JC Effects of ammoniumperfluorooctanoate on Leydig cell function in vitro in vivo and ex vivostudies Toxicol Appl Pharmacol Fuentes S Colomina MT Rodriguez J Vicens P Domingo JL Interactions indevelopmental toxicology concurrent exposure to perfluorooctanesulfonate PFOS and stress in pregnant mice Toxicol Lett Seacat AM Thomford PJ Hansen KJ Olsen GW Case MT Butenhoff JLSubchronic toxicity studies on perfluorooctanesulfonate potassium salt incynomolgus monkeys Toxicol Sci Yan S Zhang H Zheng F Sheng N Guo X Dai J Perfluorooctanoic acidexposure for days affects glucose homeostasis and induces insulinhypersensitivity in mice Sci Rep Goudarzi H Araki A Itoh S Sasaki S Miyashita C Mitsui T Nakazawa HNonomura K Kishi R The Association of Prenatal Exposure to Perfluorinatedchemicals with glucocorticoid and androgenic hormones in cord bloodsamples the Hokkaido study Environ Health Perspect Barry V Winquist A Steenland K Perfluorooctanoic acid PFOA exposuresand incident cancers among adults living near a chemical plant EnvironHealth Perspect Lind PM Salihovic S van Bavel B Lind L Circulating levels of perfluoroalkylsubstances PFASs and carotid artery atherosclerosis Environ Res Darrow LA Stein CR Steenland K Serum perfluorooctanoic acid andperfluorooctane sulfonate concentrations in relation to birth outcomes in themidOhio Valley Environ Health Perspect MatillaSantander N Valvi D LopezEspinosa MJ ManzanoSalgado CBBallester F Ibarluzea J SantaMarina L Schettgen T Guxens M Sunyer J Exposure to Perfluoroalkyl substances and metabolic outcomes inpregnant women evidence from the Spanish INMA birth cohorts EnvironHealth Perspect Zeng XW Lodge CJ Dharmage SC Bloom MS Yu Y Yang M Chu C Li QQHu LW Liu KK Isomers of per and polyfluoroalkyl substances and uricacid in adults Isomers of C8 Health Project in China Environ Int 133Pt A105160Lin CY Chen PC Lin YC Lin LY Association among serum perfluoroalkylchemicals glucose homeostasis and metabolic syndrome in adolescentsand adults Diabetes Car | 2 |
Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelialmesenchymal transition EMT and apoptosis in BC are not fully understood We ï¬nd that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4 EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8 Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspeciï¬c transmembrane protein foundin a variety of peripheral cells11 is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored at °C for western blotting and PCR analysis The othergroup of specimens was ï¬xed in formalin and parafï¬nizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and veriï¬ed for their authenticities by STR proï¬ling Epirubicin was purchased fromPï¬zer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The ï¬nal protein staining score was the percentage score multiplied by the intensity score ï¬nalprotein staining scores were divided into three categoriesas follows negative low expression and high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturers instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments à cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were ï¬xed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantiï¬edby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturers protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magniï¬cation à b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplanMeier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magniï¬cation à f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell ï¬uorescence was measured with a FACScan ï¬owcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefï¬ciency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of à stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacriï¬ced by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups à stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacriï¬ced by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were ï¬xed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were ï¬xed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunoï¬uorescenceImmunoï¬uorescence staining was performed as previously described19 In brief after corresponding treatments the cells ï¬xed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfï¬cial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by KaplanMeier analysis and thelogrank test was used to compare survival differencesbetween groups Pearsons correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically signiï¬cantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe ï¬rst measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfï¬cial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassigniï¬cantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and KaplanMeier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was signiï¬cantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot signiï¬cant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown signiï¬cantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further conï¬rm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efï¬ciency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was conï¬rmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1BE Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown signiï¬cantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunoï¬uorescence showed the same results as thoseOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magniï¬cation à f Correlation analyses of proteinexpression levels between DNER and catenin g KaplanMeier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further conï¬rm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were signiï¬cantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunoï¬uorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally KaplanMeier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfï¬cial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a speciï¬c Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were signiï¬cantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These ï¬ndingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown signiï¬cantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to conï¬rm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efï¬ciency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efï¬ciency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantiï¬cation of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see ï¬gure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightï¬eldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t | 2 |
tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patients survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m gÅodowiczp kuczyÅski k kuczyÅska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative basallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in healthy breast column means that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnccab004592 tnncab010907 tnrcab022343 all the images were taken at à magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using theδδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukeys posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was 7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukeys posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukeys posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo à na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatients outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal markervimentin followed by the upregulation of epithelial markerecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrnamediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectinsyndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a | 0 |
"This approach discovered 3 nodules that were in different lobes than the primary tumor. Nodule fluorescence was independent of size metabolic activity histology tumor grade and vascularity. Conclusions This is the first-in-human demonstration of identifying pulmonary nodules during Thoracic surgery with NIR imaging without a priori knowledge of their location or existence. NIR imaging can detect pulmonary nodules during lung resections that are poorly visualized on computed tomography and difficult to discriminate on finger palpation. Mol Cancer Mol. Cancer Molecular Cancer 1476-4598 BioMed Central 24655544 3998010 1476-4598-13-68 10.1186/1476-4598-13-68 Research Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition Sun Ming 1 sunmingnjmu.edu.cn Liu Xiang-Hua 1 liuxianghuanjmu.edu.cn Wang Ke-Ming 2 422825636qq.com Nie Feng-qi 3 957714486qq.com Kong Rong 1 31815857qq.com Yang Jin-song 4 yangjinsongmedmail.com.cn Xia Rui 1 273459189qq.com Xu Tong-Peng 3 1034045558qq.com Jin Fei-Yan 1 759729211qq.com Liu Zhi-Jun 1 sm13776403108126.com Chen Jin-fei 4 1423594097qq.com Zhang Er-Bao 1 273459189qq.com De Wei 1 deweinjmu.edu.cn Wang Zhao-Xia 2 zhaoxiawang88hotmail.com 1Department of Biochemistry and Molecular Biology Nanjing Medical University Nanjing 210029 Peoples Republic of China 2Department of Oncology Second Affiliated Hospital Nanjing Medical University Nanjing Jiangsu 210011 Peoples Republic of China 3Department of Oncology First Affiliated Hospital Nanjing Medical University Nanjing Peoples Republic of China 4Department of Oncology Nanjing First Hospital Nanjing Medical University Nanjing P. R. China 2014 21 3 2014 13 68 68 16 9 2013 13 3 2014 Copyright 2014 sun et al.; licensee BioMed Central Ltd. 2014 sun et al.; licensee BioMed Central Ltd. This is an Open Access distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons./publicdomain/zero/1.0/) applies to the data made available in this unless otherwise stated. Background Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cellular processes such as differentiation proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. The clinical significance of BANCR and its molecular mechanisms controlling cancer cell migration and metastasis are unclear. Methods Expression of BANCR was analyzed in 113 non-small cell lung cancer (NSCLC) tissues and seven NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of BANCR in NSCLC cells. The effects of BANCR on cell viability were evaluated by MTT and colony formation assays. Apoptosis was evaluated by Hoechst staining and flow cytometry. Nude mice were used to examine the effects of BANCR on tumor cell metastasis in vivo. Protein levels of BANCR targets were determined by western blotting and fluorescent immunohistochemistry. Results BANCR expression was significantly decreased in 113 NSCLC tumor tissues compared with normal tissues. Additionally reduced BANCR expression was associated with larger tumor size advanced pathological stage metastasis distance and shorter overall survival of NSCLC patients. Reduced BANCR expression was found to be an independent prognostic factor for NSCLC. Histone deacetylation was involved in the downregulation of BANCR in NSCLC cells. Ectopic expression of BANCR impaired cell viability and invasion leading to the inhibition of metastasis in vitro and in vivo. However knockdown of BANCR expression promoted cell migration and invasion in vitro. Overexpression of BANCR was found to play a key role in epithelial-mesenchymal transition (EMT) through the regulation of E-cadherin N-cadherin and Vimentin expression. Conclusion We determined that BANCR actively functions as a regulator of EMT during NSCLC metastasis suggesting that BANCR could be a biomarker for poor prognosis of NSCLC. Background Non-small cell lung cancers (NSCLCs) including adenocarcinomas and squamous cell carcinomas are the predominant forms of lung cancer and account for the majority of cancer deaths worldwide [1]. Despite recent advances in clinical and experimental oncology the prognosis of lung cancer remains poor with a 5-year overall survival rate of around 11% [2]. A continuing problem of NSCLC tumorigenesis is the metastasis of cancer cells which is the main cause of death in patients. Thus a detailed understanding of the mechanisms and molecular pathways activated in metastatic cells is crucial in identifying new treatment options for anticancer therapy that target metastasis. The invasion and metastasis of cancer cells are landmark events that involve many changes in cellular behavior and lead to different steps of the metastatic cascade [34]. One of the most crucial steps in the metastatic cascade is the acquisition of invasive capabilities including turnover of cell-cell junctions degradation of the cell matrix and activation of pathways that control cytoskeletal dynamics in cancer cells. This process is accompanied by multiple changes in gene expression such as the loss of epithelial markers and a gain in mesenchymal markers [56]. Over the past decade cell and tumor biologists have identified the key role of epithelial-mesenchymal transition (EMT) in cancer cell metastasis a biological process where epithelial cells lose their polarity and undergo transition into a mesenchymal phenotype [7]. EMT enhances tumor cell invasion in response to environmental triggers and augments invasive functions by promoting Rac-dependent mesenchymal migration and also contributes to cell growth and survival [89]. Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of non-epithelial cadherins such as N-cadherin. The loss of E-cadherin expression is a fundamental event in EMT and a crucial step in the progression of papillomas to invasive carcinomas [10]. To date substantial effort has been devoted to understanding how EMT is regulated during cancer progression. It has been verified that EMT can be initiated by external signals such as hepatocyte growth factor (HGF) epidermal growth factor (EGF) transforming growth factor (TGF)-b and fibroblast growth factor (FGF) [11]. In addition to these signaling pathways triggered by membrane receptors recent studies have highlighted the importance of noncoding RNAs in the regulation of the epithelial phenotype by controlling EMT inducers. The miR-200 family has been found to control EMT by downregulating the expression of Zeb factors [12]. Furthermore the long noncoding RNA (lncRNA) MALAT-1 promoted EMT by regulating ZEB1 ZEB2 and Slug expression and activating Wnt signaling [13]. The lncRNAs are important new members of the ncRNA family that are greater than 200 nt and are unable to be translated into proteins. These lncRNAs are often expressed in a spatial- and temporal-specific pattern. Although very few lncRNAs have been characterized in detail they have been found to participate in a large range of biological processes including modulation of apoptosis and invasion reprogramming stem cell pluripotency and parental imprinting. These findings indicate that lncRNAs play a major role in the regulation of the eukaryotic genome [14-16]. Researchers have linked the dysregulation of lncRNAs with diverse human diseases in particular cancers [17-19]. Therefore identification of cancer-associated lncRNAs and investigation of their molecular and biological functions in controlling EMT are important in understanding the molecular biology of NSCLC metastasis and progression. BRAF-activated non-coding RNA (BANCR) an 693-bp lncRNA on chromosome 9 was firstly found by Ross J. Flockhart et.al via RNA-seq screen for transcripts affected by the expression of the oncogene BRAFV600E. BANCR is overexpressed in melanoma cells and crucial for melanoma cell migration [20]. In this study we investigated the effects of BANCR expression on NSCLC cell phenotypes in vitro and in vivo. Moreover we also showed that alteration of BANCR expression can influence E-cadherin N-cadherin and Vimentin protein levels which indicated that BANCR affected NSCLC cells invasion and metastasis partly via epithelial-mesenchymal transition. This study advances our understanding of the role of lncRNAs such as BANCR as a regulator of pathogenesis of NSCLC and facilitate the development of lncRNA-directed diagnostics and therapeutics. Results BANCR expression was downregulated and correlated with poor prognosis of NSCLC BANCR expression levels were investigated in 113 paired NSCLC samples and adjacent histologically normal tissues using quantitative polymerase chain reaction (qPCR) assays. BANCR expression was significantly downregulated (P?<?0.01) in 79% (89/113) of cancerous tissues compared with normal tissues (Figure 1A). BANCR expression levels in NSCLC were significantly correlated with tumor size (p?=?0.001) advanced pathological stage (p?<?0.001) and lymph node metastasis (p?=?0.001). However BANCR expression was not associated with other parameters such as gender (p?=?0.232) and age (p?=?0.616) in NSCLC (Table 1)." | 1 |
"The cancer tissue used in this study was received from patients that had surgical resection of both the primary tumor and related metastases. None of the patients had received chemo- or radiotherapy before the resection of the primary tumor. Medical charts and pathology reports were reviewed to record clinical and pathological data. Glass slides were reviewed to determine the histological type according to the WHO classification [30]. Follow-up information including the patient outcome and the time interval between the date of surgical resection and death was collected. The cases lost to follow-up and deaths from causes other than CRC were considered censored data for the survival analysis. The median follow-up period was 37.9 months (range 0.8104.6 months). Ethical statement All human specimens were obtained from the files of surgically resected cases examined at the Department of Pathology Seoul National University Bundang Hospital for the pathologic diagnosis. The retrospective study was performed using the stored samples after the pathologic diagnosis and all of the samples were anonymized before the study. The participants did not provide written informed consent in this study. The study was approved by the Institutional Review Board of Seoul National University Bundang Hospital under the condition of anonymization (reference: B-1109/136-302). Tissue array methods To evaluate the regional stromal differences samples were taken from each patient from four areas: the center and periphery of the primary cancer distant metastases and lymph node metastases. The distant metastatic sites for the tissue arrays were as follows: liver in 83 cases (45.9%) lung in 38 cases (21.0%) seedings in 38 cases (21.0%) distant lymph nodes in 6 cases (3.3%) and ovary in 16 cases (8.3%). The representative core tissue specimens (2 mm in diameter) were taken from individual paraffin blocks and rearranged in new tissue array blocks using a trephine apparatus (Superbiochips Laboratories Seoul South Korea) [31]. Immunohistochemistry Array slides were labelled by immunohistochemistry using antibodies for CD31 (1?100 DAKO Glostrup Denmark) D2-40 (1?100 DAKO) SMA (1?1000 Neomarkers Fremont CA USA) desmin (1?300 DAKO) and PTEN (1?80 Epitomics Burlingame CA USA) after a microwave antigen retrieval procedure except SMA. Non-reactive sites were blocked using 1% horse serum in Tris-buffered saline (pH 6.0) for 3 min. Primary antibodies were applied and antibody binding was detected with diaminobenzidine (DAB). Sections were counterstained with hematoxylin. The reactivity of PTEN in each tissue section was scored as negative faint or strong and the percentage of PTEN-positive fibroblasts was quantified. For the statistical analysis the sample was deemed PTEN-positive if 5% or more CAFs were scored as strong positives. Calculation of LVD MVD and CAFs using digital pathology Slides were concurrently evaluated by two pathologists (H.E.L and H.S.L) using light microscopy to improve the accuracy of the results (Fig. 1). CRC cells were considered as internal negative controls. Medium- to large-sized vessels were considered as internal positive controls for CD31 and D2-40. Intestinal muscular layer or medium- to large-sized vessels were considered as internal positive controls for desmin and SMA. Samples showing inappropriate staining in internal negative or positive controls were considered non-informative and were excluded from the analysis. Slides were scanned using an Aperio ScanScope® CS instrument (Aperio Technologies Inc. Vista CA) at 20 magnification. Subsequently they were analyzed in ImageScope¢ using the Microvessel Analysis v1 algorithm (Aperio Technologies) and MVD and LVD were calculated. Because desmin-positive muscularis mucosa and propria are positive for SMA immunostaining the area of CAFs (mm2) was calculated by subtracting the areas of desmin staining from that of SMA staining (SMA - desmin). .0091811.g001 Representative sections from four tumor locations stained with CD31 D2-40 SMA or desmin antibodies (100). Statistical analysis A chi-squared test or Fisher's exact test (2-sided) for non-continuous variables and Mann-Whitney or Kruskal-Wallis analysis for continuous variables were used to compare each parameter with respect to the CRC site and according to its clinicopathologic features. The correlation between continuous variables was analyzed using the Pearson correlation coefficient. To determine the best cut-offs of continuous variables for predicting patient survival the maximal chi-squared method was performed using R program (http://cran.r-project./). The overall survival curves were plotted using the Kaplan-Meier product-limit method and the significance of the differences between these curves was determined using the log-rank test. A univariate and multivariate regression analysis was performed using the Cox's proportional hazards model to determine hazard ratios (HRs). P-values of less than 0.05 were considered statistically significant. All statistical analysis excluding the maximal chi-squared test was performed with the IBM SPSS statistics 20 (Armonk NY USA). Results 1. Heterogeneity of cancer-associated stroma according to examined tumor locations The clinicopathological characteristics of the advanced CRC patients are described in . The CRC patients with synchronous metastases had aggressive features including larger tumor size more advanced pT and pN stage and the presence of perineural and venous invasion than the patients with metachronous metastasis (p<0.05). .0091811.t001 Clinicopathologic characteristics of advanced colorectal cancers. Parameters Total Metachronous Synchronous P value (n?=?181) (n?=?57) (n?=?124) Age (median range) 60.00 (2893) 62.00 (3679) 60.00 (2893) 0.241 Sex 0.007 Male 97 39 (68.4%) 58 (46.8%) Female 84 18 (31.6%) 66 (53.2%) Location 0.055 Right colon 37 6 (10.5%) 31 (25.0%) Left colon 75 29 (50.9%) 46 (37.1%) Rectum 69 22 (38.6%) 47 (37.9%) Size of primary tumor 5.30 (2.013.0) 4.20 (29) 5.50 (2.513) <0.001 Histologic grade 0.227 Low grade 157 52 (91.2%) 105 (84.7%) High grade 24 5 (8.8%) 19 (15.3%) T stage <0.001 T1 0 0 0 T2 5 3 (5.3%) 2 (1.6%) T3 107 45 (78.9%) 62 (50.0%) T4 69 9 (15.8%) 60 (48.4%) N stage <0.001 N0 35 23 (40.4%) 12 (9.7%) N1 58 23 (40.4%) 35 (28.2%) N2 88 11 (19.3%) 77 (62.1%) Perineural invasion 0.011 Absent 89 36 (63.2%) 53 (42.7%) Present 92 21 (36.8%) 71 (57.3%) Venous invasion 0.028 Absent 126 46 (80.7%) 80 (64.5%) Present 55 11 (19.3%) 44 (35.5%) The heterogeneous values for LVD MVD and CAF area are shown in Fig 2. LVD was the highest in center of the primary cancers (median interquartile range (IQR); 37.00 10.5081.00) than any other site (5.00 1.0023.75 at the periphery; 2.50 1.0015.00 in lymph node metastases; 3.00 1.0020.00 in distant metastases). MVD was lower in distant metastases (median IQR; 641.50 428.001006.75) than at the periphery of the primary cancer (731.00 508.251049.75) and lymph node metastases (893.50 520.251275.25). The area occupied by CAFs was the lowest in the distant metastases (median IQR; 0.91 0.681.18) than any other site (1.12 0.881.41 in the center; 1.22 0.961.54 in the periphery1.4 1.001.71 in lymph node metastases). In addition the stromal characteristics varied in relation to the metastatic an examined. MVD and LVD were the higher in lung metastases than those in the liver peritoneum or lymph nodes (p<0.001; Fig. 3). However the amounts of CAFs were consistent among the different metastatic ans (p?=?0.180). .0091811.g002 Heterogeneity of lymphatic vessel density (LVD) microvessel density (MVD) and amount of cancer-associated fibroblasts (CAFs) with respect to tumor location. The LVD (A) MVD (B) and CAF area (C) was significantly different according to each tumor location. .0091811.g003 LVD MVD and CAF area at different distant metastasis sites. The characteristics of cancer-associated stroma differed with respect to the metastatic site. LVD (A) and MVD (B) were greater in the metastatic tumor samples collected from the lung than in samples collected from other metastatic sites (p<0.001). However the amount of CAFs was not significant different between metastatic sites (C). Despite the heterogeneity of stromal characteristics CRC cases with higher LVD MVD and CAFs in center of the primary cancers had a tendency of higher LVD MVD and CAFs in periphery (p<0.05; Table S1). However LVD in center and periphery of primary cancer were not correlated with LVD in related distant metastasis (Table S1). In addition the amount of microvasculature was significantly correlated with the amount of CAFs (Table S2). 2. Clinical significance of cancer-associated stroma in advanced CRCs The MVD LVD and amount of CAFs present at each tumor location were compared according to their clinicopathologic features (Table 2). High grade CRCs were associated with lower CAFs in samples taken from the central cancer site (p?=?0.041). When compared with synchronous metastases the patients with metachronous metastases had higher LVD in center and periphery of the primary cancer and had higher MVD in lymph node metastases. Most patients with metachronous metastases were treated by adjuvant chemotherapy before metastasectomy. LVD and MVD in the distant metastases were significantly higher in the patients who had received chemotherapy before metastasectomy than those who did not (p?=?0.011 and 0.048 respectively). .0091811.t002 Table 2 Clinicopathologic factor and LVD MVD and CAFs. Center (median) Periphery (median) LN metastasis (median) Distant metastasis (median) LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs LVD MVD CAFs Total 39 717 1.13 5 740 1.22 3 888 1.42 3 648 0.91 Histologic grade Low grade 40 717 1.15* 5 741 1.23 3 895 1.43 3 665 0.92 High grade 34 683.5 0.94* 6 643.5 1.18 2 656 1.32 6 498 0.82 pT stage pT2 34 758 1.15 16 870 1.48 6 772 0.73 pT3 47 737 1.19 5 803 1.22 2.5 884 1.43 3 724 0.92 pT4 33 639 1.09 4 630 1.22 3 895 1.41 3 520 0.93 LN metastasis Absent 49 602 1.15 8 712 1.42 4 772 0.94 Present 39 737.5 1.12 4 740 1.21 3 884 1.41 3 617 0.91 Perineural invasion Absent 41 738 1.12 6 772 1.32 5.5 931.5 1.42 4 687 0.94 Present 39 672 1.13 4 702 1.2 2 796 1.39 3 548.5 0.86 Metastasis Synchronous 34* 717.5 1.11 3.0* 741 1.21 3 797* 1.39 3 617 0.93 Metachronous 55* 716 1.21 8.0* 712 1.23 2 1117* 1.63 5 698 0.91 Chemotherapy Not done 2.0* 597.5* 0.93 Done 10.0* 684* 0.91 * p<0.05; ** p<0.01; chemotherapy prior to metastatectomy of distant metastasis. 3. Expression loss of PTEN in CAFs PTEN was expressed in cytoplasm and sometimes the nucleus of both cancer and non-neoplastic cells when examined using immunohistochemistry. Expression of PTEN was lost in 8 cases in the center 2 cases in the periphery 4 cases in lymph node metastases and 11 cases in distant metastases (Table S3). In all 11 distant metastases with PTEN loss PTEN expression was intact in both the center and periphery of primary cancer (data not shown). PTEN loss in distant metastasis was correlated with synchronous metastasis (p?=?0.018). 4. Cancer-associated stroma and patient prognosis By using the obtained cut-offs lower LVD MVD and CAFs in the center LVD and CAFs in the periphery and MVD and CAFs in distant metastases were all significantly correlated with lower survival (p<0.05; Fig. S1). Among other clinicopathologic features synchronous metastasis old age larger size high histologic grade advanced pT and pN stage and presence of perineural invasion were associated with a worse prognosis (Table 3). By multivariate Cox regression analysis the hazard ratio of synchronous versus metachronous was the highest (4.029) with the lowest p value (p<0.001). CAFs in distant metastasis LVD and MVD in the center LVD in the periphery age and perineural invasion also independently predicted patient survival. In addition loss of PTEN expression in CAFs in distant metastases was associated with a worse prognosis (p?=?0.042; Fig S2) but not in primary cancer or lymph node metastasis. .0091811.t003 Table 3 Univariate and multivariate survival analysis according to clinicopathologic features. Univariate survival analysis Multivariate survival analysis Factors HR (95% CI) P value HR (95% CI) P value Synchronous vs. Metachronous 4.617 (2.4728.624) <0.001 3.762 (1.8387.701) <0.001 Age 1.023 (1.0041.044) 0.020 1.033 (1.0111.056) 0.003 Sex (female vs. male) 1.428 (0.9202.218) 0.113 Location (left vs. right) 0.503 (0.3140.806) 0.004 0.700 (0.4131.188) NS (0.186) Size 1.073 (1.0051.146) 0.036 1.040 (0.9031.198) NS (0.584) Histologic grade (high vs. low) 1.862 (1.0613.269) 0.030 1.491 (0.7632.912) NS (0.243) pT stage (pT4 vs. pT2/3) 2.341 (1.5033.645) <0.001 1.137 (0.6741.921) NS (0.630) pN stage (pN1/2 vs. pN0) 3.848 (1.7608.411) 0.001 1.773 (0.7584.146) NS (0.186) Perineural invasion 2.628 (1.6404.211) <0.001 2.108 (1.2653.513) 0.004 Venous invasion 1.217 (0.7571.956) 0.418 Center LVD (high vs. low) 0.364 (0.1580.836) 0.017 0.298 (0.1180.753) 0.010 Center MVD (high vs. low) 0.391 (0.2330.655) <0.001 0.437 (0.2380.801) 0.007 Center CAFs (high vs. low) 0.579 (0.3520.954) 0.032 1.038 (0.6071.773) NS (0.892) Periphery LVD (high vs. low) 0.235 (0.0860.644) 0.005 0.279 (0.0960.809) 0.019 Periphery MVD (high vs. low) 1.456 (0.9112.327) 0.117 Periphery CAFs (high vs. low) 0.524 (0.3360.817) 0.004 0.813 (0.4991.326) NS (0.406) LN LVD (high vs. low) 1.646 (0.8743.100) 0.123 LN MVD (high vs. low) 0.597 (0.2941.213) 0.154 LN CAFs (high vs. low) 0.717 (0.4231.217) 0.218 Metastasis LVD (high vs. low) 0.569 (0.3141.032) 0.063 Metastasis MVD (high vs. low) 0.579 (0.3640.921) 0.021 1.262 (0.7202.211) NS (0.417) Metastasis CAFs (high vs. low) 0.492 (0.2710.894) 0.020 0.290 (0.1440.582) 0.001 Metastasis PTEN (intact vs. loss) 0.454 (0.2080.993) 0.048 0.575 (0.2391.383) NS (0.217) Discussion Carcinoma cells in different tissue areas have distinct characteristics [32]. In central areas of the tumor carcinoma cells maintain an epithelial cell phenotype but carcinoma cells in the invasive front acquire a more malignant and mesenchymal phenotype and are thought to have an increased migratory capacity and contribute to metastatic diseases. These metastatic cells may restore the epithelial phenotype at metastatic sites [33]. In addition to carcinoma cells themselves microenvironment is suggested to be uneven within a given tumor because tumor formation and progression involve the co-evolution of cancer cells and microenvironments [34]. The present study demonstrated that the cancer-associated microenvironment also had distinct characteristics in different areas. Of the sites examined LVD was highest in the center of the primary cancer. MVD was slightly higher in center than at the periphery of the primary cancer but this difference was not statistically significant. Interestingly the amount of CAFs in distant metastases was significantly lower than in center and periphery of the primary cancer. We show that the stromal microenvironment has regional heterogeneity both within the primary tumor and between the primary site and its related metastases. Furthermore our data suggests that the stromal heterogeneity might be attributable to tumor heterogeneity. Therefore it would be beneficial to consider both stromal and tumor cell heterogeneity in order to manage CRC patients better. We evaluated the MVD LVD and amount of CAFs in metastatic tissues of various ans including the liver lung peritoneal seeding distant lymph nodes and ovary. Of the metastatic ans we examined both LVD and MVD were the highest in lung. In our previous study the KRAS discordance rate was also significantly higher in matched lung metastases than in other matched metastatic ans [35]. The underlying mechanism is not known. It could be that primary CRCs with high LVD and MVD have a tendency to produce lung metastases; however our results indicated that LVD and MVD in the center and at the periphery of the primary cancers were lower in the patients with lung metastases (data not shown). Alternatively it may be due to the physiological characteristics of metastatic ans interactions between cancer cells and microenvironment within the metastatic an or the characteristics of the cancer cell clones prone to lung metastasis. However technical or sampling errors also may be possible thus further large-scale studies are required. Although numerous studies have attempted to demonstrate an association between tumor microenvironment characteristics and survival the prognostic impacts of MVD and LVD are still controversial. Some studies have been presented that active angiogenesis and lymphangiogenesis represented by high MVD and LVD are associated with poor prognosis and aggressive clinicopathologic factors [36] [37]. Recent meta-analysis has demonstrated that LVD was significantly associated with disease-free survival but not overall survival [38]. Other studies have reported no statistical significance of MVD and LVD on survival [39]. Prall et al. has reported that high MVD and LVD are related with better survival in a consecutive series and liver metastases [40]. Our results were based on patients with advanced disease with distant metastasis and we showed that high MVD and LVD were related with improved survival. This might be because all the patients in this study had confirmed to have distant metastasis and microvasculatures could influence even delivery of the chemotherapeutic drug into the tumor. However our study had some limitations in terms of the survival analysis. We enrolled the CRC patients with available surgically resected cancer tissues from both primary tumors and corresponding metastatic tumors. Not all advanced CRC patients with metastatic diseases were included and far advanced cases were not enrolled because of their inoperability. Therefore unrecognized biases might have influenced our survival results. Some studies have demonstrated an anti-tumorigenic effect of fibroblasts [20] [21]. However it has become clear that CAFs contribute to the progression of cancer and their prognostic significance in various cancers also has been raised [41] and furthermore several studies have observed genetic alterations in CAFs [26] [27]. PTEN loss of CAFs has been observed in breast cancer and prognostic association of it has been suggested [27] [28]. " | 1 |
"One limitation of this study is the small number of cases under study although 15 FISH-positive cases is comparable to most other studies. The relatively small number of FISH-negative cases may have affected our ability to identify FISH-negative IHC-positive cases. However the study design does permit an assessment of the sensitivity of the IHC assay which is the most important consideration for a possible screening test. Our comparison of the immunohistochemical assays was not directly equivalent as the D5F3 assay included a proprietary tyramide signal amplification step whereas the ALK1 and 5A4 assays were conducted using our routine diagnostic detection system. However our study design also has several strengths. In particular the use of archival diagnostic paraffin blocks and FISH testing conducted in the course of routine diagnosis make the results of the study directly relevant to clinical practice. In summary we find IHC to be a highly sensitive (86%) and specific (100%) test for ALK rearrangement in lung adenocarcinoma. We find a slight advantage of a proprietary amplified assay (D5F3 Ventana) over two other antibodies with conventional DAB staining (ALK1 Dako and 5A4 Abcam) but only in scanty samples. Intensity of staining was the most discriminating measure and the proportion of cells staining did not contribute. We identified two cases that were positive for the ALK rearrangement by FISH but negative by all immunohistochemical assays and suggest that in discordant cases the IHC test result may be more predictive of treatment response than FISH. Further discordant cases need to be examined to help guide the treatment of these cases. Immunohistochemical testing is clearly at least a useful adjunct to FISH and we feel that it is reasonable in routine practice to use a sensitive IHC assay as a screening test. The danger of missing treatable cases using this method (i.e. FISH-positive IHC-negative crizotinib-sensitive tumors) appears very small especially when specimens contain adequate material. In difficult cases further investigations such as re-biopsy and repeated IHC/FISH may be helpful. Disclosure: This project was supported by the National Institute of Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London and the NIHR RM/ICR Biomedical Research Center. All other authors declare no conflict of interest. REFERENCES 1. Inamura K Takeuchi K Togashi Y EML4-ALK lung cancers are characterized by rare other mutations a TTF-1 cell lineage an acinar histology and young onset. Mod Pathol 2009 22 508 515 19234440 2. Koivunen JP Mermel C Zejnullahu K EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008 14 4275 4283 18594010 3. Shinmura K Kageyama S Tao H EML4-ALK fusion transcripts but no NPM- TPM3- CLTC- ATIC- or TFG-ALK fusion transcripts in non-small cell lung carcinomas. Lung Cancer 2008 61 163 169 18242762 4. Soda M Choi YL Enomoto M Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007 448 561 566 17625570 5. To KF Tong JH Yeung KS Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant. J Thorac Oncol 2013 8 883 891 23625156 6. McDermott U Iafrate AJ Gray NS Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 2008 68 3389 3395 18451166 7. Kwak EL Bang YJ Camidge DR Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010 363 1693 1703 20979469 8. Lindeman NI Cagle PT Beasley MB Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists International Association for the Study of Lung Cancer and Association for Molecular Pathology. J Thorac Oncol 2013 8 823 859 23552377 9. Murakami Y Mitsudomi T Yatabe Y A Screening Method for the ALK Fusion Gene in NSCLC. Front Oncol 2012 2 24 22655265 10. Rodig SJ Mino-Kenudson M Dacic S Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009 15 5216 5223 19671850 11. Peled N Palmer G Hirsch FR Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer. J Thorac Oncol 2012 7 e14 e16 22895149 12. Eisenhauer EA Therasse P Bogaerts J New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009 45 228 247 19097774 13. Selinger CI Rogers TM Russell PA Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 2013 26 1545 1553 23743928 14. Conklin CM Craddock KJ Have C Laskin J Couture C Ionescu DN Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol 2013 8 45 51 23196275 15. Sholl LM Weremowicz S Gray SW Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas. J Thorac Oncol 2013 8 322 328 23407557 16. Savic S Bode B Diebold J Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol 2013 8 1004 1011 23689429 Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24292447 3915116 bjc2013735 10.1038/bjc.2013.735 Clinical Study A phase II multicentre study of ziv-aflibercept in combination with cisplatin and pemetrexed in patients with previously untreated advanced/metastatic non-squamous non-small cell lung cancer Ziv-aflibercept/cisplatin/pemetrexed in NSCLC Chen H 1 * Modiano M R 2 Neal J W 3 Brahmer J R 4 Rigas J R 5 Jotte R M 6 Leighl N B 7 Riess J W 3 Kuo C J 3 Liu L 8 Gao B 8 DiCioccio A T 8 Adjei A A 1 Wakelee H A 3 1Department of Medicine Roswell Park Cancer Institute Elm & Carlton Streets Buffalo NY 14263 USA 2Arizona Oncology/Arizona Clinical Research Center 1620W. St Mary's Rd Tucson AZ 85745 USA 3Department of Medicine Stanford University School of Medicine and Cancer Institute 875 Blake Wilbur Dr Stanford CA 94305 USA 4Department of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Bunting/Blaustein CRB 1650 Orleans St. G94 Baltimore MD 21231 USA 5Department of Medicine Norris Cotton Cancer Center Geisel School of Medicine at Dartmouth 1 Medical Center Drive Lebanon NH 03756 USA 6Rocky Mountain Cancer Centers 1800 Williams Street Suite 200 Denver CO 80218 USA 7Department of Medicine Princess Margaret Hospital and University of Toronto 610 University Avenue Toronto ON M5G 2M9 Canada 8Regeneron Pharmaceuticals Inc. 777 Old Saw Mill River Road Tarrytown NY 10591 USA *E-mail: hongbin.chenroswellpark. 04 02 2014 28 11 2013 110 3 602 608 29 08 2013 27 10 2013 30 10 2013 Copyright 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: This study evaluated the efficacy and safety of ziv-aflibercept in combination with cisplatin and pemetrexed in non-small cell lung cancer (NSCLC). Methods: This single arm multicentre phase II trial enrolled patients with previously untreated locally advanced or metastatic non-squamous NSCLC. Patients received intravenous ziv-aflibercept 6?mg?kg?1 pemetrexed 500?mg?m?2 and cisplatin 75?mg?m?2 every 21 days for up to six cycles. Maintenance administration of ziv-aflibercept was to continue until disease progression intolerable toxicity or other cause for withdrawal. The co-primary end points were objective response rate (ORR) and progression-free survival (PFS). Planned sample size was 72 patients. Results: The study was closed prematurely because of three confirmed and two suspected cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled. Median age was 61.5 years;" | 1 |
Detecting ulcerative colitis from a0colon a0samples a0using a0efficient a0feature selection and machine learningHanieh Marvi Khorasani1 a0Hamid a0Usefi2 Lourdes pe±acastillo1Ulcerative a0colitis a0UC a0is a0one a0of a0the a0most a0common a0forms a0of a0inflammatory a0bowel a0disease a0IBD a0characterized a0by a0inflammation a0of a0the a0mucosal a0layer a0of a0the a0colon a0Diagnosis a0of a0UC a0is a0based a0on a0clinical a0symptoms a0and a0then a0confirmed a0based a0on a0endoscopic a0histologic a0and a0laboratory a0findings a0Feature a0selection a0and a0machine a0learning a0have a0been a0previously a0used a0for a0creating a0models a0to a0facilitate a0the a0diagnosis a0of a0certain a0diseases a0In a0this a0work a0we a0used a0a a0recently a0developed a0feature a0selection a0algorithm a0DRPT a0combined a0with a0a a0support a0vector a0machine a0SVM a0classifier a0to a0generate a0a a0model a0to a0discriminate a0between a0healthy a0subjects a0and a0subjects a0with a0UC a0based a0on a0the a0expression a0values a0of a0 a0genes a0in a0colon a0samples a0We a0validated a0our a0model a0with a0an a0independent a0gene a0expression a0dataset a0of a0colonic a0samples a0from a0subjects a0in a0active a0and a0inactive a0periods a0of a0UC a0Our a0model a0perfectly a0detected a0all a0active a0cases a0and a0had a0an a0average a0precision a0of a0 a0in a0the a0inactive a0cases a0Compared a0with a0results a0reported a0in a0previous a0studies a0and a0a a0model a0generated a0by a0a a0recently a0published a0software a0for a0biomarker a0discovery a0using a0machine a0learning a0BioDiscML a0our a0final a0model a0for a0detecting a0UC a0shows a0better a0performance a0in a0terms a0of a0average a0precisionInflammatory bowel disease IBD is a chronic inflammatory condition of the gut with an increasing health burden1 Ulcerative colitis UC and Crohns disease are the two most common forms of chronic IBD with UC being more widespread than Crohns disease2 There is no cure for UC3 and people with the disease alternate between periods of remission inactive and active inflammation2 The underlying causes of UC are not completely understood yet but it is thought to be a combination of genetic environmental and psychological factors that disrupt the microbial ecosystem of the colon34 Genomewide association studies GWAS have identified risk loci for IBD5 and risk loci specifically associated with UC6 However the lower concordance rate in identical twins of in UC compared with in Crohns disease indicates that genetic contribution in UC is weaker than in Crohns disease7 Thus using gene expression data for disease diagnostic might be more appropriate for UC than using GWAS data as it has been done for Crohns disease8There are several features used for clinical diagnosis of UC including patient symptoms and laboratory endoscopic and histological findings7 Boland et at9 carried out a proofofconcept study for using gene expression measurements from colon samples as a tool for clinical decision support in the treatment of UC The purpose of Boland et a0als study was to discriminate between active and inactive UC cases even though they only considered gene expression of eight inflammatory genes instead of assessing the discriminatory power of many groups of genes they concluded that mRNA analysis in UC is a feasible approach to measure quantitative response to therapyMachine learningbased models have a lot of potential to be incorporated into clinical practice10 specially in the area of medical image analysis1112 Supervised machine learning has already proved to be useful in disease diagnosis and prognosis as well as personalized medicine1314 In IBD machine learning has been used to classify IBD paediatric patients using endoscopic and histological data15 to distinguish UC colonic samples from control and Crohns disease colonic samples16 and to discriminate between healthy subjects UC patients and Crohns disease patients using transcriptional profiles of peripheral blood171Department of Computer Science Memorial University St Johns NL A1B3X5 Canada 2Department of Mathematics and Statistics Memorial University St Johns NL A1C5S7 Canada email usefimunca lourdesmuncaScientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cAccession number of controls of UC cases Description of samplesGSE11521819GSE1122320GSE226192124GSE75214active22GSE75214inactive22Mucosal biopsies from uninflammed colonic tissuesBiopsies from uninflammed sigmoid colonMucosal colonic tissue from discordant twinsMucosal colonic biopsies from active UC patients and from controlsMucosal colonic biopsies from inactive UC patients from controlsPlatformAffymetrix Human Genome U133A Array and Affymetrix Human Genome U133B ArrayAgilent012391 Whole Human Genome Oligo Microarray G4112AAffymetrix Human Genome U133 Plus ArrayAffymetrix Human Gene ST ArrayAffymetrix Human Gene ST Array of genes features UsageModel selectionModel selectionModel selectionModel evaluationModel evaluationTable Summary of datasets used in this studyIn this study our goal was to investigate whether combining machine learning with a novel feature selection algorithm an accurate model using the expression profiles of few genes could be generated from transcriptomewide gene expression data To do this we apply a machine learning classifier on gene expression data to generate a model to differentiate UC cases from controls Unlike previous studies1617 to reduce the effect of technical conditions we combined a number of independent gene expression data sets instead of using a single data set to train our model Additionally by using feature selection we were able to identify genes out of thousands genes for which expression measurements were available The expression values of these genes is sufficient to generate a SVM model to effectively discriminate between UC cases and controls On a gene expression dataset not used during training our proposed model perfectly detected all active cases and had an average precision of in the inactive casesMethodsData a0gathering a0 We searched the NCBI Gene Expression Omnibus database GEO for expression profiling studies using colonic samples from UC subjects in active and inactive state and controls healthy donors We identified five datasets accession numbers GSE11521819 GSE1122320 GSE2261921 GSE7521422 and GSE945216 As healthy and Crohns disease subjects were used as controls in GSE945216 this data set was excluded from our study We used three of the datasets for model selection using 5fold crossvalidation and left one dataset for independent validation Table a0 We partitioned the validation dataset into two datasets Active UC vs controls and inactive UC vs controlsAll data sets were obtained from studies where the diagnoses of patients were either based on endoscopical findings GSE7521422 and GSE2261921 followed the criteria described by LennardJones23 GSE1122320 or based on clinical features as well as radiologic endoscopic and laboratory findings GSE115218 Disease state was either assessed during colonoscopy and classified into no signs of inflammation inactive low inflammation and moderatehigh inflammation active GSE22619 defined as active with a Mayo endoscopic subscore ¥ GSE75214 or graded by a gastroenterologist or gastrointestinal pathologist GSE11223 GSE1152 The control group had either normal mucosa at endoscopic level GSE75214 no significant pathological findings during endoscopic and histological examinations GSE22619 normal colonoscopies GSE1152 and GSE11223 or tissues abnormalities other than IBD GSE1152 and GSE11223For each dataset GEO2R25 was used to retrieve the mapping between probe IDs and gene symbols Probe IDs without a gene mapping were removed from further processing Expression values for the mapped probe IDs were obtained using the Python package GEOparse26 The expression values obtained were as provided by the corresponding authorsData a0preprocessing a0 We performed the following steps for data preprocessing i Calculating expression values per gene by taking the average of expression values of all probes mapped to the same gene i Handling missing values with KNearest Neighbours KNN imputation method KNNImputer from the missingpy library in Python27 KNNImputer uses KNN to fill in missing values by utilizing the values from nearest neighbours We set the number of neighbours to nneighbours2 and we used uniform weightTo get our final training datasets we merged datasets GSE1152 GSE11223 and GSE22619 by taking the genes present in all of them The merged dataset has UC samples and controls and genes These same genes were selected from GSE75214 for validation As the range of expression values across all datasets were different we normalized the expression values of the final merged dataset and validation dataset by calculating Zscores per sampleModel a0 generation a0 To create a model to discriminate between UC patients from healthy subjects we selected the features genes using the dimension reduction through perturbation theory DRPT feature selection method28 Let D [A b] be a dataset where b is the class label and A is an m n matrix with n columns genes and m rows samples There is only a limited number of genes that are associated with the disease and as such a majority of genes are considered irrelevant DRPT considers the solution x of the linear system Ax b with the smallest 2norm Hence b is a sum of xi Fi where Fi is the ith column of A Then each component xi of x is viewed as an assigned weight to the feature Fi So the bigger the xi the more important Fi is in connection with b DRPT then filters out features whose weights are very small compared to the average of local maximums over Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cSubsetSubset Subset Subset Subset Subset Subset Subset Subset Subset Subset AP of FeaturesTable Ten top subsets of genes with the highest crossvalidated average APxis After removing irrelevant features DRPT uses perturbation theory to detect correlations between genes of the reduced dataset Finally the remaining genes are sorted based on their entropySelected features were assessed using 5fold crossvalidation and support vector machines SVMs as the classifier First we performed DRPT times on the training dataset to generate subsets of features Then to find the best subsets we performed repetitions of stratified 5fold crossvalidation CV on the training dataset We utilized average precision AP as calculated by the function average_precision_score from the Python library scikitlearn29 version as the evaluation metric to determine the best subset of genes among those generated subsets The four subsets with the highest mean AP over the crossvalidation folds were chosen for creating the candidate models For each of the four selected subset of features we created a candidate SVM model using all samples in the training dataset To generate the models we used the SVM implementation available in the function SVC with parameter kernellinear from the Python library scikitlearn To evaluate the prediction performance of each of the ten models we validated it on the GSE75214active and GSE75214inactive datasets In this step we utilized the precisionrecall curve PRC to assess the performance of the candidate models on unseen data An additional candidate model was created using the most frequently selected genesBioDiscML a0 BioDiscML30 is a biomarker discovery software that uses machine learning methods to analyze biological datasets To compare the prediction performance of our models with BioDiscML we ran the software on our training dataset of the samples N52 were utilized for training and the remaining N25 for testing Since the software generates thousands of models and we required only one model we specified the number of best models as in the config file numberOfBestModels1 One best model out of all models was created based on the 10fold crossvalidated Area Under PrecisionRecall Curve numberOfBestModelsSortingMetric TRAIN10CVAUPRC on the train set We used Weka to evaluate the performance of the model generated by BioDiscML on the GSE75214active and GSE75214inactive datasets Selected features by BioDiscML are C3orf36 ADAM30 SLS6A3 FEZF2 and GCNT3 In order to be able to use the model in Weka we loaded the training dataset as it was created by BioDiscML which was one of the outputs of the software This dataset has six features including selected genes and class labels and samples We also modified our validation datasets by extracting BioDiscML selected features After loading the training and test dataset in Weka explorer we loaded the model and we entered the classifier configuration as wekaclassifiersmiscInputMappedClassifier I trim W wekaclassifierstreesRandomTree K M V S which is the classifiers set up in the generated model by BioDiscMLUse a0of a0experimental a0animals a0and a0human a0participants a0 This research did not involve human participants or experimental animalsResultsFeature a0selection a0reduced a0significantly a0the a0number a0of a0genes a0required a0to a0construct a0a a0classification a0model a0 We performed DRPT times on the training dataset to select subsets of features Then we performed 5fold crossvalidation to find the subsets with the highest mean average precision AP over the folds The range of AP for the subsets is between and with an average of ± Table a0 shows the ten subsets with the highest crossvalidated AP and the number of selected features genes on each subset On average DRPT selected ± genes per subsetTop a0 five a0 models a0 are a0 able a0 to a0 perfectly a0 discriminate a0 between a0 active a0 UC a0 patients a0 and a0 controls a0 We selected the four top subsets with the highest mean AP which are subsets and Table a0 and created candidate models based on them Each candidate model was created using all samples on the training dataset and the features of the corresponding subset To identify the genes most relevant to discriminate between healthy and UC subjects we looked at the number of times each gene was selected by DRPT On DRPT runs genes were selected at least once The upper plot on Fig a0 shows the number of times each gene was selected and the lower plot shows the normal quantilequantile QQ plot Based on this plot we Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Identifying the most frequently selected genes Top Number of times each gene was selected Genes were sorted based on the number of times they were selected by DRPT Bottom Normal QQplot Horizontal line at indicates the threshold selected to deem a gene as frequently chosensaw that the observed distribution of the number of times a gene was selected deviates the most from a Gaussian distribution above times We considered the genes selected by DRPT more than times as highly relevant and created a fifth model using genes selected by DRPT at least times over runsIn order to evaluate the prediction performance of the candidate models each model was tested on the validation datasets and PRC was plotted for model assessment Figs a0 and As the AP approximates the AUPRC34 we used AP to summarize and compare the performance of these five models All five candidate models achieved high predictive performance on the validation dataset GSE75214active with an average AP of ± while the average AP of these five models on the validation dataset GSE75214inactive was ± The models with the best performance were the model created with the most frequently selected genes and subset with an AP of and on GSE75214active and GSE75214inactive respectively However based on a Friedman test35 p value all five models have comparable performance on the validation datasets We chose the model generated with the most frequently selected genes as our final modelOur a0top a0models a0outperformed a0the a0model a0generated a0by a0BioDiscML a0 The average AUPRC achieved by the model created by BioDiscML on both GSE75214active and GSE75214inactive datasets was and respectively Comparing the performance of our candidate models and the model created by BioDiscML on the two validation datasets we observed that we achieved better AUPRC on both datasets AUPRC on the active dataset AUPRC on the inactive dataset In terms of running time subset selection by DRPT and final model creation and validation took minutes while the running time of BioDiscML to create all the models and output the best final model was minutesScientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Precisionrecall curve of top selected subsets on GSE75214activeFigure a0 Precisionrecall curve of top selected subsets on GSE75214inactiveLinks a0between a0the a0most a0frequently a0selected a0genes a0and a0UC a0 We used Ensembl REST API Version to find the associated phenotypes with each gene belonging to the subset of the most frequently selected genes Table a0 Among these genes FAM118A is the only one with a known phenotypic association with IBD and its subtypes The evidence supporting the association of some of the other genes with UC based on phenotype is more indirect For example long term IBD patients are more susceptible to develop colorectal cancer37 and one of the genes TFRC is associated with colorectal cancer IBD patients are more prone to develop cardio vascular disease which is associated with blood pressure and cholesterol38 and four of the most frequently selected genes LIPF MMP2 DMTN and PPP1CB are associated with blood pressure and cholesterolWe looked at whether some of the most frequently selected genes contained any of the known IBDassociated SNPs5 To do this we utilized Ensembls BioMart39 website Ensembl Release version September to retrieve the genomic location of the genes We then used the intersectBed utility in BEDtools40 to find any overlap between the IDB risk loci and the genomic location of the genes None of the IBDassociated SNPs was located on our genes Similarly gene set enrichment analysis found no enriched GO term or pathway among these genes Additionally these genes are not listed as top differentially expressed genes in previous studies on UC4142We searched the literature for links between the genes and UC and we found the following MMP2 expression has been found significantly increased in colorectal neoplasia in a mouse model of UC43 and MMP2 levels are elevated in IBD44 TFRC has been found to have an antiinflammatory effect on a murine colitis model45 KRT8 genetic variants have been observed in IBD patients and it was suggested that these variants are a risk factor for IBD46 DUOXA2 has been shown to be critical in the production of hydrogen peroxide within the colon and to be upregulated in active UC47Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0cGene symbolCWF19L1FCER2MMP2PPP1CBRPL23AP32ZNF624REG1BTFRCFAM118ACFHR2KRT8PRELID1ZNF92ABHD2C16orf89CAB39LSPATC1LDUOXA2MESP1MAML3PITX2DMTNASF1BPGFBEX4ODF1PTGR1ZNF35LIPFSLC25A13BARX2C2orf42Associated phenotypesSpinocerebellar ataxia autosomal recessive depressive disorder MajorBlood protein levels post bronchodilator FEV1Multicentric OsteolysisNodulosisArthropathy MONA spectrum disorders cholesterol HDL lip and oral cavity carcinoma body height winchester syndromeNoonan Syndromelike disorder with loose anagen hair Heel bone mineral density Blood pressure basophils asopathy with developmental delay short stature and sparse slowgrowing hairAttention deficit disorder with hyperactivity body HeightNoneContrast sensitivity Body Mass IndexBreast ductal adenocarcinoma esophageal adenocarcinoma thyroid carcinoma clear cell renal carcinoma prostate carcinoma pancreatic cancer gastric adenocarcinoma hepatocellular carcinoma lung adenocarcinoma rectal adenocarcinoma basal cell carcinoma colorectal adenocarcinoma squamous cell lung carcinoma head and neck squamous cell carcinoma colon adenocarcinoma iron status biomarkers transferrin levels mean corpuscular hemoglobin concentration red cell distribution width combined immunodeficiency red blood cell traits high light scatter reticulocyte percentage of red cells reticulocyte fraction of red cells Immunodeficiency Chronic inflammatory diseases ankylosing spondylitis Crohns disease psoriasis primary sclerosing cholangitis ulcerative colitis Glucose Peanut allergy maternal genetic effects Heel bone mineral densityMacular degeneration blood protein levels feeling miserable alanine aminotransferase ALT levels after remission induction therapy in acute lymphoblastic leukaemia ALL asthmaCirrhosis familial cirrhosis hepatitis C virus susceptibility to cirrhosis cryptogenic cirrhosis noncryptogenic cirrhosis susceptibility to gamma glutamyl transferase levels cancer pleiotropyBody fat distribution heel bone mineral density activated partial thromboplastin timeNoneItch intensity from mosquito bite adjusted by bite size gut microbiota Obesityrelated traits coronary artery disease advanced age related macular degeneration squamous cell lung carcinoma pulse pressureNoneHemoglobin S erythrocyte count pancreatic neoplasmsNoneFamilial thyroid dyshormonogenesis thyroglobulin synthesis defectNoneSocial science traits intelligence MTAG chronic mucus hypersecretion borderline personality disorder congenital heart malformationAxenfeldRieger syndrome ring dermoid of cornea iridogoniodygenesis type peters anomaly familial atrial fibrillation rieger anomaly stroke ischemic stroke cataract PITX2related eye abnormalities phosphorus cognitive decline rate in late mild cognitive impairment creatinine intraocular pressure incident atrial fibrillation wolffparkinsonwhite pattern parkinson disease early onset atrial fibrillation anterior segment sygenesis Total cholesterol levels LDL cholesterolNoneMood instability blood protein levelsNoneBody weight body mass index glucose IgA nephropathy Chronic lymphocytic leukaemia type diabetes erythrocyte indicesBody height menarche monocyte count blood protein levelsNoneMaximal midexpiratory flow rate blood protein levels respiratory function tests blood pressureCitrullinemia type II neonatal intrahepatic cholestasis due to citrin deficiency citrin deficiency citrullinemia type I bone mineral densityType diabetes breast cancer night sleep phenotypes response to cyclophosphamide in systemic lupus erythematosus with lupus nephritis strokeNone of times selectedTable Phenotypes associated with the most frequently selected genes by DRPT as obtained from Ensembl REST API Version DiscussionIn this study we showed the feasibility of using machine learning and feature selection to identify a reduced number of genes from microarray data to aid in the diagnosis of UC One might argue that distinguishing UC patients from Crohns disease CD patients has more clinical relevance than distinguishing UC patients from controls However we were limited on the choice of groups to classify by data availability as we could only find three gene expression data sets obtained from colonic samples of UC and CD patients in GEO GSE1152 GSE75214 and GSE126124 As children samples were transcriptionally profiled for GSE12612448 instead of adults ones we decided that the age difference could introduce extra biological variation in the expression data unrelated to UC That left us with only two data sets which were not enough to train the model with multiple data sets and have at least one holdout data set for validationAnother limitation of this study is that we used gene expression profiles of colonic samples Further research is required to assess the accuracy of our 32gene model in gene expression profiles of blood samples A recent study48 found a similar transcriptional profile between blood and colon tissue from patients with IBD If indeed Scientific RepoRtS 101038s41598020705830Vol1234567890wwwnaturecomscientificreports 0cour 32gene model is found accurate in blood samples then a less invasive procedure such as a blood test could be used to diagnose UC instead of a colonoscopy or sigmoidoscopyIn a previous study where machine learning was employed to perform a risk assessment for CD and UC using GWAS data49 a twostep feature selection strategy was used on a dataset containing Crohns disease cases UC cases and controls with SNPs In that study Wei et a0al reduced the number of features by filtering out SNPs with pvalues greater than and then applied a penalized feature selection with L1 penalty to select a subset of SNPs We decided against filtering out genes based on an arbitrary pvalue of statistical significance of differential expression as researchers are strongly advised against the use of pvalues and statistical significance in relation to the nullhypothesis5051Our 32gene model achieved AP of and discriminating active UC patients from healthy donors and inactive UC patients from healthy donors respectively We found direct or indirect links to UC for about a quarter of the most frequently chosen genes The remaining genes should be further investigated to find associations with UC To put the performance of our 32gene model into perspective we looked at previous studies applying machine learning to create models for the diagnostic of UC Maeda et a0al52 extracted features from endocystoscopy images to train a SVM to classify UC patients as active or healing This approach achieve precision at recall which is lower than the one achieved by our 32gene model Figs a0 and Yuan et a0al17 applied incremental feature selection and a SMO classifier a type of SVM on gene expression data from blood samples to discriminate between healthy subjects UC patients and Crohns disease patients The 10fold crossvalidation accuracy of their best model using the expression values of genes to classify UC patients was while our method obtained better accuracy than this with substantially less number of genes In terms of potential for clinical translation of a machine learningbased model a model requiring to quantify the gene expression levels of fewer genes is more suitable for the development of a new diagnostic test than one requiring the quantification of the expression levels of thousands of genesUsing an efficient feature selection method such as DRPT and a SVMclassifier on gene expression data we generated a model that could facilitate the diagnosis of UC from expression measurements of genes from colonic samples To avoid systematic experimental bias on the training data we used three transcriptomic datasets from three separated studies Our top model was validated with promising results on a data set not used for training however additional research is required to evaluate the genes as potential biomarkers on a external set of subjectsReceived March Accepted July References Kaplan G G The global burden of IBD from to Nat Rev Gastroenterol Hepatol 101038 Ord¡s I Eckmann L Talamini M Baumgart D C Sandborn W J Ulcerative colitis Lancet nrgas tro2015150 101016S0140 Eisenstein M Ulcerative colitis towards remission Nature S33 101038d4158 Khan I et al Alteration of gut microbiota in inflammatory bowel disease IBD cause or consequence IBD treatment targeting the gut microbiome Pathogens a0 103390patho gens8 de Lange K M et al Genomewide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease Nat Genet 101038ng3760 Anderson C A et al Metaanalysis identifies additional ulcerative colitis risk loci increasing the number of confirmed associations to Nat Genet 101038ng764 Conrad K Roggenbuck D Laass M W Diagnosis and classification of ulcerative colitis Autoimmun Rev 101016jautre v201401028 Romagnoni A et al Comparative performances of machine learning methods for classifying Crohn disease patients using genomewide genotyping data Sci Rep 101038s4159 z Boland B S et al Validated gene expression biomarker analysis for biopsybased clinical trials in ulcerative colitis Aliment Pharmacol Ther 101111apt12862 Shah P et al Artificial intelligence and machine learning in clinical development a translational perspective NPJ Digit Med 101038s4174 Esteva A et al Dermatologistlevel classification of skin cancer with deep neural networks Nature McKinney S M et al International evaluation of an AI system for breast cancer screening Nature Molla M Waddell M Page D Shavlik J Using machine learning to design and interpret geneexpression microarrays AI 101038natur e2105 101038s4158 Mag perspectives Hum Genet 101038s4159 Xu J et al Translating cancer genomics into precision medicine with artificial intelligence applications challenges and future Mossotto E et al Classification of paediatric inflammatory bowel disease using machine learning Sci Rep Olsen J et al Diagnosis of ulcerative colitis before onset of inflammation by multivariate modeling of genomewide gene expression data Inflamm Bowel Dis 101002ibd20879 Yuan F Zhang YH Kong XY Cai YD Identification of candidate genes related to inflammatory bowel disease using minimum redundancy maximum relevance incremental feature selection and the shortestpath approach Biomed Res Int 101155201757419 Moehle C et al Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease J Mol Med Berl 101007s0010 Zahn A et al Aquaporin8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis World J Gas Noble C L et al Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis Gut troenterol Scientific RepoRtS 101038s41598020705830Vol0123456789wwwnaturecomscientificreports 0c Lepage P et al Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis Gastroenterology Inflamm Bowel Dis Vancamelbeke M et al Genetic and transcriptomic bases of intestinal epithelial barrier dysfunction in inflammatory bowel disease LennardJones J E Classification of inflammatory bowel disease Scand J Gastroenterol Suppl 10310900365 discussion H¤sler R et al A functional methylome map of ulcerative colitis Genome Res Barrett T et al NCBI GEO archive for functional genomics data setsupdate Nucleic Acids Res D991D995 Gumienny R GEOparse pypiproje ctGEOpa rse Troyanskaya O et al Missing value estimation methods for DNA microarrays Bioinformatics 101093bioin forma tics176520 abs200212104 Afshar M Usefi H HighDimensional Feature Selection for Genomics Datasets KnowledgeBased Systems arxiv Pedregosa F et al Scikitlearn machine learning in Python J Mach Learn Res Leclercq M et al Largescale automatic feature selection for biomarker discovery in highdimensional omics data Front Genet Burlington Holmes G Donkin A Witten I a0H Weka A machine learning workbench In Proceedings of ANZIIS Australian New Zealand Intelligent Information Systems Conference Hall M et al The weka data mining software an update ACM SIGKDD Explor Newsl Witten I H Frank E Hall M A Pal C J Data Mining Practical Machine Learning Tools and Techniques Man Kaufmann M¼ller A C et al Introduction to Machine Learning with Python A Guide for Data scientists OReilly Media Inc California DemÅ¡ar J Statistical comparisons of classifiers over multiple data sets J Mach Learn Res Yates A et al The Ensembl REST API Ensembl data for any language Bioinformatics Kim E R Chang D K Colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis World J Gastroenterol diovasc Dis Schulte D et al Small dense LDL cholesterol in human subjects with different chronic | 2 |
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0 in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci xiaogan rr ci huanggang rr ci conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global morans index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by and respectively and the risk estimates increased to and with every μgm3 increase in average concentrations of pm25 and pm10 in respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0 was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0 was associated with a increase of daily confirmed new cases wuhan rr ci1032 xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p wuhan rr ci xiaogan rr ci huanggang rr ci wuhan r2 p xiaogan r2 p Ï2 p a μgm3 increase in pm25 was associated with a increase in fatality rate Ï2 p a μgm3 increase in pm10 was associated with a increase in fatality rate no2 a 10μgm3 no2 increase lag0 was associated with a increase in daily confirmed new cases wuhan rr ci xiaogan rr ci huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci estimate on deaths value cid0 ci kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearmans one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci and deaths cid0 ci low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0 in no2 is associated with a ci increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053 xiaogan rr ci but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop | 0 |
" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimers disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnΠinterferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinsons disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnΠfurther elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated oxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide humans nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino viruss infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap n collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra | 0 |
preoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer one e0237244 101371 pone0237244editor louise emilsson university of oslonorwayreceived february accepted july published august copyright strous this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement data cannot beshared publicly because of ethical concernspatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data data are available fromthe viecuri institutional data access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdata heart rate variability hrv represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer hrv is also believed to predict the occurrence andseverity of postoperative complications we aimed to determine the role of preoperativehrv as a prognostic factor in overall and cancer free survival in patients with colorectalcancermethodsretrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between january and december who underwent curative surgical treatment hrv was measured as timedomain parameters sdnn standarddeviation of nnintervals and rmssd root mean square of successive differencesbased on preoperative second ecgs groups were created by baseline hrv low hrvsdnn 20ms or rmssd 19ms and normal hrv sdnn �20ms or rmssd �19msprimary endpoints were overall and cancer free survivalresultsa total of patients were included in this study hrv was not significantly associated withoverall survival sdnn 20ms vs sdnn �20ms244 vs adjusted hr p rmssd 19ms vs rmssd �19ms270 vs adjustedhr p or cancer recurrence sdnn 20ms vs�20ms201 vs adjusted hr p rmssd 19ms vs�19ms vs adjusted hr p there was no one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfunding the authors received no specific fundingfor this workcompeting interests the authors have declaredthat no competing interests existsignificant association between hrv and cealevel at one year followup or between hrvand occurrence of a postoperative complication or the severity of postoperativecomplicationssheart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment these results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low hrvintroductionin there were over million newly diagnosed colorectal cancer patients worldwide andover in the netherlands alone it is the fourth most common cause of death worldwide to improve survival it is of importance to get a better insight into modifiable prognosticfactors emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityhrv could be one of these prognostic factors []hrv is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [] it has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity these mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway a higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer in addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others also patientswith normal hrv seem to live longer in different sorts of metastatic cancer independent ofconfounders in patients with colorectal cancer a low hrv at baseline has shown to beassociated with higher cea levels at months after diagnosis which predicts a poorer prognosis in patients undergoing curative treatment for colorectal cancer hrv does not only seemto influence cancer prognosis a recent study showed that patients with lower hrv have moreintraoperative blood loss and more and more severe postoperative complications identifying patients with low hrv is easy and noninvasive when its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis alsoif we could predict the occurrence and severity of postoperative complications based on hrvimproving hrv before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients prognosis recent studies focussing on improving hrv by improving physicalfitness by means of physical exercise show promising results in both older men and woman however the only previous study on colon cancer and hrv including patients receiving curative treatment included a small sample and did not examine whether hrv predicts one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancersurvival in these patients to clarify the predictive value of hrv in prognosis of patientswith colorectal cancer further exploration is needed current studies identifying hrv as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease []the aim of this study was to determine the role of preoperative hrv as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentmethodsdata collectiondata from the netherlands cancer registry ncr were used the ncr collects data on allnewly diagnosed cancer patients in the netherlands information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry anatomical site of the tumour is registered according tothe international classification of diseases oncology the tumournodemetastasis tnmclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis quality of the data is high due to thorough training of theregistration team and consistency checks for the study population additional data were collected from the medical records of thepatients this encompassed information on hrv cealevels asa classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on hrv or the endpoint being analysed severity of the postoperative complicationsaccording to the claviendindo classification was also documented medical records wereassessed between january and july and reevaluated for revision of this between the 20th and 25th of april this study was approved by the research committee and the board of directors of viecurimedical centre data was obtained under the law scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons in the netherlands unless patients objected to use of their personal medical record forscientific research data was encrypted with an encryption key provided by the ncr encryption was shortly lifted to access the patients number for accessing hisher medical record following extraction data were encrypted againstudy populationthe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between january and january at viecuri medical centre who underwent curative surgical treatment patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances hrv indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of hrv we did not exclude patients taking alphablockers calcium one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerinhibitors diuretics amiodarone aceinhibitors or arbs as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on hrv istherefore less univocal and sometimes completely absent heart rate variabilityheart rate variability was analysed using a 12lead 10second ecg 150hz used for preoperative screening in case of multiple ecgs per patient the most recent ecg before date of surgery was used for hrvanalysis in case of multiple ecgs per patient on the same date theecg with the best quality was chosen meaning an ecg without motion artefacts in case ofmotion artefacts there was always an ecg without motion artefacts available recorded on thesame date time between two consecutive rpeaks was measured in lead ii with an accuracy of02ms using museecg hrv was presented using the timedomain hrv parameters sdnnstandard deviation of nnintervals and rmssd root mean square of successive differences in milliseconds calculated using the following calculations rsdnn ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrri 00 rrmeanþ2pnn 00 rmssd ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrriþ1 00 rriþ2pn 00 n 00 ð1þð2þsdnn and rmssd obtained from 10s ecgs were found to correlate with results of ecgsof longer durations power spectral analysis hrv parameters as lf and hf can only beobtained in longer recording ecgs and were therefore not implementable in this study sdnn and rmssd were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively in case of ansdnn 20ms or rmssd 19ms hrv was classified as low and in case of sdnn �20ms orrmssd �19ms as normal these cutoff values were based on cutoff values used in otherstudies showing an association between lowhrv as sdnn 20ms and rmssd 19ms andcolorectal cancer as there is no standardised definition of low and normal hrv endpoints and definitionsthe primary endpoints of this study were overall and cancer free survival overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer patients dying without cancerrecurrence were censored on day of death secondary endpoints were elevated cealevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the claviendindoclassificationstatistical analysisin this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex bmicomorbidities and asaclassification heart rate and tumour characteristics as tnmstagetumour localisation and tumour differentiation and their association with hrv and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerprognosis normal distribution of the continuous variables heart rate age and bmi as well assdnn and rmssd were tested with a kolmogorovsmirnov test because of normal distribution heart rate age and bmi were compared between hrvgroups using unpaired ttest allother variables were categorical and were compared between hrvgroups using chisquarestatistics as groups were all of sufficient powerdifferences in overall survival and cancer free survival in months according to sdnn andrmssd were visualized by means of kaplanmeier curves and statistically tested using the logrank test multivariate coxregression analyses were conducted to calculate the prognosticassociation between hrv and overall and cancer free survival while adjusting for other prognostic variables multivariate logistic regression was used to assess the independent effect ofsdnn and rmssd on cealevels and the occurrence and severity of postoperative complications variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including crohnsdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome differences in cealevel at baseline and one year checkup between and within groups of low hrv and normal hrv were assessed with a repeatedmeasures linear model and tested using the tukey test to test the implication of a longer timebetween ecg and treatment all analyses were repeated after excluding patients with an ecgolder than months a twotailed pvalue � was considered significant in all analysesdata were analysed using ibm spss statistics version ibm corp ny armonk usaresultsof colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study reasons for exclusion are presented in fig median sdnn andrmssd were 204ms interquartile range iqr 115ms351ms and 175ms iqr 99ms299ms respectively table shows descriptive data of the included patients by hrv groupsbaseline heart rate and age were negatively associated with hrv the group of patients withlow hrv contains more patients with a history of cardiac disease regardless of the hrv defining parameter when defining low hrv by rmssd 19ms more patients in this group havehypertension as comorbidity this group also contains more patients with an asa classification greater than oneduring a median followup of months iqr all causedeath occurred in patients cancer recurrence occurred in patients during a median followup of months iqr to rule out any distort in results caused by a delay between ecg and treatment all analyseswere repeated after exclusion of ecgs older than months this did not lead to any new significant results therefore these results were not displayed in detail in this papersurvivalin low hrv groups slightly more patients died compared to normal hrv groups sdnn20ms versus �20ms versus respectively rmssd 19ms versus�19ms versus respectively these observed differences between hrvgroups in overall survival were not significant fig a sdnn p b rmssd one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig flowchart of the study101371 pone0237244g001p after adjustment for some potential confounders these associations remained notsignificant sdnn 20ms versus �20ms hr p and rmssd19ms versus �19ms hr p tables and age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival age also differed at baseline and was identified as a confounder when defining low andnormal hrvgroups by sdnn cardiac disease was identified as confounder when conducting sensitivity analyses with sdnn and rmss as continuous variables results remained thesame there was no association between hrv and overall survival sdnn hr p and rmssd hr p in low hrv groups slightly more patients had recurrence of cancer compared to normalhrv groups sdnn 20ms versus �20ms versus respectivelyrmssd 19ms versus �19ms versus respectively these observed differences between hrv groups in cancer free survival were not significant fig a sdnnp b rmssd p as in overall survival after adjustment for some potentialconfounders these associations remained not significant sdnn 20ms versus �20mshr p and rmssd 19ms versus �19ms hr p tables and in sdnnbased groups baseline heart rate was identified asa confounding variable sensitivity analyses with sdnn and rmssd as continuous variables one 101371 pone0237244 august one 0ctable descriptive data of included patients according to prespecified hrv groupssdnn 20ms n sdnn �20ms n prmssd 19ms n rmssd �19ms n phrv and prognosis in colorectal cancerheart rate�age�age n year� yearsex nmalefemale [] [] [] [] [] [] [] [] mean bmi sd [] [] [] []comorbidities ncardiac diseasehypertensiondiabetes mellitusthyroid diseasepulmonary diseasevascular diseaseneurological diseaseotherasa nasa1asa2asa34localisation tumour nright colonleft colonrectumtumour stage niiiiii differentiation grade nwellmoderate poorundifferentiated � non normaldistributed data presented as median with interquartile range101371 pone0237244t001did not alter these results there was no association between hrv and cancer free survivalsdnn hr p and rmssd hr p cealevelcealevel at baseline and one year checkup was registered in patients and elevated in of these patients this was elevated at one year checkup in only patients low hrv was notsignificantly associated with elevated cealevels at one year checkup as shown in table sensitivity analyses with sdnn and rmssd as continuous variables did not alter these resultssdnn hr p and rmssd hr one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for overall survival in groups of low hrv and normal hrv presented as a sdnn andb rmssd101371 pone0237244g002 one 101371 pone0237244 august one 0ctable multivariate analyses of associations of sdnn and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancersdnn20ms�20msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t002p adjusting for covariates was not possible because of the small number of patientswith an elevated cealeveldifferences between cealevel at baseline and one year checkup were compared betweenand within hrvgroups results were displayed in fig there were no significant differencesin cealevel at baseline and one year checkup between the low hrv group and normal hrvgroup defined by sdnn and rmssd p and p respectively also there were nosignificant differences in cealevel at baseline and at one year checkup within the low hrvgroup and normal hrv group defined by sdnn and rmssd p and p respectivelypostoperative complicationsin patients one or more postoperative complications occurred within days after surgerythe occurrence of a postoperative complication was not significantly associated with lowhrv defined as sdnn 20ms or rmssd 19ms even after adjustment for some potentialconfounders table heart rate age cardiac disease and hypertension were identified asconfounding covariates when conducting sensitivity analyses with sdnn and rmss as continuous variables the association between occurrence of a postoperative complication with one 101371 pone0237244 august one 0ctable multivariate analyses of associations of rmssd and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancerrmssd19ms�19msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t003baseline hrv remained not significant sdnn hr p andrmssd p the same applied when postoperative complicationswere graded according to the claviendindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationstable discussionin this observational cohortstudy we determined the heart rate variability in preoperativeecgs of patients with primary colorectal cancer who underwent curative surgical treatment hrv refers to physiological variations in beattobeat intervals it was presented intimedomain parameters sdnn and rmssd hrv was not significantly associated with overall survival or cancer free survival independent of some risk factors also this study showedno significant differences in cealevels at one year checkup between patients with low hrvand patients with normal hrv patients with low hrv did not have more or more severepostoperative complications compared to patients with normal hrvtumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing dnadamage and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for cancer free survival in groups of low hrv and normal hrv presented as a sdnnand b rmssd101371 pone0237244g003 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable univariate analyses of low hrv and risk of elevated cealevel at one year checkupcea μglor cipcea μglor ciindependent of baseline ceaindependent of baseline ceasdnn 20ms n rmssd 19ms n sdnn �20ms n normal baseline cea � μglsdnn 20ms n sdnn �20ms n elevated baseline cea μglsdnn 20ms n reference reference rmssd �19ms n normal baseline cea � μglrmssd 19ms n rmssd �19ms n elevated baseline cea μglrmssd 19ms n reference reference sdnn �20ms n referencermssd �19ms n referencep101371 pone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression it has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation this is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway malfunctioning of this route may play part in the onset of cancer this theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [] the vagus nerve is also believed to modulate tumour progression an active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [] absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls epidemiologically low hrv has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [] however the results of the present study do not support thesefindingsto our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment de couck studied the relationship between hrv and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer independent of confounders the hypothesised inverse relationship of hrv and the tumour markerpsa at and months after diagnosis was only significant in patients with stage iv prostatecancer not stage ii and iii in colorectal cancer mouton found that low hrv definedas sdnn ms predicted significantly higher levels of the tumour marker cea at months after diagnosis again these results were only found in patients receiving palliativetreatment not curative only one previous study showed a significant inverse relationshipbetween hrv and mortality in cancer in general independent of stage this study of guo had a large study population of patients with various types of cancer low hrv wasdefined as sdnn 70ms and was significantly associated with shorter survival times thissuggests that hrv is a predictive indicator of survival time not only in palliative but also incurative patients however results were not controlled for cancer type which could affect bothhrv and survival and should therefore be interpreted with caution the fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in hrv in this sample one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig bar chart for cealevel at baseline and one year checkup in groups of low hrv and normal hrv presented as asdnn and b rmssd101371 pone0237244g004 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and risk of occurrence of a postoperative complication within dayspostoperative complicationor ciunadjustedsdnn 20ms n sdnn �20ms n referenceadjusted�sdnn 20ms n sdnn �20ms n referenceunadjustedrmssd 19ms n rmssd �19ms n referenceadjusted�rmssd 19ms n rmssd �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371 pone0237244t005psome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer however based on current evidence on this subject we cannot supportthis hypothesis the positive association between low hrv and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low hrv midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased hrv a study of de couck showed that cancerpatients in general have a significantly lower hrv than healthy people the same resultswere found in studies of bijoor where rmssd was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group when comparing patients with advanced stage cancer tnm iii and iv to patients with anearly stage of cancer tnm i and ii rmssd was found to be significantly lower in patientswith advanced stages of cancer thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased hrv besides the proposed influence of low hrv on survival of colorectal cancer patientsthrough development and increased progression of cancer reimer suggested that lowhrv could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications however the results found in this study were notconcurrent with those of reimer who included patients with asa undergoingelective surgery their analysis of hrv was through powerspectrum parameters based on longer ecgrecordings instead of the timedomain parameters based on 10s ecgs used in thisstudy but the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatrmssd and sdnn based on ecg recordings of 10s are in substantial agreement with those of45min and a 10s ecg therefore suffices to determine time domain hrv parameters howeverreimer did not correct for possible confounders in their study patients with low hrvwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low hrv group correcting for this comorbidity may change the significance of their findings a study of one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and severity of postoperative complications according to claviendindo classificationunadjustedminor grade i and iior cipmajor grade iii iv vor cisdnn 20msn n sdnn �20msn referencen referenceadjusted�sdnn 20msn sdnn �20msn referenceunadjustedrmssd 19msn rmssd �19msn referenceadjusted�n n referencen n referencermssd 19msn n rmssd �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371 pone0237244t006scheffler did not show any significant preoperative differences in hrv between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels thus also in relation to predicting postoperative complications results are not uniformthis study was subject to a number of limitations due to its retrospective character confounding may have occurred we attempted to correct for all possible confounders withindatabase availability with regression analyses also not all possible confounding factors werewithin database availability reasons for diagnostic procedure smoking alcohol abuse are presumed to have an effect on hrv and outcomes but were not documented in enough patientsto be taken into account when conducting multivariate analyses analysed ecgs were thoseperformed before or at time of preoperative screening in this cohort ecgs were madewithin the year before surgery only ecgs were older than five years at time of surgery theprecise effects of these differences in time from ecg to cancer treatment on hrv areunknown to test the implication o | 0 |
"metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes Numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors Aspartate hydroxylase ASPH is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion ASPH also promotes tumor growth by stimulation ofangiogenesis and immunosuppression These effects are mainly achieved via the activation of Notch and SRCsignaling pathways ASPH expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact Therefore small molecule inhibitors of ASPH enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models ASPH can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation CD and CD4 T cells in mice The PAN3011 vaccine against ASPH has already been tested in aphase clinical trial in patients with prostate cancer In summary ASPH is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapyKeywords ASPH Small molecule inhibitor Metastasis ImmunotherapyBackgroundCancer is a multifactorial disease with an approximate million fatalities in Worldwide it is the secondleading cause of death [] The complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression Despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis [] delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer Since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential In thisapproachesthat Correspondence smahelmnaturcunicz1Department of Genetics and Microbiology Faculty of Science CharlesUniversity BIOCEV Vestec Czech RepublicFull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsAspartate hydroxylase ASPH has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] ASPH belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] The overexpressed ASPH is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the Cterminal regionto the extracellular environment where it is accessible toantibody binding Recently molecular targeted therapyhas been developed against this target using small molecule inhibitors SMI that can inhibit the catalytic site The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKanwal Journal of Experimental Clinical Cancer Research Page of in the Cterminal region Moreover as antigenic epitopes that reside on the ASPH protein can efficientlystimulate cluster of differentiation CD and CD8 Tcell responses unique to tumor cells harboring ASPHthis enzyme can be used as a tumor associated antigenTAA in immunotherapy [ ]ThedioxygenasesStructure of the ASPH gene and isoformsASPH is a type II transmembrane protein of approxito the family of αmately kDa that belongsketoglutaratedependenthydroxylated products of ASPH hydroxylation were firstdetected in blood coagulation proteins [] ASPHwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbEGFlike domains of various proteins[] Fig Thereafter the human ASPH gene wascloned and characterized [] This gene spanning base pairs long region of genomic DNA and containing exons is located at the position q123 of thehuman chromosome The ASPH sequence is highlyconserved in mammalian evolution The sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species[] The whole ASPH protein consists of five domainsan Nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a Cterminal catalytic domain [] Tissue specific transcription is directed from two putative promoters P1 and P2which differ in their regulation sequences [ ] Whilethe transcription from the P1 promoter was observed inmost human tissues the P2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor MEF2 particularly in muscle tissues []The ASPH gene undergoes extensive alternative splicingresulting in four protein isoforms ie ASPH humbugjunctate and junctin [ ] These proteins vary in theCterminal region which affects their function [ ]The two longest ASPH transcript variantsthat aretranscribed from the P1 and P2 promoters and differ inthe length of the ²untranslated region encode the fulllength ASPH protein This protein contains the catalyticCterminal domain that catalyzes the posttranslationalhydroxylation in the cbEGFlike domains of numerousproteins Supplementary Fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] Thetruncated isoforms humbug junctate and junctin sharethe Nterminal part with the ASPH protein but lackcatalytic function They are involved in calcium homeostasis [] Humbug has a potential role in cell adhesionand calcium flux and similar to ASPH its overexpressionhas been correlated with aggressive tumorcell behavior[]reticulummembranebound protein that is known for its functionin the regulation of the intracellular Ca2 concentrationJunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicJunctateisaLocalization in cells tissue distribution andexpression regulationASPH is predominantly a cellsurface protein [] that isalso localized in the endoplasmic and sarcoplasmicreticulum [] Furthermore a recent study identifiedmitochondriallocalization of ASPH in hepatocellularcarcinoma HCC In that study ASPH overexpressioncorrelated with an instability of mitochondrial DNA andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in HCC []ASPH is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands [] and has a potential role in placentalimplantation and fetal growth [] On the contrary theASPH expression in normal adult tissues is relativelylow or negligible However ASPH expression is inappropriately activated during oncogenesis when ASPH is required for generation of malignant and metastaticphenotypes The elevated expression of ASPH at bothFig ASPH catalytic reaction Aspartyl and asparaginyl residues in cbEGFlike domains are hydroxylated 0cKanwal Journal of Experimental Clinical Cancer Research Page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer Table The first study that demonstrated thesignificantly higher expression of both ASPH mRNAand protein in HCC and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by Lavaissiere [] Subsequently they verified the role of upregulated ASPH protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the NIH3 T3 cell line and animal models []The level of ASPH also correlated with cell motility andinvasiveness in in vitro experiments [ ] In thestudy by Maeda [] the overexpression of theASPH protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients Similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung [] and colon carcinomas[] and glioblastoma multiforme [] Recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples ASPH has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes [] In glioblastoma the prognostic significance ofASPH was suggested by profiling of alternative mRNAsplicing []ASPH gene expression is upregulated via Wntcatenin [] and insulininsulinlike growth factor IGF1insulin receptor substrate IRS1 signaling [ ]through extracellular signalregulated kinaseERKmitogenactivated protein kinase MAPK andphosphatidylinositol3kinaseprotein kinase B PI3KAkt pathways Fig for review see ref [] InsulinIGF1IRS1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorsTable Summary of the studies which have identified the elevated ASPH expression in human tumor tissuesStudyPositive cases of studied samples nnTumor tissuesDetectionmethodIHCAntibody recognized region ofASPH proteinFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab Nterminus or 15C7 Abcatalytic domainFB50 Ab NterminusmAb G3 hybridomaPolyclonalFB50 mAb NterminusIHCIHCIHCIHCRTqPCRIHCRTqPCRIHCIHCIHCRTqPCRIHCFB50 Ab NterminusLavaissiere []HepatocellularCholangiocarcinomaBreastColonPalumbo []Pancreatic adenocarcinomaSepe et al[]Primitive neuroectodermalmedulloblastoma neuroblastomaMaeda et al[]Cantarini []CholangiocarcinomaHepatocellularMonte et al[]HepatocellularYang et al[]Wang et al[]Dong et al[]Tang et al[]Lin et al[] types of tumor tissuesaHepatocellularPancreatic cancerHepatocellularBreast 75fold higher level of mRNAcompared to normal tissue 7fold higher level of mRNAcompared to normal tissuePancreatic ductal adenocarcinomaOgawa []aLiver kidney breast cervical ovarian Fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomaFB50 Ab NterminusIHC 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Regulation of ASPH expression and ASPH involvement in signaling pathways The expression of the ASPH protein can be regulated atseveral levels The ASPH gene can be amplified in tumor cells and its transcription activated by INIGF1 and Wnt catenin pathways or inducedby hypoxia At the posttranscriptional level miR200a and miR135a can downregulate ASPH expression Stability of the ASPH protein can bereduced by phosphorylation with GSK3 Conversely ASPH can enhance GSK3 activity by inhibition of its phosphorylation with AKT and p38kinases ASPH also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the Notch receptor and ligands ex JAG and interaction with pRb vimentin and ADAMs Finallyinactivation of NK cells by ASPH has been demonstrated Green arrow activation signal red bar inhibitory signal[] The catenindependent Wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies Upon aberrant activation of Wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus [] where an interaction between catenin and Tcellfactorlymphoidenhancerbinding factor TCFLEF proteins forms atranscriptional regulatory complex which enhances theexpression of Wnt target genes including IRS1 []ASPH was proposed as a common link between Wntcatenin and insulinIGF1IRS1 pathways and downstream signaling []The regulation of ASPH gene expression in tumorsmight also be affected by a copy number variation Inthe study by Kadota [] the ASPH gene locus hasbeen identified as one of the DNA regions with focalamplification in primary breast cancer In colorectal cancer ASPH gain or amplification was found in ofsamples [] Next a suppressant role of the microRNAmiR200a in posttranscription regulation of the ASPHexpression in hepatoma cells has been found [] MiR200a belongs to miR200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref[] Similarly miR135a has beenshown to suppress ASPH in endometrial cancer []Moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 GSK3casein kinase CK2 protein kinase A PKA and protein kinase C PKC on ASPH [] several studies demonstrated that phosphorylation can regulate the ASPHprotein expression [ ] Inhibition of the GSK3activity did not modify mRNA expression but increased 0cKanwal Journal of Experimental Clinical Cancer Research Page of the ASPH protein level [] Direct phosphorylation ofASPH by GSK3 probably decreases ASPH stability andthus reduces cell mobility [] ASPH protein expressionwas also increased by inhibitors of PKA PKC and CK2[] Mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofASPH phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] ThereforeASPH phosphorylation probably regulates the functionof this protein by various mechanismsASPH expression can also be regulated by hypoxia andoxidative stress In human neuronal cells this effect wasmediated by hypoxia inducible factor alpha HIF1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain PHD proteins and factorinhibiting HIF FIH are inactivated Consequently theHIF1 heterodimer made up of subunits HIF1α andHIF1 functions as a transcription factor likely enhancing ASPH expression by binding to hypoxiaresponsiveelements [] In hypoxic regions of glioblastoma bothHIF1α and ASPH were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of ASPH in mesenchymal transition [] Brewitz showed reducedASPH hydroxylation activity at low oxygen concentrations and suggested an ASPH role in oxygen hypoxiasensing ASPH upregulation induced by hypoxia couldcompensate for reduced enzymatic activity [] Moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon ASPHoverexpression which was reversed by silencing ASPHexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion []ASPH protein interactions and signaling pathwaysThe ASPH hydroxylation consensus sequence is confined within cbEGFlike domains that are found in proteins of diverse function including Notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3Axl family ofreceptor tyrosine kinases []The Notch signaling cascade is a remarkably conservedpathway Notch proteins Notch1 Notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment [] Notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells Dysregulation of the Notch pathway is directly linkedto cancer vascular disorders and congenital defects [] In mammals Notch signaling activated by binding ofone of two families of canonical Notch ligandsjaggedJAG1 and JAG2 and delta like DLL1 DLL3 and DLL4leads to the generation of the cleaved Notch intracellulardomain NICD fragment and its nuclear translocation Inthe nucleus the NICD fragment interacts with the DNAbinding complex CSL CBF1RBPjκ SuH Lag1 Thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bHLH transcription factor HES1 HESwith YRPW motif HEY1 CD44 epithelial cell adhesionmolecule EPCAM cmyc protooncogene matrix metallopeptidase MMP29 cyclin D1 cyclooxygenase vascular endothelial growth factor VEGF and proliferatingcell nuclear antigen PCNA [ ]Upregulation of ASPH results in enzymatic modification of the cbEGFlike repeats in the Notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof Notch signaling [ ] Furthermore the interactionof ASPH with a disintegrin and metallopeptidase domainADAM stabilizes this complex and enhances theS2 cleavage ofthe Notch receptors and subsequentNICD fragment release [] The activation of the targetgenes in malignant cells increases cell proliferation migration and invasion []through the epithelialtomesenchymal transition EMT that is probably upregulated by the interaction of ASPH with vimentin []Consequentlythis activation supports tumor growthand metastasis The ASPHNotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]The SRC kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis []ASPH has been identified as an SRC pathway activatorOverexpressed ASPH directly interacts with ADAM12 and strengthens the SRC activation by these proteinswhich promotes MMPmediated extracellular matrixdegradation and tumor invasiveness []ASPH can also contribute to malignant phenotype ofcells by interaction with other proteins Iwagami et alrevealed the interaction of ASPH with GSK3 that prevents GSK3 inactivation by phosphorylation with upstream kinases [] This mechanism was confirmed ina castrationresistant prostate cancer model [] GSK3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination Some types of tumors aresensitive to GSK3 inhibitors [] Recently Huang elucidated a direct binding of ASPH with retinoblastoma protein pRb leading to pRb phosphorylation[] They also showed that this effect was mediated byincreased binding of cyclindependent kinase CDK CDK4 and cyclins D1 and E with pRb and wasdependentAsASPH enzymaticonactivity 0cKanwal Journal of Experimental Clinical Cancer Research Page of phosphorylation of pRb inactivates its tumorsuppressorfunction ASPH can contribute to the progression of cellcycle via interaction with pRbEffect of ASPH on an immune systemTumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system [] In theseprocesses various mechanisms of both innate and adaptive immunity are included [] Immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties []As potential targets of ASPH hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when ASPH is overexpressed on cancercells Indeed such effect was demonstrated for humannatural killer NK cells by using recombinant ASPHwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively []Antibodies against ASPH inhibited these effectsInteraction of ASPH with other immune cells has notbeen studied However we suppose possible influence ofASPH on different tumorinfiltrating cells This assumption comes from the involvement of Notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells Forinstance Notch activation contributed to stimulation ofproinflammatoryantitumorigenic M1 polarization inboth bone marrowderived primary macrophages [] and tumorassociated macrophages[] WhenNotch signaling was abrogated protumorigenic M2polarization was induced even by stimulators of M1polarization [] miR125a has been identified as adownstream mediator of Notch signaling in macrophages [] Similarly the Notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of CD4 and CD8 T cells[] Different Notch receptors and their interactionwith different ligands contribute to these processes []Moreover noncanonical Notch signaling is implicatedin regulation of immune cells [] While activation ofNotch signaling in some cells eg T helper cells cytotoxic CD8 T cells and M1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory T cellsit leads to immunosuppression [] Thus immunostimulatory effect of Notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity [] Therapeutics affecting Notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedASPH as a therapeutic targetOncogenic abilities of ASPH have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with ASPHoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma [] pancreatic cancer [ ] glioma [] breast carcinoma [] castrationresistant prostatecancer [] and colorectal cancer [] In studies analyzingASPH function various approaches were utilized to revealsignaling pathways affected by ASPH Particularly ASPHexpression was diminished by using small interfering RNAs[ ] short hairpin RNAs [ ] or theCRISPRCas9 system [ ] The importance of ASPHenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by SMIs [ ] In vitro assays showed ASPH involvement in cell proliferation migration and invasion Cellularalterations included EMTinhibition of apoptosis andstemness acquisition Tumor growth and invasivenesscould further be supported by ASPHinduced extracellularmatrix degradation angiogenesis and transendothelial migration Notch and SRC signaling are probably major pathways influenced by ASPH Fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models Thus these studies also demonstrated thatASPH is a suitable target for cancer treatment especially bySMIs or immunotherapySmall molecule inhibitorsSMIs of ASPH Fig have been developed and used totest the role of ASPH in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] A small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches Not only can these inhibitors inhibit the catalytic activity of ASPH unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit ASPHcatalytic activity in the endoplasmic reticulum Differentcancers have different ASPH expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed []The first ASPH SMIs published were the tetronimidesMOI500 and MOI1100 Tetronimides were originallysynthesized in by Dahn [] and are redoxactivemimics of ascorbic acid and 2oxoglutarate MOI500 isa mixed inhibitor that inhibits both ASPH and the FatMass and Obesity protein FTO [] and is not onlyorally bioavailable but also can penetrate the blood 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Small molecule inhibitors of ASPHthereand kinasesbrain barrier MOI1100 is a more potent inhibitor ofASPH and is also more selective [] Despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for MOI1100 Enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup [] as in MOI1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inMOI1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters MOI1182 isreported to have the ability to suppress invasive activityat a concentration of nM [] SMIs of ASPH have acharacteristic in vitro concentration dependent profilewhere the activity of the SMI plateaus at value around viability [] emphasizing the noncytotoxic properties of this class of inhibitorsNatural products and inhibitors of other enzymes thathave been repurposed as ASPH inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthineCN2019101414327 and guaianolides related to nortrilobolideCN2019104575886CN2019101414219CN2019101414187 0cKanwal Journal of Experimental Clinical Cancer Research Page of sesquiterpene with complex anticancerBosutinib is a wellknown inhibitor of BCRABL and SRCtyrosine kinases approved for the treatment of chronic myelogenous leukemia [] Cepharanthine is a natural productactivityincluding AMPactivated protein kinase AMPK activationand nuclear factor kappa B NFκB inhibition [] Nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium ATPase SERCA inhibition [] Recently a family of potent pyridine dicarboxylates have alsobeen published [] utilizing a mass spectrometrybasedinhibition assay [] These compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate The synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include PHD2 FIH and lysinespecific demethylase 4EKDM4E in addition to ASPH with varying degrees of selectivity However while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface ASPH inhibitors that may nothave cell penetrating activity []immunity As a target of humoralImmunotherapyASPH can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression Since ASPH is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityASPH on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity ADCC complement dependent cytotoxicity CDCor antibodydependent cellular phagocytosis ADCP []When the ASPH antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen HLA class Ior class II molecules and recognized by CD8 or CD4 Tlymphocytes respectively [] that can be stimulated byimmunization breaking tolerance to selfantigens []Induction of ASPHspecific CD4 and CD8 T cells wasexamined in blood samples of HCC patients Using synthetic peptides derived from ASPH after prediction of HLAclass I and HLA class IIrestricted epitopes it has beenfound that ASPH is a highly immunogenic protein that activates both types of analyzed T cells [] Thus efficientantitumor reactions could be stimulated by immunizationThe first vaccine against ASPH was based on matureddendritic cells DC loaded with the ASPH protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma [] This study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis BothCD8 and CD4 cells contributed to an antitumor effectinduced in a mouse model of HCC by immunizationwith ASPHloaded DCs []The next antiASPH vaccine was based on a bacteriophage lambda display system The viral capsid proteingpD was fused with the N or Cterminus of ASPH andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models []The vaccine PAN3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer[] This study demonstrated safety and immunogenicity of PAN3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen PSAor PSA doubling time ASPHspecific immune responseswere mediated by both antibodies and T lymphocytesAs ASPH is a type II transmembrane protein its Cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes Development ofASPHspecific antibodies has been described in several s [] The human IgG1 PAN622 recognizesthe catalytic domain of ASPH This antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells [] In the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results [] MouseIgG1 monoclonal antibody binding to the CterminalASPH domain mediated ADCC by human NK cells []Recently a secondgeneration antibody approach hasbeen disclosed The prepared antibody binds to the extreme Cterminus of ASPH US that is involved in specific substrate recognition [] Thereforethis antibody has direct ASPH inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activityPAN622 wasbioimagingusedforConclusionsASPH is an important enzyme in malignant transformationof cells It stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment Two main pathways Notch and SRCthrough which ASPH promotes the tumor growth havebeen identified It has also been shown that ASPH expression is induced by some growth factors and hypoxia and isregulated at various levels The overexpression of ASPHand its downstream targets has been detected in numeroushuman malignancies Since ASPH is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsFig Small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig ASPH as a therapeutic target ASPH expression is upregulated by growth factors and hypoxia Its enzymatic activity can be inhibited bySMIs or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionConsequently tumor growth and metastasis are also reduce | 2 |
predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cellsmonocytes and macrophagesas they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung ï¬brosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389ï¬mmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid ï¬rstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a ï¬ner repertoireof self and nonselfantigens that facilitate pathogenspeciï¬c actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adeï¬ned enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have proammatoryproperties and androgens are thought to have antiammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory ï¬ow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that deï¬ne howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exempliï¬ed by tuberculosisor uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterones immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insuï¬cientcontrol of tissuedamaging ammatory responses seen inCOPD and pulmonary ï¬brosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantiï¬ed serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting ï¬nding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ¼ times those of DHEADHEA became of interest to the asthma ï¬eld because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled Eï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the ï¬rst second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary ï¬brosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both ï¬broblasts and alveolarepithelial cells Inï¬uenzaThe lungs are also the target of respiratory viruses such asuenza A ï¬u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to uenza A virus and to the uenzavaccine In general women have a more robust protectiveimmune response to uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identiï¬edthat high testosterone was correlated with a blunted responseto the ï¬u vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalï¬u is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how ï¬uctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune systemsresponse to the uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these ï¬ndings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during uenzainfection are lackingTuberculosisLike uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is deï¬nitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent speciï¬ctypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers oftheammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofproammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identiï¬ed as large highly autoï¬uorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary ï¬brosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to diï¬culties in their identiï¬cation and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of proammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentiï¬ed two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and deï¬ne thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to diï¬culties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited intoamed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and nonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into nonamed and amed tissues from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more eï¬cient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of tissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneï¬cialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16 are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identiï¬cation ofmonocytes in the lung compartments in humans has been achallenge because monocytic contamination from the bloodvessels Overcoming this challenge Desch et alperformed a ï¬ow cytometric phenotyping study and identiï¬edtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206 CD1c CD1a intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1c CD1a monocytes were described as tissue monocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erentammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erentammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a proammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as classical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or alternative activation of macrophages M2 macrophages produce ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models SimilarlyM2 macrophages are the predominant subset seen in pulmonaryï¬brosis and are responsible for ï¬brogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage ï¬uid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage ï¬uid of patients withCOPD Unlike in allergic asthma and pulmonary ï¬brosismacrophages in COPD are polarized toward an M1 proï¬le In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with deï¬ned cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldeï¬ned opposing phenotypes or does not ï¬tthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice and humans Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent proammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate | 2 |
the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second peoples hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second peoples hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at a0 °c for further experiments this research was approved by the ethics committee of lianyungang second peoples hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbeccos modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0 with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative realtime polymerase chain reaction qrtpcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit cck assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of à a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandualluciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenolchloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells à transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by students t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second peoples hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of à a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi | 0 |
mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5 | 0 |
" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus piv respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecaloral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled toilet plume in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as the quintessential perpetrator of inflammation to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1 generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a gray zone about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a one size fits all consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should never worry about action only inaction 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o | 0 |
" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteams experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as claysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest highly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [] They havetherefore been a potential alternate medicine for several diseases [] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof singlesingle type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as claysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterialsMoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately à à microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethodsMolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CEDCED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors previous research[] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvygreneBAlraopcilihpordyHaCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study to mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[] we propose that nanoclays may be injected asclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature httpsdoi101038nature02145Li F Structural analysis of major species barriers between humansand palm civets 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objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anticancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell contextdependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0csainzcort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoys 5a medium alamarblue® ab invitrogen were bought leibovitzs l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillinstreptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoys 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research lhospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillinstreptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapirowilk test to assess data normality and nonparametrical independent samples kruskalwallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainzcort a0et a0al bmc res notes page of fig a ht cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht cells incubated with or without μm cbd for h representative images are shown bar µm c ht cell viability according to dapi staining see the materials and methods section ht cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapipositive cells dead cells showing a higher percentage in cbdtreated cells a representative experiment c is shown p viability and a0proliferation of a0ht cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainzcort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainzcort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd a0μgml reduced the viability of cancer cells and also had effects on other survival variables [ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu bromo²deoxyuridine cbd cannabidiol dapi ²diamidinophenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag waldsch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltecub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implications in cancer metabolism trends cancer https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtnerfeigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w muellerklieser w renner k kreutz m ldhaassociated lactic acid production blunts tumor immunosurveillance by t and nk cells cell metab https 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the european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identiï¬ed a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identiï¬ed no reference point for chronic exposure could be identiï¬ed from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufï¬cient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientiï¬c output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientiï¬c outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientiï¬c opinion risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodiï¬cations or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientiï¬c opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientiï¬c opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when ï¬nalising the scientiï¬c opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic proï¬les of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspeciï¬c effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identiï¬ed from these studies reduced postnatal survival of pups due to insufï¬cientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufï¬cient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identiï¬edin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufï¬ciency cardiac failure coma and death have beenreported doses in the range of mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufï¬cient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufï¬cient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identiï¬edregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentiï¬edoccurrence data were only available for asolanine and achaconine and mostly for maincroppotatoes and new potatoes few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence ofnew potatoes with that of maincrop potatoes and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the ï¬nal processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby boiling in water and blanching of peeled potatoes by and frying in oil of peeledpotatoes by microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by and by respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the conï¬dence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identiï¬ed uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identiï¬ed due to an insufï¬cient database on the adverse effects of gas inruminants pigs poultry rabbits and ï¬sh an acute reference dose could not be derivedpotatoes are not grown speciï¬cally as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufï¬cient to perform an exposure assessmentthus no risk characterisation could be performed due to insufï¬cient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identiï¬ed to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identiï¬ed to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classiï¬cation methodology for exposure assessment methodology for risk characterisation assessment hazard identiï¬cation and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed inï¬uence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the inï¬uence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identiï¬cation and characterisation summary of uncertainties conclusions hazard identiï¬cation and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a major glycoalkaloids and their aglycones present in solanum species appendix b identiï¬cation and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c details of the study design of the toxicokinetic studies appendix d comparison of developmental toxicity of single dose studies appendix e inhibition of cholinesterases by gas appendix f rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h additional scenario for the human risk characterisation annex a occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are times greater in the peel than in the ï¬esh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include ï¬ulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientiï¬c opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid proï¬le ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of speciï¬c groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including speciï¬c groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no speciï¬cation on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the ï¬rst alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well deï¬nedtheconï¬guration is 22r 25stheitconï¬guration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversiï¬cation is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe preï¬x alpha a refers to the intact glycoside while the preï¬xes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s | 0 |
to machine learning with python a guide for data scientists oreilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res yates a the ensembl rest api ensembl data for any language bioinformatics kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet amrhein v greenland s mcshane b scientists rise up against statistical significance nature wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond p maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial universitys school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c' | 0 |
Purpose of Review Recognize which are the elements that predict why a person is aging faster or slower and which interventionwe can arrange to slow down the process which permits to prevent or delay the progression of multimorbidity and disabilityRecent Findings Aging is a complex process that leads to changes in all the systems of the body and all the functions of theperson however aging develops at different rates in different people and chronological age is not always consistent withbiological ageSummary Gerontologists are focused not only on finding the best theory able to explain aging but also on identifying one or moremarkers which are able to describe aging processes These biomarkers are necessary to better define the agingrelated pathologies manage multimorbidity and improve the quality of life The aim of this paper is to review the most recent evidence onaging biomarkers and the clusters related to them for personalization of treatmentsKeywords Biomarker of aging Frailty syndrome Aging phenotype Quality of life Multimorbidity Life expectancy SocialneedsIntroductionMost people dont grow up Most people age They findparking spaces honour their credit cards get married havechildren and call that maturity What that is is agingMaya Angelou One of the biggest megatrends impactingthe world today is population aging Aging is a topic thathas captivated both scientists and philosophers throughouthistory but aging as a population scenario emerged on aThis is part of the Topical Collection on Geriatric Oncology Beatrice Di Capuabeatricedicapuagmailcom UOC di Radioterapia Oncologica Dipartimento Diagnostica perImmagini Radioterapia Oncologica e Ematologia FondazionePoliclinico Universitario A Gemelli IRCCS Rome Italy Dipartimento di Scienze dellinvecchiamento neurologicheortopediche e della testacollo Fondazione Policlinico UniversitarioA Gemelli IRCCS Rome Italy Moffitt Cancer Center Tampa FL USAworldwide scale for the first time in the last century Thus itis hard to really identify a definition of aging It is a decrease infitness with chronological age it is a developmental phasebeyond the normal life trajectory and it is a time of the increased risk of physical and psychological disabilities testingthe limits of resilienceAging occurs at a different rate in varying geographic regions of the worldEurope is currently the oldest region with of thetotal population aged and older However the Asia andLatin America older population is growing fast with Asiasolder population almost tripling in size from million in to million in []All these data do not consider aging as an epiphenomenonbut an individual data of the global population just a chronological number Aging is intrinsically a complex scenariocharacterized by changes that take place at different levels ofbiological systems Biological age is of course influenced bychronological age but chronological age is by itself not representative of biological age biological age is determined byphysiological reserve and functional status Assessing biological age is essential to predict life expectancy and resilience to 0c Page of Curr Oncol Rep stressors [] If any definition of aging may appear incompleteand insufficient much more difficult and complex is to findthe marker or biomarker that can identify itMany theories currently trying to explain aging processesand many biomarkers are identified to measure aging and itsevolutionary stages Theories and biomarkers are not studiedto extend life span but to guide therapeutic choices and optimize patient management and personalization of careThe purpose of this paper is not purely to list which biomarkers are able to identify the various stages of aging ratherexplain how an epiphenomenon natural and physiological isso complex [] how many factors are protagonists in its development and how many actors and characters play in maximizing its individual features taking into account social andmorbidity biomarker These factors such as frailty loss ofautonomy essential needs and comorbidities influence theaging process and are able to justify why the biological ageof a person living in a country does not correspond to the ageof another person living in a country with better sociosanitaryconditionsClinical and Biological Aging PhenotypesThe aging phenotype can be described as a complex mosaicresulting from the interaction of a variety of environmentalstochastic and geneticepigenetic eventsstimuli impinginglifelong on our body [ ]There is no clear evidence which molecular cellular orphysiological changes are the most important drivers of theaging process andor how they influence one another [] In itsbroadest sense aging merely refers to the changes that occurduring an anisms life span though the rate at which thesetake place varies widely [] Despite its enormous complexityinvolving combinations of these variables a small number ofbasic molecular mechanisms underpin the aging process including a set of evolutionary highly conserved basic biological mechanisms responsible for body maintenance and repairOne of the key mechanisms is inflammation a typical featureof the aging process is the development of a chronic lowgrade inflammatory status named inflammaging [8cid129] whichemerged as critical in the pathogenesis of major agerelatedchronic diseases such as atherosclerosis type diabetes and neuro degeneration Inflammaging plays a pivotal role in themost important geriatric conditions such as sarc ia [9cid129cid129]osteoporosis [] frailty and disability thus contributing tomortality [] Interestingly a variety of tissues adipose tissue muscle ans brain liver systems immune systemand ecosystems gut microbiota of the body indicated assubsystems can contribute to the onset and progressionof such a systemic inflammatory state [] by increasing theproduction of several proinflammatory mediators or loweringthat of the antiinflammatory ones [8cid129]To differentiate the innocuous changes from those leadingto increased risk of disease disability or death biogerontologists tend to use a more precise termsenescencewhen describing aging [] Senescence is thereforethe progressive deterioration of bodily functions over time andnormal human aging has been associated with a loss of complexity in a wide range of physiological processes and anatomic structures [] including blood pressure [] strideintervals [] respiratory cycles [] and vision [] amongothers such as postural dynamics [] ultimately leading todecreased fertility and increased risk or mortality []Systemic consequences of aging are widespread but theycan be clustered into four domains Fig Changing in body compositionThe balance between energy availability and energydemandSignaling networks that maintain homeostasis NeurodegenerationThese changes develop in parallel and affect each otherthrough many feedforward and feedback loopThe phenotype that results from the aging process is characterized by increased susceptibility to disease high risk ofmultiple coexisting diseases impaired response to stress theemergence of geriatric syndromes altered response to treatment high risk of disability and loss of personal autonomywith all its psychological and social consequences On theother hand all these factors influence aging itself in a dynamic and parallel way so that they can be considered as not onlya consequence of aging but also an integral part of the agingprocessTheories of AgingHuman aging is currently defined as a dynamic process involving the continual adaptation of the body to lifelong exposure tointernal and external damaging as conceptualized in the remodelling theory of aging [21cid129cid129] Theories of aging are generallyclassified as either program or damage theories Programmedaging theories suggest that there is a deliberate deterioration withage because a limited life span results in evolutionary benefits[] This plan could be a result of aging genes The firstdescribed mutation to yield a significant extension in the life spanof Caenorhabditis elegans was in the ageI gene which wasshown to result in a increase in mean life span and a increase in maximum life span of this anism []Evolutionary biologists may argue that aging occurs due to theabsence of natural selection at the postreproductive stage of life[] Although such aging theories are subjectively appealing asthey convey a cure for aging the accumulation of damage is aspontaneous entropydriven process [] Among the damage 0cCurr Oncol Rep Fig Systemic consequences ofagingPage of theories a prevailing idea is that of oxidative damage Reactiveoxygen species ROS are generated during metabolism throughseveral interrelated reactions The supposition that aging may becaused by ROS has been further substantiated by studies involving transgenic animals for genes encoding antioxidants The lifespan of Drosophila melanogaster has been extended by overexpression of both superoxide dismutase SOD and catalase bothantioxidant enzymes [] Since mitochondria are the major producer of ROS in mammalian cells mitochondrial DNAmtDNA is therefore particularly susceptible to oxidative damage [] Mitochondrial maintenance is therefore essential topreserve cellular homeostasis and impaired mitochondrial maintenance has been described as a shared hallmark of numeroushuman pathologies and aging [] Mitochondrial DNA varieswith age and it is commonly considered that DNA hypomethylation is a typical aspect of the aging process [] ROS are activeintermediates of DNA methylation as well as histone modification These reactive oxygen species may play a role in epigeneticprocesses physiological phenotypic variations caused by external or environmental factors that switch genes onoff throughreactions of nucleophilic substitution at the DNA levelConsequently it has been suggested that better preservation ofDNA methylation levels slower cell metabolism and improvedcontrol in signal transmission through epigenetic mechanismscould be key processes involved in human longevity Oxidativedamage to proteins is irreversible and irreparable [] and mustbe degraded by the proteasome The proteasome is the mostimportant proteolytic machinery in eukaryotic cells largely responsible for the removal of oxidized proteins and the preventionof its aggregation [] However it has been shown that theactivity of proteasome is impaired during aging leading to theaccumulation of oxidizing proteins aggresome and lipofuscinsocalled the age pigment Similarly to oxidative damage nitrosamine damagethat caused by reactive nitrogen speciesRNS such as nitric oxidehas been suggested to also contribute to agerelated diseases namely hepatic steatosis and apoptosis [] as well as functional and structural changes in the cardiovascular system [ ] sleep homeostasis [] psychological disorders [] and dementia []Most supporters of the genomic instability theory of agingrefer to telomere shortening [] and mutation in DNA mitochondrial Telomeres are the repeated DNA sequences at theends of linear chromosomes which are unable to be fullyreplicated by DNA polymerasesMutations in mtDNA cause a wide range of human mitochondrial diseases and have been implicated in agerelateddiseases and agingBiomarker FeaturesFinding the biomarker of aging is one of the most importantgoals of medicine The National Institutes of HealthBiomarkers Definitions Working Group defined a biomarkeras a characteristic that is objectively measured and evaluatedas an indicator of normal biological processes pathogenicprocesses or pharmacologic responses to a therapeutic intervention []The American Federation for Aging Research AFAR recommends the following criteria for biomarkers of aging [39cid129]It must predict a persons physiological cognitive andphysical function in an agerelated way independentlyof chronological age 0c Page of Curr Oncol Rep It must be testable and not harmful to test subjects forexample a blood test or an imaging technique it mustalso be technically simple to perform and it must be accurate and reproducibly without the need for specializedequipment or techniquesIt should work in laboratory animals as well as humanssince preliminary testing is always done in nonhumansubjectsFerrucci et al reviewed the biomarkers proposed as elements of a theory based on the balance between resiliencemechanisms and accumulated damages where biomarkersact in reducing resilience mechanisms or increasing damages[40cid129] Tables and The pathways eligible to become biomarkers are thefollowingGenomic Instability Endogenous and exogenous agents continuously challenge the integrity of DNA when DNA repairmechanisms cannot manage the repeated damage the result isan accumulation of DNA somatic mutations This phenomenon causes dysregulation of gene expression and the production of altered proteins that lead to cellular damage Somaticmutation accumulation has been observed in skeletal musclecells neurons and lymphocytes B related to aging []nevertheless quantification of DNA repair capacity in humanshas yet to be finalized []Telomere Attrition Telomeres are the DNA sequences that areplaced at the end of the DNA chain and protect theTable Biological changesunderlying agingGenomic instabilityTelomere attritionEpigenetic alterationscid129 DNA methylationcid129 Histone modificationcid129 Noncoding RNALoss of proteostasisMitochondrial dysfunctionCellular senescenceDeregulated nutrientsensingSteam cell exhaustionAltered intercellular communicationchromosome ends from damage During each replicationtelomeres are reproduced but not completely so with agingthey become shorter and contribute to cellular senescence[] To date different techniques are available to detecttelomere length in circulating cells however no techniqueshave been validated for evaluating aging because of the heterogeneity between different cells between individuals andhigh measurement errors that make these techniques not yetvalid in clinical practice []Epigenetic Alterations Epigenetics refers to those mechanismsexternal to DNA that modulate gene expression in cells theregulation of gene expression determines the phenotypic characteristics of the different cells and tissues The main mechanismsare DNA methylation histone modification and noncodingRNA While DNA methylation is easily measured in circulatingcells and seems to be correlated to aging [ ] measuringhistone modification or noncoding RNA is difficult and expensive Recent evidence correlates DNA methylation with agingand agerelated chronic diseases in humans [ ]Individuals with higher levels of DNA methylation have a higherrisk of developing several agerelated diseases and prematuremortality for all causes and cardiovascular diseases [] as wellas physical and cognitive functions [ ]Loss of Proteostasis The repair of damaged structures or theirelimination is fundamental to maintain cell integrity and function [] Studies suggest that proteostasis becomes defectivewith aging and contributes to immunosenescence [] and thatautophagy appears to be more functional in longlived peopleAccumulation of DNA somatic mutationsDysregulation of gene expressionAltered proteins productionTelomere shortening contribute tocellular senescenceAltered gene expressionRelated to agerelated chronic diseasesAccumulation of damaged structuresAltered energy productionIncreased ROS productionApoptosisprogrammed cell deathActivation of pathways leading to apoptosisProduction of SASPIncrease of life span in dietary restrictionDecline of regenerative potentialInflammagingDysfunction of endocrine neuronaland immune systems 0cCurr Oncol Rep Page of Table Measurable biomarkers classified by respective hallmarksPathways measuredMeasurable biomarkersHallmarkGenomic instabilityTelomere shorteningCellular senescencecid129 DNA repair mechanismscid129 DNA modificationscid129 Telomere lengthcid129 Markers of DNA damage responsecid129 Telomerase activitycid129 Senescent markers in blood and tissueEpigenetic changes or epigenetic clockcid129 DNA methylationcid129 Histone acetylationcid129 Noncoding RNAMitochondrialDecreased autophagy proteostasiscid129 Mitochondrial volumenumbershapecid129 Mito respirationcid129 Markers of biogenesiscid129 mtDNA copy number and haplotypescid129 Autophagy markerscid129 Chaperon proteinsStem cell exhaustionDeregulated nutrientsensingAltered intercellular communicationcid129 Proliferative capacity in vitrocid129 Resistance to stresscid129 Growth hormone GH axiscid129 Metabolism alterationscid129 Measures of inflammationcid129 yH2AX immunohistochemistrycid129Leukocyte telomere lengthcid129MIR31HGcid129 p16INK4acid129 Senescenceassociated secretoryphenotype SASP proteinscid129 Measures of DNA methylationcid129 SIRT1 SIRT2 SIRT3 SIRT6 SIRT7cid129 Dosage of circulating microRNAs miR34aMiR21 miR1263p miR151a3pmiR181a5p miR1248cid129 p31 MRI spectroscopycid129 Growth differentiating factor GDF15cid129 NADcid129 Target of rapamycin TORcid129 Protein carbamylationcid129 Advanced glycation end productscid129 Insulinlike growth factor IGF1cid129 HGBA1ccid129 IL6cid129 TNFαcid129 CRP Creactive proteincid129 TNFRII tumor necrosis factorα RII[] Measuring the loss of proteostasis mechanism could be agood biomarker but to date there are no valid techniques forthis purposeMitochondrial Dysfunction The main role of mitochondria isto guarantee energy for the cell through the production ofATP They are also involved in signaling by the productionof ROS and in apoptosisprogrammed cell deathMitochondrial dysfunction is a good biomarker of aging andis associated with disability in older persons through the reduction of muscle strength [65cid129]Many techniques are measuring oxidative phosphorylationand ROS generation that have been associated with chronicdisease [ ] nevertheless the relation with aging is notcompletely validatedCellular Senescence Genomic instability telomere shorteningand other endogenous and exogenous mechanisms can inducethe cell to activate specific pathways that lead to apoptosis[] This process is called cellular senescence and is characterized by structural and functional changes in the cell []Senescent cells produce proinflammatory cytokines andchemokines growth factors and matrix proteases called senescenceassociated secretory phenotype SASP [ ]which may induce some agerelated diseases [] Thedetection of SASP has been proposed as a biomarker of aging[]Deregulated NutrientSensing Genetic mutations in growthhormone and the insulinlike growth factor have been linkedto longevity [] Moreover dietary restriction showed to increase life span in primates [ ] For these reasons thispathway has been proposed as biomarkers of agingSteam Cell Exhaustion The decline in the regenerative potential is one of the elements at the base of aging [] Despitepharmacological interventions being explored to counteractthis phenomenon [] evidences are still poor 0c Page of Curr Oncol Rep Altered Intercellular Communication With aging we also observe changes in intercellular communication as inflammatory reaction increases the other communication ways becomedysfunctional endocrine neuronal immune system []As we discussed earlier inflammation can be inappropriately increased in aging and this has been related to agerelated disease [ ]Indeed the pathways described as potential biomarkers ofaging are strongly related to inflammation for this reasonmeasuring circulating levels of cytokines is considered anew field of research [ 84cid129 ]Aging and Life ExpectancyAging and life expectancy are closely related In a broadsense determining an individuals life expectancy is also away of schematizing his or her aging process Life expectancyis a statistical measure of the average time an anism isexpected to live based on the year of its birth LEB itscurrent age and demographic factors including gender []In the last decades life expectancy has increased in high income country the rise in human life expectancy has involveddeclines in intrinsic and extrinsic mortality processes associated respectively with senescence and environmental challenges []In association to this increased longevity there are diseasescalled agerelated that increase quadratically with age andcause a progressive loss of physical mental and cognitiveintegrities leading to impaired function and increased vulnerability to morbidity mortality [] and disability in additionto increasing care needs and agerelated burden measuredthrough the sum of disabilityadjusted life years DALYs ofthese diseases among these adults Fig Ninetytwo of the of the Global Burden of Disease causes were identified asagerelated diseases In particular cardiovascular diseaseneoplasm and chronic respiratory disorders are those withhigher agerelated disease burden []Determinants of Frailty Syndrome as AgingBiomarkerFrailty can be defined as a state of increased vulnerabilityto stressors or a loss of capacity to resolve homeostasisperturbation Frailty condition is closely related to aging[88cid129cid129] and the frailty indexes can consequently be considered biomarkers of aging themselves In frail individuals it is possible to find both changing in body composition and balance between energy availability and energydemand Moreover in the definition of frailty it is welldescribed how signaling networks maintain homeostasisand association with neurodegeneration These fouraspects all refer to the hallmarks of aging Frailty is associated with adverse clinical outcomes including falls institutionalization and death [88cid129cid129]Two principal models emerged in the last decades that areable to conceptualize and consequently measure frailty in everyday clinical practice and research the frailty phenotypemodel and the cumulative deficits modelThe frailty phenotype was first described by Fried and colleagues in analyzing data from the CardiovascularHealth Study CHS involving men and women aged years and older In this study it was investigated whichcharacteristics of the population were predictive of falls disability hospitalization and death Their operational definitionof frailty included a cluster of at least three of the followingvariables unintentional weight loss selfreported exhaustionlow energy expenditure slow gait speed and weak gripstrength This model does not take into consideration cognitive impairment as a cause of increased vulnerability as thiscould contribute to functional decline and adverse events inolder people [ ]The cumulative deficits model was developed byRockwood and colleagues as part of the prospectiveCanadian Study of Health and Aging CSHA involvinga cohort of older adults [] The authors identified parameters including diseases disabilities signssymptoms and laboratory values which were defined asdeficits The sum of the deficits in a single individualallowed for the calculation of a frailty index ie thenumber of deficits divided by Frailty in this modelis not considered as a cluster of symptoms but is conceptualized as a gradable syndrome with a higher number ofdeficits implying an increased vulnerability state The twomodels of frailty show significant overlap although theycapture slightly different sides of the same problem It isimportant to notice that physical frailty is frequently associated with multimorbidity [ 93cid129 ]It has been observed that the frailty phenotype construct is intrinsically related to mobility issues Indeedin older adults physical performance measures are a robust and consistent predictor for disability hospitalization institutionalization and death both in the researchand in the clinical setting Lower physical performance isfrequently associated with loss of skeletal muscle massand quality causing reduced strength and functional impairment [95cid129cid129] This process has been called sarc iaEven though sarc ia has been long associated withaging it has to be acknowledged that it can develop muchearlier in life [] Different definitions exist for this condition for the operational definition of sarc ia both inthe clinic and for research purposes that prioritize theassessment of muscle strength over muscle mass to identity sarc ic patients Strength is more closely related tosurvival and functional decline compared with muscle 0cCurr Oncol Rep Page of CARDIOVASCULAR DISEASESAtrial ï¬brillaÆon and ï¬uÆ©er endocardiÆs hypertensive heart disease intracerebralhaemorrhage ischaemic heart disease ischaemic stroke myocardiÆs nonrheumaÆc valve disease other cardiomyopathy other cardiovascular and circulatory diseases peripheralartery diseaseNEOPLASMSLeukaemia lymphoma mulÆple myeloma myelodysplasÆc syndroms and other hematopoieÆc neoplasms brain and nervous system cancer breastcancer prostate cancer larynx cancer lip and oral cavity cancer oesophagealcancer stomach cancer colon and rectum cancer liver cancer gallbladder and biliary tract cancer pancreaÆc cancer kidney cancer bladder cancer melanoma and nonmelanoma skin cancer ovarian cancer uterine cancer thyroid cancer tracheal bronchus and lung cancer mesothelioma othermalignant neoplasms other benign and insitu neoplasmsGASTROINTESTINAL ENDOCRINE AND KIDNEY DISEASESChronic kidney disease type diabetes mellitus cirrhosis due to nonalcoholicsteatohepaÆÆs pancreaÆÆs paralyÆc ileus and intesÆnal obstrucÆon pepÆculcer disease vascular intesÆnal disorders diarrhoeal diseasesSKIN AND SUBCUTANEOUS DISEASESCelluliÆs decubitus ulcer fungal skin diseases pyoderma other skin and subcutaneousdiseasesFig Agerelated diseases adapted from Chang et al []mass [95cid129cid129] According to EWGSOP criteria sarc iais defined by the presence of low muscle strength criterion and either or low muscle quantity or quality criterion or low physical performance criterion [95cid129cid129]The physical performance parameters used in the identification of frailty syndrome both integrated eg SPPB andalone walking speed handgrip strength can be used as agingperformance biomarkersDetermination of Medical and Social NeedsWhy consider medical and social needs aging biomarkersIn Robert J Havighurst said In considering theneeds of older people it is well first to remember that olderpeople have the needs that are common to all people andsecond that they have special needs due to the fact that theyare old people This sentence describes everything there is toknow about the need for the elderly and answers the questionbeforeIn every society and age there is what is meant by normality An elderly person in this scenario needs what is needed tomaintain this level of normalcy Activity of daily living andinstrumental activity of daily living ADL and IADL aloneremodelled according to the context and gender can identifythe minimum necessary Conducting needs assessment various areas must be considered including physical health mental health emotional care social cultural economic nutritional service security legal and educationalCHRONIC RESPIRATORY DISEASESAsbestosis chronic obstrucÆve pulmonary disease coal worker pneumoconiosis intersÆÆallung disease and pulmonary sarcoidosis other pneumoconiosis silicosis lower respiratoryinfecÆonsNEUROLOGICAL DISORDERSAlzheimers disease and other demenÆas motor neuron disease Parkinsonsdisease encephaliÆs pneumococcal meningiÆsAGE RELATED DISEASESENSE AN DISEASESHearing loss vision loss ex agerelated macular degeneraÆon cataract glaucoma other sense an diseases refracÆon disorders trachomaINJURIESDrowning environmental heat and cold exposure falls foreign body in other body part other transport injuries other unintenÆonal injuriesOTHER DISEASESCongenital musculoskeletal and limb anomalies digesÆve congenital anomalies endocrine metabolic blood and immune disorders other haemoglobinopathies and haemolyÆcanaemiasMany tools are used to evaluate peoples needs The majority of these tools are focused on physical performance ableto maintain autonomy few studies focus on social needs andthe costs of care In the West World of patients accountfor of total health care expenditures This is represented by older people individuals with multiple chronic conditions many medications frequent hospitalizations and limitations on their ability to perform basic daily functions due tophysical mental or psychosocial challenge []Since the health care and social needs of older adults differfrom that of other adults it is necessary to identify the needs ofthe elderly to make proper plans that will promote their healthCurrently most of the conducted studies had mainly focused on the elderly physical health needs and had neglectedto take into account other needs such as social and health careneeds Furthermore in addition to quantitative studies discovering the older adults perceptions of their own health needsis also necessaryConclusionThere is a large interest of researchers in biomarkers of agingand despite some of them seem to be very promising biological biomarkers are still far from a clinical application to datethere is no technique that meets the mentioned criteria of theideal biomarker [40cid129] Moreover we know that the biologicalpathways are the final agents of aging but on one side theycan be influenced by social economic and environmental factors and on the other side they express in various disease and 0c Page of Curr Oncol Rep disabilities of the person physical and cognitive impairmentsagerelated disease systems functions sensory functions etcFig To date more than a single biomarker to assess agingwe should consider a cluster of biomarkers that comprisethe various elements that we analyzed social and educational aspects economic factors country of origin presence of agerelated disease presence of dependence indaily activities physical capability cognitive functionlung and cardiovascular function and presence of sensorydysfunctions In Table we propose several clinical andlaboratory biomarkers that can be used in clinical practiceand researchThe geriatric assessment GA can currently be considered a system capable of monitoring multiple biomarkersclinical and laboratory of aging and at the same timeable to relate them to each other Through the GA it ispossible to make a prediction of the risk of toxicity of atreatment of life expectancy of social needs and of compliance with the treatments GA is composed indeed byseveral evaluations made through standardized toolswhich examine various aspects of the person a multidimensional assessmentAlthough it seems difficult to imagine a geriatric assessment as a biomarker currently for its characteristicsand for the high predictivity it has it can be consideredthe gold standard in the management of the older individual and instrumenttoward which other biomarkersshould be evaluatedThe purpose of this paper was to evaluate the multipleaspects that distinguish the aging process Aging must noFig Mechanisms connectingdifferent clusters of biomarkerslonger be described as a simple demographic event butas a complex mosaic in which several tesserae relate toeach other some in a very evident way others often in amore subdued but all fundamental way Each aging theory has attempted to justify this process effectively however there is no single biomarker to date that has beenfound able to identify the stage of this process At thesame time clinical clusters have been added to purelybiological markers and social ones should certainly beconsidered It therefore becomes important not to consider biomarkers only as life span but to try to overcomethis link and focus on the set of factors that influencingeach other are able to guide aging in good health andgood quality of life towards a lived aging as a slowdecline At the time we are writing this paper COVID infection is reaping victims especially in Italy Thehighest mortality is observed among the older adultsbut surprisingly it seems to maintain similar values between the youngest and oldest old over yearsCurrently no plausible justification is provided for thesedata In frailty the number of comorbidities the reducedfunctional reserve was the most used reasons Indirectlythis infection is highlighting the need to use parametersthat can more easily identify the aging process regardlessof chronological ageThe studies analyzed in the literature show that if on theone hand there are physiological biomarkers able ofhighlighting some features of aging other functionalmarkers performance social and economic status somepathologies and the presence of addiction are able ofspeed it up or slow it | 2 |
"Ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages The 5year diseasefree survival is below MicroRNAs subset of thenoncoding RNA molecules regulate the translation in post transcriptional level by binding to specific mRNAs topromote or degrade the target oncogenes or tumor suppressor genes Abnormal expression of miRNAs were foundin numerous human cancer including ovarian cancer Investigating the miRNAs derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer We aimed to findbiological markers for early diagnosis of ovarian cancer by investigating BRCA1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals miRNAsMethods The study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of miRNAs was performed on mononuclear cells that were isolated from the peripheral blood samplesThe miRNA expression profile changes in the study were found by using microarray analysis miRNA isolationprocedure performed from the lymphocyte in accordance with the kit protocol The presence and quality of theisolated miRNAs screened by electrophoresis Raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values Target proteins were detected for each miRNA byusing different algorithmsResults After the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the miRNAs miR6131 miR1305 miR1973p miR3651 and downregulation of miRNAs miR3135b miR4430 miR664b5p miR7663p were found statically significantContinued on next page Correspondence hy2188istanbuledutrDepartment of Cancer Genetics Istanbul Faculty of Medicine OncologyInstitute Istanbul University Istanbul Turkey The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cTuncer Journal of Ovarian Research Page of Continued from previous pageConclusions The detected miRNAs out of miRNAs might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies The miRNA expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology BRCA1 mutationstatus and ovarian cancer risk in accordance with the obtained dataThere is a need for validation of the miRNAs which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualsKeywords Monozygotic twins miRNA expression profiles BRCA1 and BRCA2 BiomarkersBackgroundOvarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality [] In Turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide Globocan datas showthat each year more than women are diagnosedwith ovarian cancer OC worldwide and approximately women die from it The data of Globocan for Turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy The 5year survival rate was given as These data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate [] The epithelial ovarian cancersEOCoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy [] Themajority of EOC patients are diagnosed in advanced stages Stage III and IV and year freesurvivalrate is below [] The standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy Significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance [] Ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsBRCA1 and BRCA2 genes are the most common breastovarian cancer syndrome associated genes Both BRCA1and BRCA2 have roles in the control of the genomic stability cell cycle and apoptosis The mutations occurringin these genes result with the inability of DNA repairand therefore results in the accumulation of the mutations in the cell The rate of the breast cancer susceptibility of women with BRCA1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with BRCA2 gene mutation until the age of years is and ovarian cancer development risk is [] Twin studies became important on genetics by theendandcentury Geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently [] When a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies Because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation In addition diversity may be revealed with a very limited patient population Therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies Monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and Mendelian disorders There are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution [] Therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins Phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease The most striking study is the oneconducted with monozygotic twins in Canada and evidencing that multiple sclerosis MS was a genetic disease [] MicroRNAs are one of the subset of the noncoding RNAs generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mRNAs [ ] The regulatoryroles of miRNAs were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] miRNA genes are known to locate in thechromosomal breaks This DNA breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] The noninvasive biologicalindicators have been used for the 0cTuncer Journal of Ovarian Research Page of treatment resistance of ovarian cancer The most common of this indicators are the cancer antigen125 CA and cancer antigen153 CA153 These biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease [] Therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period Thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsDifferentially synthesize miRNAs such as miR miR141 miR125b miR2223p or let7 family has beenshown in studies with ovarian cancer patients [] However the use of these miRNAs as a biomarker in ovariancancer is not yet available In order to clearly define therole of miRNAs in the pathogenesis of ovarian cancerwe planned to investigate the BRCA mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation In thisstudy miRNAs which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andBRCA wild type healthy siblingsMethodsPatients recruitmentThe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study The patient diagnosed with ovarian cancer and all family members applied to the Cancer Genetics Clinic of OncologyInstitute of Istanbul University for BRCA breast cancersusceptibility gene testing were examined for BRCAgene mutation All family members in the study consisted of highrisk individuals with Hereditary Breast andOvarian Cancer HBOC syndrome and the people included in the study were given as BR codes according topatient file number The monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have BRCA1 gene mutation c5266dupC pGln1756Profs74 rs397507247 on exon Thepatients brother and daughter were found negative forBRCA1BRCA2 gene mutations In this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by miRNA microarray method Thepedigree of the family included in the study and theirhierarchical cluster analaysis via Euclidean method isshown on Fig The study was approved by the Ethics Committee ofthe Istanbul Faculty of Medicine Following InstitutionalEthics Committee approval informed consents were obtained from all participants before enrollment into thestudy Ethics Committee Approval Number atstoredthey wereLymphocyte and miRNA isolationFicoll SigmaAldrich Darmstadt Germany density gradient was used to separate white blood cells mononuclear cells from other blood components miRNAisolation procedure was performed from the lymphocytein accordance with the kit protocol using the miRNeasyMini Kit Qiagen cat NoID The proceduresteps in accordance with the protocol are as follows μL QIAzol solution was included on the cells storedin nitrogen tank Cell fractionation was enabled by mixturing using the vortex For complete nucleoproteinfractionation °C roomtemperature for min By adding μL chloroformthey were shacked and mixed on hand The tubes wereincubated for min at °C room temperature thenwere centrifuged for min at °C and g Thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μL ethanol The supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μL ethanol Seven hundred microliters was taken from the obtained mixture and was transferred to the RNeasyMiniElute spin colon placed on mL collection tubeThe tubes were centrifuged for s at g at °Croom temperature Seven hundred microliters RWT buffer was added to spin colons and the colons werewashed by centrifuging at g for sThe centrifuging procedure was repeated by including μL RPE buffer twice consecutively to colons The colons placed into clean tubes with mL were dried bycentrifuging min in maximum speed The colonsplaced in mL sterile tubes were included μL distilled water by centrifuging at g in min and themiRNAs were collectThe quality control of the miRNAsThe presence and quality of the isolated miRNAs werescreened by electrophoresis at V on Agarose gelThen the purity and concentrations of the miRNAswere measured on Thermo Scientific NanoDrop spectrophotometer NanoDrop Technologies Wilmington DE USA device The miRNA purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe NanoDropin 0cTuncer Journal of Ovarian Research Page of Fig The pedigree of the family included in the study and their hierarchical cluster analysis Legend The pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by Hierarchical clustering Euclidean distance Complete Linkage BR Healthy Brother BRCA1 negative nonBRCA1 mutation carrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR Healthy Monozygotic twin BRCA1 positive BRCA1 mutation carrier BR Monozygotic twindiagnosed with ovarian cancer BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths The measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for RNA measurementswere included in the study The purity of miRNAs wereevaluated using a Bioanalyser device BioanalyserAgilent Technologies Santa Clara CA USA AgilentRNA Nano Kit Agilent Technologies Santa ClaraCA USA for confirming whether the miRNAs were appropriate and in adequate level for microarray analysisThe evaluated sample concentrations and results wereanalyzed The samples with RNA concentrations between ngμL and rRNA rate over and RNA integrity number values between and were evaluated asthe appropriate samples for array study 0cTuncer Journal of Ovarian Research Page of Microarray trial protocolMicroarray protocol was performed by preparing theSpikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides The slide scanning procedure was performedusing the Agilent Microarray Scanner Agilent Microarray Scanner with Surescan High Resolution Technology Agilent Technologies Santa Clara CA USAdevice The scanning procedure of the slides were performed on SurePrint G3 Human miRNA MicroarrayRelease 8x60K Agilent Inc Santa Clara CA platform and using the Agilent Technologies G2600D scanning protocol The analysis of the TIFF Tagged ImageFile Format extensioned files obtained after scanningprocedure was performed using the Agilent Feature Extraction v11011 programThe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedThen Bioinformatic Analysis procedure was performedData analysisRaw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values Then the miRNAs demonstrating differentexpression profile among the samples were filteredUsing the Fold change rates and independent twosample T test the possible difference between the compared groups were evaluated All evaluations were performed to enable the cutoff values as the foldchangerates FC ¥ and pvalue Hierarchical clusteranalysis was performed using the Euclidean method Fig and Complete Linkage cluster method The control ofthe experimental errors and the detection of the erroneous finding rate were identified using the HochbergmethodBioinformatic analysisTarget proteins were detected for each miRNA by usingtwo algorithms Targetscan71 httpswwwtargetscanvert_71 and MirdbV5 httpsmirdbmiRDBThe targeted genes thought for each miRNA wereconfirmed by also both algorithms and the miRNAtarget relations were also experimentally confirmed mirTarbase70httpsmirtarbasembcnctuedutwphpindexphp databaseComparison groupsIn the study miRNA analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer The miRNA data was evaluated by comparing different groups in order to investigate the effectof BRCA mutation in ovarian malignancy developmentand determine the miRNAs that can be important in theovarian cancer pathogenesis In Group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of miRNAs in the formationof ovarian cancer In Group the family members withBRCA1 mutation were compared with family memberswithout BRCA1 mutation to identify the changes ofmiRNAs expression levels according to BRCA positivityIn Group the monozygotic ovarian cancer patient withBRCA1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand BRCA positivity on miRNAs expression level InGroup all family members were compared with ovarian cancer monozygotic twin in order to find the miRNAs that might be important in the predisposition ofovarian cancer The comparison groups also showed inTable ResultsWe identified differentially expressed comparisonof miRNAs between the groups The raw data obtainedafter experimental studies were filtered before the comparisons between the groups The upregulated or downregulated miRNAs expression levels more than foldFC and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values Allthese comparisons were evaluated for ovarian cancer etiology BRCA1 mutation carriage and the ovarian cancerrisk Hierarchical cluster analysis of the expression of miRNAs represents sharp separations of upregulatedyellow from downregulated blue in Fig miRNAs total of miRNAs were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings The miRNAsmiR1273 g3p miR1305 miR1973p miR3651 miR and miR92a3p expressions were found to haveupregulated and the other miRNAs let7i 5p miR125a5p miR15b5p miR22 3p miR3135b miRTable Comparison groups and cases in the groupsCaseGroup BR1639ControlBR1447Group BR1639BR1447BR1547BR2030BR1546BR1861BR1850BR2028Group BR1639Group BR1639BR1447BR1447BR1547BR2030BR1546BR1861BR1547BR2030BR1546BR1861BR1850BR2028 0cTuncer Journal of Ovarian Research Page of Fig Hierarchical cluster analysis of the expression of miRNAs Legend BR Healthy Brother BRCA1 negative nonBRCA1 mutationcarrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR HealthyMonozygotic twin BRCA1 positive BR Monozygotic twin diagnosed with ovarian cancer BRCA1 positive BR Healthy Sister BRCA1positive BR Healthy Sister BRCA1 positive BR Healthy Sister BRCA1 positive The miRNAs that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d miR3423p miR4430 miR451a miR664b5pand miR7663p expressions were found to have downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are given in Table and Fig Different miRNAs level were compared between group in order to determine the effect of BRCA1 gene mutation Group was consisted after the comparison of theBRCA1 gene mutation carrier family members and individuals not carrying BRCA12 gene mutation accordingto miRNAs expression profiles After the comparisonsdownregulated and upregulated miRNAsrelated toBRCA1 gene mutation carrier were determined The expression of a total of miRNAs including miR4449miR46533p miR4865p miR5739 miR6165 andmiR 8743p associated with the BRCA1 gene mutationcarrying were upregulated and the expression of a totalof miRNAs including miR1263p miR320a miR320b miR320c miR320d miR320e miR3243p miR miR miR4428 miR4516 miR4741 miR miR564 miR6089 miR68695p miR68915pmiR71075p and miR78473p were found downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are shown in Table and Fig AftercomparisonDifferent miRNA levels were compared between group in order to determine the relation with ovarian cancerdevelopment and BRCA positivity Group consists ofcomparison of miRNAs of BRCA1 positive ovarian cancer patient with all other BRCA1 positive healthy individualsanddownregulated miRNAs related to mutation carriage inBRCA1 gene and epithelial ovarian cancer etiology weredetermined The expression of miRNAs includingmiR1260a miR1260b miR165p miR175p miR181b5p miR 26b5p miR4281 miR4286 miR5100miR68403p miR71145p miR7975 and miR7977were found to have upregulated and the expression of miRNAs including miR12255p miR1423p miR 26a5p miR miR29a3p miR30d5p miR3196upregulated 0cTuncer Journal of Ovarian Research Page of Table Ovarian cancer etiology related upregulated and downregulated miRNAs and target proteins in monozygotic twinsmiRNAsSequence of miRNAmiRNAStatusTarget genesFold change FCvaluesACCACUGCACUCCAGCCUGAGUpregulatedZNF138 TMEM239 BMP3UUUUCAACUCUAAUGGGAGAGAUpregulatedPTPN4 PRKAA1 PAPD7FRAT2 DEPDC1 FBXO41UUCACCACCUUCUCCACCCAGCUpregulatedCD82 PMAIP1 MTHFD1 CHECK1 AGO1 CASP10CAUAGCCCGGUCGCUGGUACAUGA UpregulatedGGCUGGUCAGAUGGGAGUGUpregulatedRACGAP1 OLA1 TEX261PTGS1 NFIC ZNF200PAGR1 IGF2BP1 CACNG8UAUUGCACUUGUCCCGGCCUGUUGAGGUAGUAGUUUGUGCUGUUUCCCUGAGACCCUUUAACCUGUGA Downregulated ERBB3 CDKN1A TP53 ERBB2 EGFR STAT3MYCSTAT3 PTEN ATM NOTCH2 CDH1 NFKB1UpregulatedDownregulated TLR4 BMP4 EIF2C1 NEUROG1 SOCS1 IGF1VEGFAmiR1273 g3pmiR1305miR1973pmiR3651miR6131miR92a3plet7i5pmiR125a5pmiR15b5pUAGCAGCACAUCAUGGUUUACAmiR223pAAGCUGCCAGUUGAAGAACUGUDownregulated BCL2 VEGFA CCND1 CCNE1 CDK1 CDK4 CDK6 E2F3MAPK1Downregulated CDKN1A WNT1 ERBB3 MYCBP HMGB1 E2F2 PTENPOTED SOD2miR3135bmiR320dmiR3423pmiR4430miR451amiR664b5pGGCUGGAGCGAGUGCAGUGGUGAAAAGCUGGGUUGAGAGGAUCUCACACAGAAAUCGCACCCGUAGGCUGGAGUGAGCGGAGAAACCGUUACCAUUACUGAGUUUGGGCUAAGGGAGAUGAUUGGGUADownregulated BIRC5 ABL2 MAPK1 MYCNDownregulated DCTN5 SYNCRIP FBXO28Downregulated GEMIN4 DNMT1 ID4 SREBF1SREBF2 BMP7Downregulated ZNF485ABL2 MAPK1 MSH5 PTENDownregulated CPNE3 RAB5A IL6R AKT1 MMP2Downregulated CD55MSN RHOBTB3 PLAG1miR7663pACUCCAGCCCCACAGCCUCAGCDownregulated COX1 MAPK1 NF2 RAD51 STK4 STK24 VEGFCFig The upregulated and downregulated miRNAs and foldchanges associated with ovarian cancer etiology in monozygotic twins 0cTuncer Journal of Ovarian Research Page of Table BRCA1 gene mutation positivity related upregulated and downregulated miRNAs and target molecules An additional tablefile shows this in more detail [see Additional file ]miRNAsSequence of miRNATarget genesmiRNAStatusFold changeFC valuesmiR4449miR46533pmiR4865pmiR5739miR6165miR8743pmiR1263pmiR320amiR320bmiR320cmiR320dmiR320emiR3243pmiR3656miR4284miR4428miR miR4741miR484miR564miR miR6869 5pmiR68915pmiR71075pmiR7847 3pCGUCCCGGGGCUGCGCGAGGCAUpregulatedZFHX3UGGAGUUAAGGGUUGCUUGGAGAUpregulatedATG2A CREBL2 MAT2A FRS2 TMED4 UBN2UCCUGUACUGAGCUGCCCCGAGGCGGAGAGAGAAUGGGGAGCCAGCAGGAGGUGAGGGGAGCUGCCCUGGCCCGAGGGACCGAUpregulatedUpregulatedUpregulatedUpregulatedOLFM4CD40ARHGAP5 IGF1R DOCK3 CADM1DLX6CD207CHIC1PPL2A PLXDC1PER1TFAP2AFADS1 AMER1LUZP1 COX6B1HDAC1 AQP3 STAT3 CDK9UCGUACCGUGAGUAAUAAUGCGDownregulatedTOM1 CRK VEGFA SOX2 TWF1 PITPNC1 IGFBP2 KRASAAAAGCUGGGUUGAGAGGGCGADownregulatedMCL1 BANP ITGB3 BMI1 NRP1 NFATC3 TRPC5AAAAGCUGGGUUGAGAGGGCAADownregulatedCDK6DCTN5SYNCRIPARF1 BCL9L ZNF600AAAAGCUGGGUUGAGAGGGUAAAAGCUGGGUUGAGAGGAAAAGCUGGGUUGAGAAGGACUGCCCCAGGUGCUGCUGGGGCGGGUGCGGGGGUGGDownregulatedDownregulatedSYNCRIPFBXO28SMARCC NPM3DCTN5 SYNCRIP FBXO28DCTN5 NPM3 ZNF275 DDX19A NCAPD2 TXNL1DownregulatedDownregulated WNT9B CREBBP DVL2 WNT2BDownregulatedMRPL12 LSP1 MNT PRDM2ZNF770 CECR1GGGCUCACAUCACCCCAUDownregulatedBCL2L11RBBP5HNRNPA1 ZNF264 TRIB3 CRTAPCAAGGAGACGGGAACAUGGAGCDownregulatedMSL1MAPRE3MYH14CASP2 CCND2 CDK14TP63GGGAGAAGGGUCGGGGCDownregulatedSTAT3M6PRGPR137CCCND2 CCNT1 CDKN1A SCOC TP53CGGGCUGUCCGGAGGGGUCGGCUDownregulatedDDX39BMAPK1 ZBTB39 HMGA1UCAGGCUCAGUCCCCUCCCGAUDownregulatedFIS1 PAGR1 ZEB1 SLC11A2SMAD2 ANAPC7 TBRG1AGGCACGGUGUCAGCAGGCDownregulatedGID4 CNBP E2F3 RCAN3 AKT2 APPL1 SLC1A2 GPR155GGAGGCCGGGGUGGGGCGGGGCGGDownregulatedNKX2 TPT1 KCTD5 BBX SGCD CDH7 CCNB1GUGAGUAGUGGCGCGCGGCGGCDownregulatedTUBB2AMAPK1NRBF2 WEE1HMGA2 MAPK1 STAG2UAAGGAGGGGGAUGAGGGGDownregulatedCHD4 CD207 DDX6 CHRDL1CCND2 TP63UCGGCCUGGGGAGGAGGAAGGGDownregulatedVAV3 CASP16 CCND1 CASP16 MAPK14CGUGGAGGACGAGGAGGAGGCDownregulatedHAVCR1 POTED DNAJC10 SOD2 M6PR CDK19miR3423p miR3665 miR3960 miR4466 miR4530miR46873p miR47875p miR4943p miR50015pmiR50065p miR5787 miR6068 miR6087 miR miR6090 miR6124 miR6125 miR638 miR65105p miR68005p miR7704 miR8063 and miR were found to have downregulated After bioinformatic analysis a total of upregulated and downregulated statistically significant miRNAs and the targetmolecules are given in Table and Fig For identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group The upregulated ordownregulated miRNAs in association with the epithelialovarian cancer risk were identified after the comparisonThe expression of the miRNAs consisting of let7a5plet7b5p miR181a5p miR1973p miR215pmiR2233p miR23a3p miR27a3p miR36533pmiR4255p miR572 miR5745p miR6127 and miR were detected to be upregulated The expression ofthe miRNAs consisting of let7i5p miR 125a5pmiR15b5p miR1505p miR22 3p miR3283pmiR4430 miR451a miR46975p miR664b5p andmiR7663p were detected to have downregulated Atotal of upregulated and downregulated statisticallysignificant miRNAs and the target molecules are givenin Table and Fig DiscussionWomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients Although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 CA125 CA15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0cTuncer Journal of Ovarian Research Page of Fig The upregulated and downregulated miRNAs and foldchanges associated with BRCA1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations [] Therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary The genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism [] The changes in genesthat result in the occurrence of tumors such as miRNAexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis We aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the miRNAs in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the miRNA molecules of the other healthy members in the family Thus that may cause less bias thanthe controls to be selected from the population Ninetynine different miRNA molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals Seventeen differentmiRNAs were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study The association between out of miRNAs and ovarian carcinoma is beingreported for the first time in this study Due to the highnumber of newly detected miRNAs in our study the discussion and comparison were only made between thecandidate miRNAs Although miR1973p miR1305miR6131 miR3651 miR3135b miR4430 miR664b5p and miR7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancersWang found the elevated level of miR1973pinthe same way as we do The upregulated miR1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyResearchers reported that LINC00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the miR1973p expression Similar resultswere detected in thyroid cancer in the study of Liu et al[ ] Jin reported that increased expressionlevel of miR1305 caused pluripotent stem cells to accelerate the cell cycle G1S transfer in addition to causingthe cellular differentiation with the increased miR1305expression [] The expression levels ofreducedmiRNA125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature Langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer [] The miRNA125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of Qin in human cervical carcinomas [] and miR125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0cTuncer Journal of Ovarian Research Page of Table BRCA1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated miRNAs targetmolecules An additional table file shows this in more detail [see Additional file ]miRNAsSequence of miRNAmiRNAStatusTarget genesAUCCCACCUCUGCCACCAAUCCCACCACUGCCACCAUUAGCAGCACGUAAAUAUUGGCGUpregulatedUpregulatedUpregulatedCAAAGUGCUUACAGUGCAGGUAGUpregulatedPSAT1UNC13A RPS27 BRD7SFRP1 DKK2 SMAD4 PSAT1UNC13A RPS27CCNE1 ARL2 BCL2 HMGA1 CDK6 CCND1VEGFA RECK PRDM4TGFBR2 PTEN CDKN1A BCL2L11E2F1TP53STAT3AACAUUCAUUGCUGUCGGUGGGUUpregulatedTCL1A TIMP3 PLAG1 BCL2RNF2VSNL1 ATMFold changeFC valuesmiR1260amiR1260bmiR16 5pmiR175pmiR181b5pmiR26b5pmiR4281miR4286miR5100UUCAAGUAAUUCAGGAUAGGUUpregulatedGGGUCCCGGGGAGGGGGGACCCCACUCCUGGUACCUpregulatedUpregulatedUUCAGAUCCCAGCGGUGCCUCUUpregulatedPTGS2 EPHA2 CHORDC1 EZH2CCNE1ABCA1 GATA4NCDN CDKN1A BCL3LDLR ZNF354B NSD1 RABGAP1TAOK1 MKNK2COX10 DEK KCNN3 RAB11FIP1DYNLT1 NOTCH2SLFN12L CTC1 GXYLT2GDE1FADS1PER1 ATG9AmiR68403pGCCCAGGACUUUGUGCGGGGUGUpregulatedmiR71145pUCUGUGGAGUGGGGUGCCUGUUpregulatedM6PR HNRNPUL1 SHMT1 ZNF529ACVR2B PAICS TAF8miR7975miR7977AUCCUAGUCACGGCACCAUUCCCAGCCAACGCACCAUpregulatedUpregulatedmiR12255pGUGGGUACGGCCCAGUGGGGGGDownregulatedKBTBD8GULP1 CASZ1 RAD51HSPA1B ZNF703 TMEM185BSF3B3COX6B1 CCDC9 CDH7ORC4 ODF2L MTRNR2L7PSMG2 MTRNR2L3miR1423pmiR26a5pmiR2861miR29a3pmiR30d5pmiR3196miR 3423pmiR3665miR3960miR4466miR4530miR46873pmiR47875pmiR4943pmiR50015pmiR50065pmiR miR6068miR6087miR6088miR6090miR6124UGUAGUGUUUCCUACUUUAUGGADownregulatedARNTLTGFBR1 RAC1 ROCK2 CCNT2 TAB2 PTPN23UUCAAGUAAUCCAGGAUAGGCUDownregulatedEZH2RB1ADAM17 HMGA2CCND2 CPEB3 DNMT3BGGGGCCUGGCGGUGGGCGGUAGCACCAUCUGAAAUCGGUUAUGUAAACAUCCCCGAC | 2 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l zhang l wang l zhan y hedyotis diï¬usawilld inhibits colorectal cancer growth in vivo via inhibition of stat3 signalingpathway int j mol sci doi 103390ijms13056117chen q shu c laurence a d chen y peng b g zhen z j eï¬ect of huaier granule on recurrence after curative resection of hcca multicentre randomised clinical trial gut doi 101016s0618chen w zheng r baade p d zhang s zeng h bray f cancerstatistics in china ca cancer j clin doi 103322caacchoi j b kim j h lee h pak j n shim b s kim s h reactive oxygen species and p53 mediated activation of p38 and caspases iscritically involved in kaempferol induced apoptosis in colorectal cancer cellsj agric food chem doi 101021acsjafc8b02656dai s liu j sun x and wang n ganoderma lucidum inhibitsproliferation of human ovarian cancer cells by suppressing vegf expressionand upregulating the expression of connexin bmc complement alternmed doi darband s g kaviani m youseï¬ b sadighparvar s pakdel f g attari j s quercetin a functional dietary ï¬avonoid with potential chemopreventive properties in colorectal cancer j cell physiol doi 101002jcp26595du z x jia x y and lin k colorectal cancer mortality characteristicsand predictions in china asian pac j cancer prev doi 107314apjcp201516177991gu z f zhang z t wang j y and xu b b icariin exerts inhibitoryeï¬ects on the growth and metastasis of kyse70 human esophageal carcinomacells via pi3kakt and stat3 pathways environ toxicol pharmacol doi 101016jetap201706004han m song y and zhang x quercetin suppresses the migrationand invasion in human colon cancer caco2 cells through regulating tolllikereceptor 4nuclear factorkappa b pathway pharmacogn mag s237s244doi huang c and hu g shikonin suppresses proliferation and inducesapoptosis in endometrioid endometrial cancer cells via modulating mir106bptenaktmtor signaling pathway biosci rep 38bsr20171546 doi101042bsr20171546jung y jerng u and lee s a systematic review of anticancer eï¬ectsof radix astragali chin j integr med doi 101007s116550152324xkerwin s m chembiooï¬ce ultra suite j am chem soc doi 101021ja1005306kuo y t liao h h chiang j h wu m y chen b c chang c m complementary chinese herbal medicine therapy improves survivalof patients with pancreatic cancer in taiwan a nationwide populationbasedcohort study integr cancer ther doi lee y w chen t l shih y r v tsai c l chang c c liang h h adjunctive traditional chinese medicine therapy improves survival inpatients with advanced breast cancer a populationbased study cancer doi 101002cncr28579li c yang x chen c cai s and hu j isorhamnetin suppresses coloncancer cell growth through the pi3kaktmtor pathway mol med rep doi 103892mmr20141886li x chen c dai y huang c han q jing l cinobufaginsuppresses colorectal cancer angiogenesis by disrupting the endothelialmammalian target of rapamycinhypoxiainducible factor 1α axis cancer sci doi 101111cas13988liu h liu h zhou z parise r a chu e and schmitz j c herbalformula huang qin ge gen tang enhances 5ï¬uorouracil 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" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | 0 |
"as metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors aspartate hydroxylase asph is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion asph also promotes tumor growth by stimulation ofangiogenesis and immunosuppression these effects are mainly achieved via the activation of notch and srcsignaling pathways asph expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact therefore small molecule inhibitors of asph enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models asph can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation cd and cd4 t cells in mice the pan3011 vaccine against asph has already been tested in aphase clinical trial in patients with prostate cancer in summary asph is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapykeywords asph small molecule inhibitor metastasis immunotherapy cancer is a multifactorial disease with an approximate million fatalities in worldwide it is the secondleading cause of death the complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential in thisapproachesthat correspondence smahelmnaturcunicz1department of genetics and microbiology faculty of science charlesuniversity biocev vestec czech republicfull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsaspartate hydroxylase asph has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] asph belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] the overexpressed asph is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the cterminal regionto the extracellular environment where it is accessible toantibody binding recently molecular targeted therapyhas been developed against this target using small molecule inhibitors smi that can inhibit the catalytic site the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ckanwal of experimental clinical cancer research page of in the cterminal region moreover as antigenic epitopes that reside on the asph protein can efficientlystimulate cluster of differentiation cd and cd8 tcell responses unique to tumor cells harboring asphthis enzyme can be used as a tumor associated antigentaa in immunotherapy [ ]thedioxygenasesstructure of the asph gene and isoformsasph is a type ii transmembrane protein of approxito the family of αmately kda that belongsketoglutaratedependenthydroxylated products of asph hydroxylation were firstdetected in blood coagulation proteins [] asphwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbegflike domains of various proteins fig thereafter the human asph gene wascloned and characterized this gene spanning base pairs long region of genomic dna and containing exons is located at the position q123 of thehuman chromosome the asph sequence is highlyconserved in mammalian evolution the sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species the whole asph protein consists of five domainsan nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a cterminal catalytic domain tissue specific transcription is directed from two putative promoters p1 and p2which differ in their regulation sequences [ ] whilethe transcription from the p1 promoter was observed inmost human tissues the p2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor mef2 particularly in muscle tissues the asph gene undergoes extensive alternative splicingresulting in four protein isoforms ie asph humbugjunctate and junctin [ ] these proteins vary in thecterminal region which affects their function [ ]the two longest asph transcript variantsthat aretranscribed from the p1 and p2 promoters and differ inthe length of the ²untranslated region encode the fulllength asph protein this protein contains the catalyticcterminal domain that catalyzes the posttranslationalhydroxylation in the cbegflike domains of numerousproteins supplementary fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] thetruncated isoforms humbug junctate and junctin sharethe nterminal part with the asph protein but lackcatalytic function they are involved in calcium homeostasis humbug has a potential role in cell adhesionand calcium flux and similar to asph its overexpressionhas been correlated with aggressive tumorcell behavior reticulummembranebound protein that is known for its functionin the regulation of the intracellular ca2 concentrationjunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicjunctateisalocalization in cells tissue distribution andexpression regulationasph is predominantly a cellsurface protein that isalso localized in the endoplasmic and sarcoplasmicreticulum furthermore a recent study identifiedmitochondriallocalization of asph in hepatocellularcarcinoma hcc in that study asph overexpressioncorrelated with an instability of mitochondrial dna andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in hcc asph is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands and has a potential role in placentalimplantation and fetal growth on the contrary theasph expression in normal adult tissues is relativelylow or negligible however asph expression is inappropriately activated during oncogenesis when asph is required for generation of malignant and metastaticphenotypes the elevated expression of asph at bothfig asph catalytic reaction aspartyl and asparaginyl residues in cbegflike domains are hydroxylated 0ckanwal of experimental clinical cancer research page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer table the first study that demonstrated thesignificantly higher expression of both asph mrnaand protein in hcc and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by lavaissiere subsequently they verified the role of upregulated asph protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the nih3 t3 cell line and animal models the level of asph also correlated with cell motility andinvasiveness in in vitro experiments [ ] in thestudy by maeda the overexpression of theasph protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung and colon carcinomas and glioblastoma multiforme recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples asph has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes in glioblastoma the prognostic significance ofasph was suggested by profiling of alternative mrnasplicing asph gene expression is upregulated via wntcatenin and insulininsulinlike growth factor igf1insulin receptor substrate irs1 signaling [ ]through extracellular signalregulated kinaseerkmitogenactivated protein kinase mapk andphosphatidylinositol3kinaseprotein kinase b pi3kakt pathways fig for review see ref insulinigf1irs1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorstable summary of the studies which have identified the elevated asph expression in human tumor tissuesstudypositive cases of studied samples nntumor tissuesdetectionmethodihcantibody recognized region ofasph proteinfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminus or 15c7 abcatalytic domainfb50 ab nterminusmab g3 hybridomapolyclonalfb50 mab nterminusihcihcihcihcrtqpcrihcrtqpcrihcihcihcrtqpcrihcfb50 ab nterminuslavaissiere hepatocellularcholangiocarcinomabreastcolonpalumbo pancreatic adenocarcinomasepe primitive neuroectodermalmedulloblastoma neuroblastomamaeda cantarini cholangiocarcinomahepatocellularmonte hepatocellularyang wang dong tang lin types of tumor tissuesahepatocellularpancreatic cancerhepatocellularbreast 75fold higher level of mrnacompared to normal tissue 7fold higher level of mrnacompared to normal tissuepancreatic ductal adenocarcinomaogawa aliver kidney breast cervical ovarian fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomafb50 ab nterminusihc 0ckanwal of experimental clinical cancer research page of fig regulation of asph expression and asph involvement in signaling pathways the expression of the asph protein can be regulated atseveral levels the asph gene can be amplified in tumor cells and its transcription activated by inigf1 and wnt catenin pathways or inducedby hypoxia at the posttranscriptional level mir200a and mir135a can downregulate asph expression stability of the asph protein can bereduced by phosphorylation with gsk3 conversely asph can enhance gsk3 activity by inhibition of its phosphorylation with akt and p38kinases asph also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the notch receptor and ligands ex jag and interaction with prb vimentin and adams finallyinactivation of nk cells by asph has been demonstrated green arrow activation signal red bar inhibitory signal the catenindependent wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies upon aberrant activation of wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus where an interaction between catenin and tcellfactorlymphoidenhancerbinding factor tcflef proteins forms atranscriptional regulatory complex which enhances theexpression of wnt target genes including irs1 asph was proposed as a common link between wntcatenin and insulinigf1irs1 pathways and downstream signaling the regulation of asph gene expression in tumorsmight also be affected by a copy number variation inthe study by kadota the asph gene locus hasbeen identified as one of the dna regions with focalamplification in primary breast cancer in colorectal cancer asph gain or amplification was found in ofsamples next a suppressant role of the micrornamir200a in posttranscription regulation of the asphexpression in hepatoma cells has been found mir200a belongs to mir200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref similarly mir135a has beenshown to suppress asph in endometrial cancer moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 gsk3casein kinase ck2 protein kinase a pka and protein kinase c pkc on asph several studies demonstrated that phosphorylation can regulate the asphprotein expression [ ] inhibition of the gsk3activity did not modify mrna expression but increased 0ckanwal of experimental clinical cancer research page of the asph protein level direct phosphorylation ofasph by gsk3 probably decreases asph stability andthus reduces cell mobility asph protein expressionwas also increased by inhibitors of pka pkc and ck2 mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofasph phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] thereforeasph phosphorylation probably regulates the functionof this protein by various mechanismsasph expression can also be regulated by hypoxia andoxidative stress in human neuronal cells this effect wasmediated by hypoxia inducible factor alpha hif1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain phd proteins and factorinhibiting hif fih are inactivated consequently thehif1 heterodimer made up of subunits hif1α andhif1 functions as a transcription factor likely enhancing asph expression by binding to hypoxiaresponsiveelements in hypoxic regions of glioblastoma bothhif1α and asph were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of asph in mesenchymal transition brewitz showed reducedasph hydroxylation activity at low oxygen concentrations and suggested an asph role in oxygen hypoxiasensing asph upregulation induced by hypoxia couldcompensate for reduced enzymatic activity moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon asphoverexpression which was reversed by silencing asphexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion asph protein interactions and signaling pathwaysthe asph hydroxylation consensus sequence is confined within cbegflike domains that are found in proteins of diverse function including notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3axl family ofreceptor tyrosine kinases the notch signaling cascade is a remarkably conservedpathway notch proteins notch1 notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells dysregulation of the notch pathway is directly linkedto cancer vascular disorders and congenital defects in mammals notch signaling activated by binding ofone of two families of canonical notch ligandsjaggedjag1 and jag2 and delta like dll1 dll3 and dll4leads to the generation of the cleaved notch intracellulardomain nicd fragment and its nuclear translocation inthe nucleus the nicd fragment interacts with the dnabinding complex csl cbf1rbpjκ suh lag1 thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bhlh transcription factor hes1 heswith yrpw motif hey1 cd44 epithelial cell adhesionmolecule epcam cmyc protooncogene matrix metallopeptidase mmp29 cyclin d1 cyclooxygenase vascular endothelial growth factor vegf and proliferatingcell nuclear antigen pcna [ ]upregulation of asph results in enzymatic modification of the cbegflike repeats in the notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof notch signaling [ ] furthermore the interactionof asph with a disintegrin and metallopeptidase domainadam stabilizes this complex and enhances thes2 cleavage ofthe notch receptors and subsequentnicd fragment release the activation of the targetgenes in malignant cells increases cell proliferation migration and invasion through the epithelialtomesenchymal transition emt that is probably upregulated by the interaction of asph with vimentin consequentlythis activation supports tumor growthand metastasis the asphnotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]the src kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis asph has been identified as an src pathway activatoroverexpressed asph directly interacts with adam12 and strengthens the src activation by these proteinswhich promotes mmpmediated extracellular matrixdegradation and tumor invasiveness asph can also contribute to malignant phenotype ofcells by interaction with other proteins iwagami revealed the interaction of asph with gsk3 that prevents gsk3 inactivation by phosphorylation with upstream kinases this mechanism was confirmed ina castrationresistant prostate cancer model gsk3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination some types of tumors aresensitive to gsk3 inhibitors recently huang elucidated a direct binding of asph with retinoblastoma protein prb leading to prb phosphorylation they also showed that this effect was mediated byincreased binding of cyclindependent kinase cdk cdk4 and cyclins d1 and e with prb and wasdependentasasph enzymaticonactivity 0ckanwal of experimental clinical cancer research page of phosphorylation of prb inactivates its tumorsuppressorfunction asph can contribute to the progression of cellcycle via interaction with prbeffect of asph on an immune systemtumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system in theseprocesses various mechanisms of both innate and adaptive immunity are included immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties as potential targets of asph hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when asph is overexpressed on cancercells indeed such effect was demonstrated for humannatural killer nk cells by using recombinant asphwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively antibodies against asph inhibited these effectsinteraction of asph with other immune cells has notbeen studied however we suppose possible influence ofasph on different tumorinfiltrating cells this assumption comes from the involvement of notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells forinstance notch activation contributed to stimulation ofproinflammatoryantitumorigenic m1 polarization inboth bone marrowderived primary macrophages and tumorassociated macrophages whennotch signaling was abrogated protumorigenic m2polarization was induced even by stimulators of m1polarization mir125a has been identified as adownstream mediator of notch signaling in macrophages similarly the notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of cd4 and cd8 t cells different notch receptors and their interactionwith different ligands contribute to these processes moreover noncanonical notch signaling is implicatedin regulation of immune cells while activation ofnotch signaling in some cells eg t helper cells cytotoxic cd8 t cells and m1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory t cellsit leads to immunosuppression thus immunostimulatory effect of notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity therapeutics affecting notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedasph as a therapeutic targetoncogenic abilities of asph have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with asphoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma pancreatic cancer [ ] glioma breast carcinoma castrationresistant prostatecancer and colorectal cancer in studies analyzingasph function various approaches were utilized to revealsignaling pathways affected by asph particularly asphexpression was diminished by using small interfering rnas[ ] short hairpin rnas [ ] or thecrisprcas9 system [ ] the importance of asphenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by smis [ ] in vitro assays showed asph involvement in cell proliferation migration and invasion cellularalterations included emtinhibition of apoptosis andstemness acquisition tumor growth and invasivenesscould further be supported by asphinduced extracellularmatrix degradation angiogenesis and transendothelial migration notch and src signaling are probably major pathways influenced by asph fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models thus these studies also demonstrated thatasph is a suitable target for cancer treatment especially bysmis or immunotherapysmall molecule inhibitorssmis of asph fig have been developed and used totest the role of asph in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] a small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches not only can these inhibitors inhibit the catalytic activity of asph unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit asphcatalytic activity in the endoplasmic reticulum differentcancers have different asph expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed the first asph smis published were the tetronimidesmoi500 and moi1100 tetronimides were originallysynthesized in by dahn and are redoxactivemimics of ascorbic acid and 2oxoglutarate moi500 isa mixed inhibitor that inhibits both asph and the fatmass and obesity protein fto and is not onlyorally bioavailable but also can penetrate the blood 0ckanwal of experimental clinical cancer research page of fig small molecule inhibitors of asphthereand kinasesbrain barrier moi1100 is a more potent inhibitor ofasph and is also more selective despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for moi1100 enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup as in moi1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inmoi1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters moi1182 isreported to have the ability to suppress invasive activityat a concentration of nm smis of asph have acharacteristic in vitro concentration dependent profilewhere the activity of the smi plateaus at value around viability emphasizing the noncytotoxic properties of this class of inhibitorsnatural products and inhibitors of other enzymes thathave been repurposed as asph inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthinecn2019101414327 and guaianolides related to nortrilobolidecn2019104575886cn2019101414219cn2019101414187 0ckanwal of experimental clinical cancer research page of sesquiterpene with complex anticancerbosutinib is a wellknown inhibitor of bcrabl and srctyrosine kinases approved for the treatment of chronic myelogenous leukemia cepharanthine is a natural productactivityincluding ampactivated protein kinase ampk activationand nuclear factor kappa b nfκb inhibition nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium atpase serca inhibition recently a family of potent pyridine dicarboxylates have alsobeen published utilizing a mass spectrometrybasedinhibition assay these compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate the synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include phd2 fih and lysinespecific demethylase 4ekdm4e in addition to asph with varying degrees of selectivity however while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface asph inhibitors that may nothave cell penetrating activity immunity as a target of humoralimmunotherapyasph can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression since asph is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityasph on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity adcc complement dependent cytotoxicity cdcor antibodydependent cellular phagocytosis adcp when the asph antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen hla class ior class ii molecules and recognized by cd8 or cd4 tlymphocytes respectively that can be stimulated byimmunization breaking tolerance to selfantigens induction of asphspecific cd4 and cd8 t cells wasexamined in blood samples of hcc patients using synthetic peptides derived from asph after prediction of hlaclass i and hla class iirestricted epitopes it has beenfound that asph is a highly immunogenic protein that activates both types of analyzed t cells thus efficientantitumor reactions could be stimulated by immunizationthe first vaccine against asph was based on matureddendritic cells dc loaded with the asph protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma this study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis bothcd8 and cd4 cells contributed to an antitumor effectinduced in a mouse model of hcc by immunizationwith asphloaded dcs the next antiasph vaccine was based on a bacteriophage lambda display system the viral capsid proteingpd was fused with the n or cterminus of asph andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models the vaccine pan3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer this study demonstrated safety and immunogenicity of pan3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen psaor psa doubling time asphspecific immune responseswere mediated by both antibodies and t lymphocytesas asph is a type ii transmembrane protein its cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes development ofasphspecific antibodies has been described in several s [] the human igg1 pan622 recognizesthe catalytic domain of asph this antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells in the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results mouseigg1 monoclonal antibody binding to the cterminalasph domain mediated adcc by human nk cells recently a secondgeneration antibody approach hasbeen disclosed the prepared antibody binds to the extreme cterminus of asph us that is involved in specific substrate recognition thereforethis antibody has direct asph inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activitypan622 wasbioimagingusedforconclusionsasph is an important enzyme in malignant transformationof cells it stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment two main pathways notch and srcthrough which asph promotes the tumor growth havebeen identified it has also been shown that asph expression is induced by some growth factors and hypoxia and isregulated at various levels the overexpression of asphand its downstream targets has been detected in numeroushuman malignancies since asph is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsfig small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0ckanwal of experimental clinical cancer research page of fig asph as a therapeutic target asph expression is upregulated by growth factors and hypoxia its enzymatic activity can be inhibited bysmis or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionconsequently tumor growth and metastasis are also reducedmodels but they have not yet advanced into clinical trialsadditionally as asph was identified as a tumorassociatedantigen immunotherapy approaches vaccines and monoclonal antibodies were tested with promising results in preclinical experiments and results of pha | 0 |
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir15a5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a wellknown oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir15a5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at Ëc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at Ëc with co2reverse transcriptionquantitative pcr rtqpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at Ëc for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at Ëc for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions Ëc for min followed by cycles at Ëc for sec and Ëc for sec and a final extension at Ëc for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir15a5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at Ëc for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 siyap1 and the negative control targeting a nonspecific sequence siscramble were provided by thermo fisher scientific inc siha and c33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of siyap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck solution was added to each well and cultured for h at Ëc the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin vfitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdualluciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h posttransfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at Ëc overnight each membrane was probed with primary antibodies against yap1 cat no and βactin cat no at Ëc overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir15a5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir15a5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir15a5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir15a5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir15a5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir15a5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir15a5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir15a5p inhibits the invasion and migration of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c33a cells were significantly inhibited by mir15a5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir15a5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir15a5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcriptionquantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase activity was detected by a commercial caspase activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir15a5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdnayap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir15a5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells cotransfected with firefly luciferase constructs containing the yap1 wt or mut 'utrs and mir15a5p mimics mimics nc mir15a5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir15a5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir15a5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir15a5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s | 0 |
RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomens Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the seed and soil theory was ï¬rst proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells seeds leave their primary site of origin and spreador metastasize the microenvironment soil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve assoil for tumor development and might also serve as a seed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on theï¬eld of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identiï¬ed receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieï¬yintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and eï¬cienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentiï¬ed as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong TDC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deï¬ciency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKLRANK interactionsand blocking bone resorption The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identiï¬edas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a speciï¬c fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The eï¬cacy of Denosumab has been conï¬rmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspeciï¬c Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeï¬cient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyers plaque structures Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeï¬cient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inï¬ammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL speciï¬cally in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinï¬ammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a signiï¬cant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespeciï¬c deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspeciï¬c rankldeï¬cient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deï¬ciency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a speciï¬c antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to speciï¬cally eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the ï¬rst to reportthat during pregnancy RANKL deï¬ciency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκBcyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and ampliï¬edproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also signiï¬cantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identiï¬ed SNPs which were signiï¬cantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in signiï¬cantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Ampliï¬cation of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also signiï¬cantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which speciï¬cally inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will beneï¬t notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was signiï¬cantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no signiï¬cant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinï¬ltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand signiï¬cantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports ï¬ndings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thismicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas ï¬broblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a vicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs ï¬broblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer prostate cancer and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANKs cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinï¬ltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are signiï¬cantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a signiï¬cantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassigniï¬cantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs Moreover the beneï¬t of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the vicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a signiï¬cantimprovement in overall survival In these patients nostatistically signiï¬cant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab signiï¬cantly improved progressionfree survivalPFS Whether this survival beneï¬t is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the ï¬rst clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a signiï¬cant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ··] p A median followup of months showeda signiï¬cant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI Cox p These data suggest that adjuvant Denosumabcan signiï¬cantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratiï¬cations eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial conï¬rmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso signiï¬cantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment signiï¬cantly reduced the time to bonemetastasis at the site of ï¬rst occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe ï¬eld of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the bodys own immunesystem to seek out speciï¬cally target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as | 2 |
"(B) E-cadherin was membrane positive and vimentin was negative in p120ctn membrane-positive lung cancer cells. (C) E-cadherin was negative and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells. .0088064.t001 Correlation between E-cadherin vimentin and lymph node metastasis and p120ctn. p120ctn N membrane cytolymph/nucleolus X2 p E-cadherin negative 56 9 47 30.166 <0.01 positive 22 18 4 Vimentin negative 53 23 30 5.633 0.022 positive 25 4 21 Lymph node metastasis No 41 19 22 5.251 0.032 Yes 37 8 29 Localization of p120ctn is consistent with E-cadherin in lung cancer cells We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn (A). Although the protein expression levels of p120ctn were not related to E-cadherin the localization (membrane or cytoplasm) of p120ctn was always consistent with that of E-cadherin. We then screened cells expressing high levels of p120ctn and E-cadherin in the membrane (H460 cells) or cytoplasm (SPC cells) as well as those expressing low levels of p120ctn and E-cadherin in the membrane (H4299 cells) or cytoplasm (LK2 cells) for further study (B). .0088064.g002 Expression and localization of p120ctn and E-cadherin in H460 SPC H1299 and LK2 cells. (A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells whereas they both were confined to the cytoplasm in SPC and LK2 cells. Different functions of p120ctn isoform 1A in EMT are dependent on E-cadherin subcellular localization Knockdown of endogenous p120ctn isoform 1A by siRNA-p120ctn-1A resulted in decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells (A). However knockdown of endogenous p120ctn-1A by siRNA-p120ctn-1A showed opposite results in SPC cells where we found increased E-cadherin expression and decreased N-cadherin snail and vimentin expression (B). In comparison with the control the ablation of p120ctn isoform 1A also enhanced the H460 cells invasiveness (17.33±1.25 vs. 36.33±1.70 P<0.01) (C) whereas reduced the SPC cells invasiveness (23.0±0.82 vs. 13.0±0.82 P<0.01) (D). These results revealed that the p120ctn isoform 1A plays a different role in EMT and cell invasiveness in different E-cadherin subcellular locations. .0088064.g003 p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells. (A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01). Inhibitory function of p120ctn isoform 3A on EMT is not affected by differences in E-cadherin subcellular localization To verify whether p120ctn isoforms 1A and 3A play different roles in regulating EMT their expression plasmids were transiently transfected into lung cancer cells with low expression of p120ctn (H1299 with membrane E-cadherin expression and LK2 with cytoplasmic E-cadherin expression). The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin vimentin and snail expression (A); on the contrary the decreased E-cadherin expression and increased N-cadherin vimentin and snail expression were observed in LK2 cells (B). Overexpression of the p120ctn isoform 1A also reduced the H1299 cell invasiveness (52.0±2.65 vs. 33.33±2.64 P<0.01) (C) while enhanced the LK2 cell invasiveness (18.0±0.82 vs. 39.66±2.05 P<0.01) (D).. Overexpression of p120ctn isoform 3A led to increased E-cadherin expression decreased N-cadherin vimentin and snail expression (A 4B) and reduced cell invasiveness (52.0±2.65 vs. 29.66±1.53 P<0.01; 18.0±0.82 vs. 8.33±expression 0.47 P<0.01) (C 4D) in both of these cell lines. These results further confirmed that the p120ctn isoform 1A had a different effect on EMT depending on the subcellular localization of E-cadherin. They also revealed that the p120ctn isoform 3A maintained an inhibitory role in the EMT of lung cancer cells whether E-cadherin was localized to the membrane or the cytoplasm. .0088064.g004 p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization. (A B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin vimentin and snail expression. (C D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid. Discussion The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin vimentin and other mesenchymal biomarkers [3] [4] [5] [6] [7]. As an important factor for stabilizing E-cadherin p120ctn plays a role in inhibiting or promoting tumor cell proliferation and invasion that is dependent on whether E-cadherin is expressed or not [16] [17]. Furthermore p120ctn isoforms 1A and 3A have shown different effects on E-cadherin expression and tumor cell invasiveness which are based on differences in the localization of E-cadherin [18]. These results strongly suggest that p120ctn most likely regulates the EMT of tumor cells by affecting E-cadherin expression and that p120ctn isoforms 1A and 3A play different roles in EMT expressing E-cadherin in different subcellular locations. We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies [13] [14] they further suggested that p120ctn likely affects the EMT by influencing the expression of E-cadherin and vimentin and thereby the cell invasion and metastasis in non-small cell lung cancer (NSCLC). To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A [24] [25] we could not design an interference sequence specifically for p120ctn isoform 3A. Therefore we had to further study the impact of the two isoforms on EMT and cell invasiveness in lung cancer cells with different E-cadherin locations specifically by knocking down p120ctn isoform 1A in H460 and SPC cells with high p120ctn expression and transfecting cDNA plasmids for exogenous p120ctn isoforms 1A and 3A into H1299 and LK2 cells with low expression of p120ctn. Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor p120ctn isoform 1A which was consistent with previous studies [20] [26]. Decreased E-cadherin expression and disrupted cell-cell adhesion may induce EMT [27] [28] [43] [44] which results in increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. On the other hand overexpressed p120ctn isoforms 1A and 3A was shown to bind E-cadherin located on the membrane proactively in tumor cells [29] and then inhibit the degradation of E-cadherin and stabilize its expression contributing to the formation of effective epithelial cell adhesion complexes [30] [31] [32]. " | 1 |
"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Deï¬ning advance care planning for adults Aconsensus deï¬nition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneï¬ciaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conï¬ict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" | 2 |
the diagnostic platform utilizing the detection of biomarkers in various body ï¬uids called liquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes eï¬cient and reliable markerswithin the body ï¬uids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspeciï¬c to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue speciï¬c and reï¬ect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the ï¬eldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reï¬ect the pattern speciï¬c for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentiï¬cation of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artiï¬cialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas speciï¬c toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modiï¬cations of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and speciï¬city in liquid biopsybasedmethods for disease detection and classiï¬cationthe patientthe5methylcytosine5mc modiï¬cationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespeciï¬c methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor deï¬nitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeproï¬le of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodiï¬ed at amino acid residues located on their n andcterminal tails these modiï¬cations act as epigenetic marksthat can speciï¬cally distinguish diseaserelated ccfdnas in bloodsamples various types of histone modiï¬cations are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspeciï¬c pcr methylationsensitivehighresolution melting and pyrosequencing garcagimnez dna methylation speciï¬c to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modiï¬ers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identiï¬ed the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modiï¬cations on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodiï¬cations in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulï¬te sequencing rrbs meissner is an eï¬cient method for absolute quantiï¬cation ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulï¬te sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identiï¬ed in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized proï¬lingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodiï¬cations the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantiï¬cation of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body ï¬uids are of clinical signiï¬cance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the bloodbrain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 speciï¬cfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patients serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this ï¬eld to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a liquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations speciï¬c to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modiï¬cations patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identiï¬ed as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic ï¬rmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspeciï¬c mirnas zhang mir was found to be a signiï¬cant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was signiï¬cantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was signiï¬cantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentiï¬cation ofrapidly developingsigniï¬cant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticeï¬cacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnampliï¬ed neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be signiï¬cantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularproï¬ling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound beneï¬ts recentlynovel immunological approaches produced insightful responsessnyder in addition the identiï¬cation of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually beneï¬t the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises ofthe maternal ccfdna wang andcan be eï¬ciently detected atthe ï¬fth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy downs syndrome edwardssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands eï¬cient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modiï¬cations are speciï¬c to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the ï¬rst reported use of fetalderivedepigenetic marker in maternal body ï¬uids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspeciï¬c methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspeciï¬c dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the ï¬rst such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been conï¬rmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulï¬te treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna ï¬nds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microï¬uidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identiï¬cation of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identiï¬ed manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and | 0 |
peripheral serum metabolomic profiles inform central cognitive impairmentJingye Wang1 Runmin Wei12 Guoxiang Xie1 Matthias Arnold Alexandra KueiderPaisley Gregory Louie Siamak Mahmoudian Dehkordi3 colette Blach5 Rebecca Baillie Xianlin Han7 Philip L De Jager David A Bennett9 Rima KaddurahDaouk Wei Jia The incidence of Alzheimers disease AD increases with age and is becoming a significant cause of worldwide morbidity and mortality However the metabolic perturbation behind the onset of AD remains unclear In this study we performed metabolite profiling in both brain n and matching serum samples n to identify differentially expressed metabolites and metabolic pathways associated with neuropathology and cognitive performance and to identify individuals at high risk of developing cognitive impairment The abundances of metabolites glycolithocholate GLCA petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and the deoxycholatecholate DCACA ratio along with the dysregulation scores of metabolic pathways primary bile acid biosynthesis fatty acid biosynthesis and biosynthesis of unsaturated fatty acids showed significant differences across both brain and serum diagnostic groups Pvalue Significant associations were observed between the levels of differential metabolitespathways and cognitive performance neurofibrillary tangles and neuritic plaque burden Metabolites abundances and personalized metabolic pathways scores were used to derive machine learning models respectively that could be used to differentiate cognitively impaired persons from those without cognitive impairment median area under the receiver operating characteristic curve AUC for the metabolite level model median AUC for the pathway level model Utilizing these two models on the entire baseline control group we identified those who experienced cognitive decline in the later years AUC sensitivity specificity for the metabolite level model AUC sensitivity specificity for the pathway level model and demonstrated their preAD onset prediction potentials Our study provides a proofofconcept that it is possible to discriminate antecedent cognitive impairment in older adults before the onset of overt clinical symptoms using metabolomics Our findings if validated in future studies could enable the earlier detection and intervention of cognitive impairment that may halt its progressionAlzheimers disease AD one of the top leading causes of death in the United States is an increasing challenge for health care systems and will result in increased economic burden as increasing numbers of new cases are diagnosed annually12 Currently there is no therapy to prevent or slow AD progression which may be due to the inability to detect AD before its progression into evident cognitive decline Identification of early 1University of Hawaii Cancer Center Ilalo Street Honolulu HI USA 2Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI USA 3Department of Psychiatry and Behavioral Sciences Duke University Durham NC USA 4Institute of Computational Biology Helmholtz Zentrum M¼nchen German Research Center for Environmental Health Neuherberg Germany 5Duke Molecular Physiology Institute Duke University Durham NC USA 6Rosa Co LLC San Carlos CA USA 7University of Texas Health Science Center at San Antonio San Antonio TX USA 8Center for Translational Computational Neuroimmunology Columbia University College of Physicians and Surgeons Department of Neurology New York NY USA 9Rush Alzheimers Disease Center Rush University Medical Center Chicago IL USA 10Institute of Brain Sciences Duke University Durham NC USA 11Department of Medicine Duke University Durham NC USA email kaddu001mcdukeedu wjiacchawaiieduScientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cbiomarkers associated with preclinical symptoms would allow early intervention or preventive strategies to be developed3 Research has identified multiple neurochemical perturbations in AD including amyloid precursor protein metabolism phosphorylation of tau protein and a wide range of metabolic perturbations4 Unfortunately current biomarkers for early disease including cerebrospinal fluid CSF betaamyloid and tau levels5 structural and functional magnetic resonance imaging6 the recent use of brain amyloid imaging7 or inflammaging8 and CSF markers to track brain atrophy and deposition of cortical betaamyloid and neurofibrillary tangles are limited because they are either invasive timeconsuming or expensiveRecent studies have focused on obtaining biomarkers to identify features that differentiate persons a0with cognitive impairment from persons without cognitive impairment Molecular markers sensitive to the underlying pathogenic factors would be highly relevant to early disease detection and facilitation of disease monitoring and treatment responses Metabolomics is an unbiased approach to study smallmolecule metabolites that offers hope for the discovery of more biomarkers for AD This profiling technology has already been used to identify differential metabolites that can distinguish mild cognitive impairment MCI subjects who will develop AD from stable MCI9 Mounting evidence suggests that AD is closely accompanied with the abnormal bile acid BA metabolism10 free fatty acid FFA metabolism14 lipid metabolism1718 and neurotransmitter metabolism19 BAs have become increasingly recognized as important metabolic signaling molecules that modulate lipid glucose and energy metabolism20 More importantly BAs in brain act as neuroactive steroids21 Different classes of BAs can either inhibit or potentiate GABAα a0and inhibit NMDA receptors while also exerting neuroprotective effects Recent crosssectional studies have shown differences in blood BAs in AD compared with noncognitively impaired individuals2425 Additionally researchers found an accumulation of FFAs in the hippocampus and cortex of AD mice compared to control mice2627 Another animal study examined the role of elevated FFA in the pathogenesis of AD and established a potential mechanism of FFA causing hyperphosphorylation of tau through astrogliamediated oxidative stress28 Alterations of FFAs have also been detected in postmortem AD brains tissues14 and serum samples16 which may indicate an alternative fuel source before the onset of clinical symptoms29 These observations have given rise to the possibility that metabolic perturbations could presage the onset of cognitive impairment and therefore aid in the identification of individuals with higher risks by providing additional information to use with standard clinical markersIn this study we performed metabolomic profiling in participants from a large longitudinal cohort with the goal of identifying metabolic changes as well as key metabolic pathways that might serve as new predictors of future cognitive impairment in older adultsMaterials and methodsParticipants The Religious Orders Study ROS which began in is a longitudinal clinicalpathologic cohort study of risk factors of cognitive decline and incident dementia run from the Rush Alzheimers Disease Center that is comprised of individuals from religious communities eg Catholic brothers nuns and priests across the USA3031 The Rush Memory and Aging Project MAP which began in includes participants from northeastern Illinois USA with a broader range of socioeconomic status and life experiences31 Participants in both studies enroll without known dementia agree to annual clinical evaluation and an donation Both studies were approved by an Intuitional Review Board of Rush University Medical Center All subjects signed an informed consent an Anatomic Gift Act and a repository consent to allow their biospecimens and data to be used for ancillary studies All research was performed in accordance with relevant guidelinesregulations set forth by the Rush University Medical Center Both studies are conducted by the same team of examiners and share a large common core of data collection at the item level to allow for efficient merging of dataCognitive performance tests Cognitive performance was measured using a battery of cognitive performance tests of which could be summarized in five cognitive domains ie episodic memory working memory semantic memory perceptual orientationvisuospatial ability and perceptual speed Table a0S1 Domains are created by averaging the zscores based on mean and standard deviation from all baseline data for tests in each domain The global cognitive function score is calculated by averaging zscores for all tests to yield a global measure of cognitive function Additionally the MiniMental State Examination was also administered to characterize the cohortClinical diagnoses Medical conditions were documented via selfreport and clinical evaluation Clinical diagnoses each year were determined blinded to previously collected data A threestep process starts with an actuarial decision tree based on the history of cognitive decline and impairment ratings in five cognitive domains based on cutoffs for cognitive tests32 a0followed by clinical judgment by a neuropsychologist for cognitive impairment and determination of dementia and its causes by a clinician ie neurologist geriatrician second neuropsychologist geriatric nurse practitioner33 The diagnosis of AD follows the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimers Disease and Related Disorders Association NINCDSADRDA34 Participants were categorized as a AD b MCI if diagnosed cognitive impairment by the neuropsychologist but not diagnosed dementia by the clinician32 and c no cognitive impairment NCI if diagnosed without AD or MCI35 At the time of death brain autopsies and histopathological exams were performed by clinicians to confirm the diagnosis After an autopsy was completed a spectrum of neuropathologic diagnoses was obtained such as a pathologic diagnosis of AD as defined using the modified NIA Reagan criteria However many other pathologies were present in the brains of older individuals the mean age of death is a0years old in ROSMAP and they were catalogued for each participantScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cNCI converters were those participants who were cognitively normal at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followupNeuropathology Upon death a postmortem neuropathological evaluation was implemented and the procedures follow those outlined by the pathologic dataset recommended by the National Alzheimers Disease Coordinating Center Brains of deceased subjects were removed weighed cut into one cmthick coronal slabs and stored Each brain was examined for the neuropathological indices of common pathologies that contribute to cognitive impairment The location age and volume of all macroscopic infarcts were recorded and tissue was obtained for histological confirmation in addition to the identification of microscopic infarctions as previously described3637 AD pathology was identified using the modified Bielschowsky silver stain technique and by use of the Consortium to Establish a Registry for Alzheimers Disease CERAD criteria38 and NIAReagan criteria39 while the assessment of neurofibrillary tangles was based on Braak criteria40 as described previously41 The CERAD score a semiquantitative measure of neuritic plaque burden is made of levels no AD possible AD probable AD and definite AD As recommended CERAD scores were reclassified to a binary level score score Seven categories of Braak stages were based on the region and severity of neurofibrillary tangles pathologyMetabolites quantification Using targeted metabolomics protocols42 and profiling protocols43 established in previous studies BAs were quantified by ultraperformance liquid chromatography triple quadrupole mass spectrometry UPLCTQMS Waters XEVO TQS Milford USA and other metabolites were quantified by gas chromatography timeofflight mass spectrometry GCTOFMS Leco Corporation St Joseph USA Details are described in the Supporting InformationStatistical analysis Stratifying by clinical diagnosis continuous demographic variables were expressed as mean [standard deviation SD] and tested by Wilcoxon ranksum test while categorical demographic variables were expressed as n percentage and tested by Chisquare test Missing values in quantitative metabolites due to limits of quantification were regarded as leftcensored missing and imputed by GSimp4445 Individual BA concentrations were normalized to the total BAs concentration ie the proportion of total BAs Metabolites were reported as median quantile quantile and tested by univariate analysis Wilcoxon ranksum test Due to the limited sample size of the AD group participants in serum samples we combined MCI and AD participants into an aggregate group MCIAD for the following data analysis Logtransformed abundances were used in the following data analysis We additionally generated BA ratios based on the BA metabolic pathway topologyTo identify metabolites differentially expressed in participants with cognitive decline we used ordinal logistic regression to compare metabolites across three groups NCI MCI AD for brain samples and logistic regression across two groups NCI MCIAD for serum samples To control the positive false discovery rate Qvalues were calculated based on Pvalues Additionally for serum samples we adjusted for potential confounders eg fasting status supplements diabetic and lipid lowering medications using logistic regressions The relationships between logtransformed brain metabolites levels with neurofibrillary tangle burden and neuritic plaque burden were expressed as boxplots across Braak scores KruskalWallis test and CERAD scores Wilcoxon ranksum test respectively Using Spearmans rank correlation test we further evaluated the associations between the abundances of each identified metabolite and the global cognitive function score in both brain and serum samples Linear regression models with each individual metabolite used as the predictor and each cognitive test as the response variable adjusted for age gender years of education and presence of APOE ε4 were used to test the associations between metabolite and cognitive function Similar analyses with an additional adjustment of BMI were conducted for serum samples The Wilcoxon ranksum test was carried out to explore whether identified variables were differentially expressed between NCI converters vs NCI nonconverters and between NCI converters vs MCIAD in sera Then we built a random forest RF predictive model to differentiate NCI nonconverters vs MCIAD using glycolithocholate GLCA deoxycholatecholate DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and age as the predictorsTo differentiate MCIAD vs NCI nonconverters we randomly split the data into training set and testing set times Each time we trained an RF model on the training set to differentiate the MCIAD from NCI nonconverters and evaluated it on the testing set using the area under the receiver operating characteristic curve AUROC sensitivity SE and specificity SP A final model was built on the whole NCI nonconverters and MCIAD dataTo investigate the preclinical predictive potentials as well as to validate the classification performance of our model we utilized this model on the entire baseline NCI group to identify those NCI converters from NCI nonconverters The differences of RF scores between NCI nonconverters vs NCI converters and NCI nonconverters vs MCIAD groups were tested by the Wilcoxon ranksum test To determine whether RF scores could independently differentiate NCI converters from NCI nonconverters in the presence of potential confounders we used the logistic regression method with RF scores as the predictor adjusting for gender years of education APOE ε4 and BMI Additionally we fit linear mixed effects models to evaluate correlations between RF scores with global cognitive function and each of the five cognitive domains separately with a random effects term for education and BMI and fixed effects terms for RF score gender and APOE ε4For the personalized pathway level analyses we extracted metabolite information from the Human Metabolome Database HMDB46 and metabolic pathway information from the Kyoto Encyclopedia of Genes and Genomes KEGG database47 to map affiliated metabolites to metabolic pathways We used the pathifier Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cOverallNCIMCIADBrain samplesNAge mean SDMale n Education mean SDAPOE ε4carrier n Serum samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Serum NCI samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Overall Overall NCI MCIAD NCI nonconverters NCI converters Table Detailed demographic characteristics of study samples Chisquare test Pvalue comparing AD vs NCI Wilcoxon rank sum test Pvalue comparing MCIAD vs NCI Wilcoxon rank sum test Pvalue comparing NCI converters vs NCI nonconverters Chisquare test Pvalue comparing NCI converter vs NCI nonconverter NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease APOE ε4 apolipoprotein E epsilon allele SD standard deviation algorithm48 to transfer metabolic level information of each sample to pathway level by generating a pathway dysregulation score PDS For each pathway each sample was projected onto a directed principal curve49 which was yielded depending on leading principal components of the pathway to optimally pass through the cloud of samples PDS was the distance along the curve between the projection of each sample and that of NCI Thus PDS could capture the pathwaylevel extent of abnormality increments or decrements for each participant relative to those with NCI We performed similar data analysis on pathway level data to what we did on metabolomics level data We tried to identify differential pathways using ordinal logistic regression across NCI MCI and a0AD groups in brain samples and logistic regression for NCI and MCIAD in serum samples Next we explored the associations between identified pathways with neuropathology KruskalWallis test for Braak scores Wilcoxon ranksum test for CERAD scores and cognitive performance Spearmans rank correlation test for the global cognitive function linear regression with adjustments for each cognitive test Then we examined the predictive potential of identified pathways in serum samples using univariate analysis Wilcoxon ranksum test for NCI converters vs NCI nonconverters NCI converters vs MCIAD Finally we built RF models on training sets and tested them on testing sets according to times random splitting on the model construction data and applied the final model on the validation data using ROC SE SP as evaluation methods The overall workflow chart of the data and the analysis are shown in Fig a0S9Data were analyzed using R version with packages including pROC pathifier randomForest ggplot2 ggsignif and MASS The statistically significance was determined by a threshold of unadjusted Pvalues and Qvalues ResultsParticipants and characteristics For the joint analyses of the ROSMAP study we measured metabolomics of serum samples NCI MCI and AD at the blood draw and postmortem brain tissues from dorsolateral prefrontal cortex NCI MCI and AD at the time of death Among brain samples and serum samples a total of participants had both brain and blood metabolomics data NCI participants n were further categorized into NCI converters and NCI nonconverters NCI converters were those participants who were cognitively normal NCI at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followup Among NCI participants the time between the blood draw and conversion ranged from to a0years with a median of a0years Fig a0S10 Detailed demographic characteristics of the serum samples and postmortem brain samples are included in Table a0 Among participants with postmortem brain samples AD patients tended to have at least one APOE ε4 allele compared to the NCI group as expected The mean age of NCI and MCIAD group at the time of blood draw among serum samples was a0years SD and SD respectively Similarly the age and the percentage of APOE ε4 Scientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cFigure a0 Brain metabolome and serum metabolome composition and alterations a Left panel the brain metabolome composition Right panel a0log10 Pvalue across clinical groups of brain tissues NCI MCI AD b Left panel the serum metabolome composition Right panel a0log10 Pvalue across clinical groups of serum tissues NCI MCIADcarriers were higher in the NCI converters group than the NCI nonconverters group We did not observe other significant demographic characteristics differences across clinical groups Table a0Identifying metabolites differentially expressed in participants with cognitive impairment In this study metabolites and metabolites overlapping metabolites were detected in brain tissues and serum samples respectively Tables a0S7 S8 Amino acids BAs carbohydrates anic acids and fatty acids were the predominant types of annotated metabolites accounting for of all the metabolites in brain tissues and in serum samples Fig a01ab left panel A total of seven a0metabolites BA BA ratio anic acid known as a longchain fatty acid fatty acids showed significant differences across clinical groups in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a01ab right panel Tables a0S14 S15 After adjusting for confounders ie fasting status supplement use diabetic and lipid lowering medications most of serum metabolites remained statistically significant Table a0S18 In brain tissues increments of the levels of GLCA DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid followed the pattern NCI MCI AD We observed increments of GLCA DCACA ratio and decrements of petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid in sera of MCIAD compared to controls Table a0 The trend of increments of identified metabolites in brain samples increments of BAs and decrements of FFAs in serum samples were further validated within individuals with both brain and serum samples From NCI to MCI and AD groups increments of identified metabolites were observed in brain samples Table a0S11 The increasing trend of GLCA DCACA ratio and decreasing trend of FFAs among MCIAD group relative to NCI group were detected in sera Table a0S11The seven brain metabolites were all negatively correlated with global cognitive function where higher scores indicate better cognitive performance Ï a0 for GLCA Ï a0 for DCACA ratio Ï a0 for petroselinic acid Ï a0 for linoleic acid Ï a0 for myristic acid Ï a0 for palmitic acid and Ï a0 for palmitoleic acid using Spearmans rank correlation analysis Fig a02a Similarly after adjusting for age gender years of education and APOE ε4 all identified metabolites remained negatively correlated with tests in five Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cBrain samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]Serum samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]NCIMCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ]NCI [ ] [ ] [ ] [ ] [ ] [ ] [ ]MCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Table Levels of metabolites differentially expressed in participants a0by diagnostic group Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing AD vs NCI Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing MCIAD vs NCI NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease IQR interquartile rangeFigure a0 Associations between metabolites level and global cognitive function a Boxplots showing group differences and P values for identified metabolites across Braak groups for brain tissue abundances b Boxplots showing group differences and significances for identified metabolites across CERAD groups for brain tissue abundances Ï correlation coefficient of Spearmans rank correlation testScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0ccognitive domains and the MiniMental State Exam see Table a0S2 for significant correlation pairs The serum concentration of two BAs showed negative correlations with global cognitive function Ï a0 for GLCA Ï a0 for DCACA ratio conversely fatty acids demonstrated positive correlations Ï for petroselinic acid Ï for linoleic acid Ï for myristic acid Ï for palmitic acid and Ï for palmitoleic acid Fig a02b Linear regression revealed similar consistent results in serum samples with adjustment for age gender years of education APOE ε4 and BMI see Table a0S2 Results of associations between identified metabolitesratio and each cognitive performance domains are shown in Table a0S13 Correlations with global cognitive function were further validated in individuals with both brain and serum samples and the directions were consistent with our previous findings among the a0entire cohort Seven identified metabolites were all negatively correlated with global cognitive function in brain samples while two BAs showed negative correlations and five FFAs showed positive correlations in serum samples Fig a0S6 Additionally the serumbrain ratio of identified FFAs were positively correlated with global cognitive function ie lower levels of identified FFAs in serum and higher levels of identified FFAs in brain were associated with worse cognition Fig a0S7Identified metabolites predicted antecedent cognitive impairment before the manifestation of clinical symptoms The concentrations of GLCA and DCACA were significantly lower in the NCI nonconverters group than in the NCI converters group By contrast the abundances of petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid were higher in the NCI nonconverters group than in the NCI converters group Fig a03a There were no significant differences in these metabolites between participants in NCI converters group vs MCIAD group Fig a03a Using the seven metabolites and age we built RF models on the training set according to 100times randomly splitting approach to differentiate MCIAD patients from NCI nonconverters group The median of times AUC on testing set was CIs with SE CIs and SP CIs using Youdens index to maximize the sum of SE and SP Fig a03b RF models showed decent classification performances in differentiating MCIAD group from NCI nonconvertersNext we were interested in studying the models early diagnostic capability for predicting NCI converters before clinical diagnosis The model was thus applied on the entire NCI group at baseline to differentiate NCI converters from NCI nonconverters We achieved an AUC of CIs SE SP at the cutoff value of Fig a03c with significant differences in RF scores between NCI converters vs NCI nonconverters between NCI nonconverters vs MCIAD group using the Wilcoxon ranksum test Pvalue Fig a03d After additional adjustment for gender years of education APOE ε4 and BMI fasting status and medications supplements diabetes lipid lowing the RF scores remained significant as an independent predictor with a coefficient of Pvalue Table a0S3 Additionally the RF scores showed significant negative correlations with global cognitive function and the five cognitive domains with the same adjustment in mixed effects models Table a0S12Personalized metabolic pathwaybased study for the association and prediction of cognitive impairment Considering altered metabolite levels were significantly associated with cognitive impairment and showed early predictive value of clinical symptoms onset we then employed the pathifier algorithm to summarize metabolite information to pathways level for further examinations All PDS scores ranged from to where larger scores represent the higher extent of the abnormality in the corresponding metabolic pathway out of metabolites detected in brain tissues and out of metabolites detected in sera were successfully mapped to the KEGG metabolic pathways This method identified metabolic pathways in brain tissues and metabolic pathways in serum samples overlapping pathways Figs a0S1ab left panel Table a0S9 Table a0S10 three of which ie primary BAs biosynthesis FFAs biosynthesis and biosynthesis of unsaturated FFAs were significantly shifted in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a0S1ab right panel Table a0S16 Table a0S17 We noted increased PDS for all three identified pathways from NCI to MCIAD that suggested dysregulation of these metabolic pathways in MCIAD patients compare to NCI Detailed PDS of these pathways stratified by diagnostic groups are described in Table a0S4 Results also indicated that higher PDS were significantly associated with lower global cognitive function ie worse cognitive performance in both brain Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S4 and serum samples Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S5 respectively In Table a0S5 we show the significant negative associations between each cognitive test and PDS of three pathways after adjusting for age gender years of education and APOE ε4 additional adjustment for BMI in serum samples Two fatty acid pathways showed significantly different PDS between the NCI nonconverters group and the NCI converters group Pvalue and Pvalue respectively A gradually increasing trend was noted for the BAs pathway across groups ie NCI nonconverters NCI converters and MCIAD Fig a04aWe then constructed a discriminant RF model in training data and tested on testing data based on three identified metabolic pathways along with age to differentiate MCIAD from NCI nonconverters in model construction data using 100times randomly splitting approach The median AUC on the a0testing set was CIs with SE CIs and SP CIs Fig a04b Applying the RF model to the whole NCI data at baseline could successfully discriminate NCI converters from NCI nonconverters with an AUC of CIs SE SP cutoff value Fig a04c Similarly predictive RF scores were significantly different between NCI converters vs NCI nonconverters and NCI nonconverters vs MCIAD group Pvalue Fig a04d After adjusting for gender Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cFigure a0 The identified panel of metabolites and its predictive performance a Boxplots showing group differences and P values for identified metabolites across NCI nonconverters NCI converters and MCIAD for serum abundances b ROC curves of metabolite models trained on the training data and tested on the testing data according to 100times randomly trainingtesting splitting c The ROC curve of the final metabolite model on the validation data d RF scores of the final metabolite model across NCI nonconverters NCI converters and MCIAD Pvalue Pvalue Pvalue Wilcoxon rank sum test The optimal cutoff was determined by the Youden index AD Alzheimers disease AUC area under | 2 |
clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the respiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the viruss genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this acute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the 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reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clÃnica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c' | 0 |
glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city peoples hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city peoples hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage yearsgendermalefemalegradeiiiiiiivtumor size cmlow n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturers instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq¢ ii takara japan gapdh was the normalized control relative expression was calculated through the δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturers instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturers instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using students ttest or oneway anova survival rate was analyzed by the kaplanmeier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells be proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4cf therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearsons correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1 v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalcancer management and research submit your 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preoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer one e0237244 101371 pone0237244editor louise emilsson university of oslonorwayreceived february accepted july published august copyright strous this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement data cannot beshared publicly because of ethical concernspatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data data are available fromthe viecuri institutional data access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdata heart rate variability hrv represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer hrv is also believed to predict the occurrence andseverity of postoperative complications we aimed to determine the role of preoperativehrv as a prognostic factor in overall and cancer free survival in patients with colorectalcancermethodsretrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between january and december who underwent curative surgical treatment hrv was measured as timedomain parameters sdnn standarddeviation of nnintervals and rmssd root mean square of successive differencesbased on preoperative second ecgs groups were created by baseline hrv low hrvsdnn 20ms or rmssd 19ms and normal hrv sdnn �20ms or rmssd �19msprimary endpoints were overall and cancer free survivalresultsa total of patients were included in this study hrv was not significantly associated withoverall survival sdnn 20ms vs sdnn �20ms244 vs adjusted hr p rmssd 19ms vs rmssd �19ms270 vs adjustedhr p or cancer recurrence sdnn 20ms vs�20ms201 vs adjusted hr p rmssd 19ms vs�19ms vs adjusted hr p there was no one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfunding the authors received no specific fundingfor this workcompeting interests the authors have declaredthat no competing interests existsignificant association between hrv and cealevel at one year followup or between hrvand occurrence of a postoperative complication or the severity of postoperativecomplicationssheart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment these results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low hrvintroductionin there were over million newly diagnosed colorectal cancer patients worldwide andover in the netherlands alone it is the fourth most common cause of death worldwide to improve survival it is of importance to get a better insight into modifiable prognosticfactors emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityhrv could be one of these prognostic factors []hrv is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [] it has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity these mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway a higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer in addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others also patientswith normal hrv seem to live longer in different sorts of metastatic cancer independent ofconfounders in patients with colorectal cancer a low hrv at baseline has shown to beassociated with higher cea levels at months after diagnosis which predicts a poorer prognosis in patients undergoing curative treatment for colorectal cancer hrv does not only seemto influence cancer prognosis a recent study showed that patients with lower hrv have moreintraoperative blood loss and more and more severe postoperative complications identifying patients with low hrv is easy and noninvasive when its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis alsoif we could predict the occurrence and severity of postoperative complications based on hrvimproving hrv before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients prognosis recent studies focussing on improving hrv by improving physicalfitness by means of physical exercise show promising results in both older men and woman however the only previous study on colon cancer and hrv including patients receiving curative treatment included a small sample and did not examine whether hrv predicts one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancersurvival in these patients to clarify the predictive value of hrv in prognosis of patientswith colorectal cancer further exploration is needed current studies identifying hrv as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease []the aim of this study was to determine the role of preoperative hrv as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentmethodsdata collectiondata from the netherlands cancer registry ncr were used the ncr collects data on allnewly diagnosed cancer patients in the netherlands information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry anatomical site of the tumour is registered according tothe international classification of diseases oncology the tumournodemetastasis tnmclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis quality of the data is high due to thorough training of theregistration team and consistency checks for the study population additional data were collected from the medical records of thepatients this encompassed information on hrv cealevels asa classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on hrv or the endpoint being analysed severity of the postoperative complicationsaccording to the claviendindo classification was also documented medical records wereassessed between january and july and reevaluated for revision of this between the 20th and 25th of april this study was approved by the research committee and the board of directors of viecurimedical centre data was obtained under the law scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons in the netherlands unless patients objected to use of their personal medical record forscientific research data was encrypted with an encryption key provided by the ncr encryption was shortly lifted to access the patients number for accessing hisher medical record following extraction data were encrypted againstudy populationthe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between january and january at viecuri medical centre who underwent curative surgical treatment patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances hrv indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of hrv we did not exclude patients taking alphablockers calcium one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerinhibitors diuretics amiodarone aceinhibitors or arbs as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on hrv istherefore less univocal and sometimes completely absent heart rate variabilityheart rate variability was analysed using a 12lead 10second ecg 150hz used for preoperative screening in case of multiple ecgs per patient the most recent ecg before date of surgery was used for hrvanalysis in case of multiple ecgs per patient on the same date theecg with the best quality was chosen meaning an ecg without motion artefacts in case ofmotion artefacts there was always an ecg without motion artefacts available recorded on thesame date time between two consecutive rpeaks was measured in lead ii with an accuracy of02ms using museecg hrv was presented using the timedomain hrv parameters sdnnstandard deviation of nnintervals and rmssd root mean square of successive differences in milliseconds calculated using the following calculations rsdnn ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrri 00 rrmeanþ2pnn 00 rmssd ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðrriþ1 00 rriþ2pn 00 n 00 ð1þð2þsdnn and rmssd obtained from 10s ecgs were found to correlate with results of ecgsof longer durations power spectral analysis hrv parameters as lf and hf can only beobtained in longer recording ecgs and were therefore not implementable in this study sdnn and rmssd were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively in case of ansdnn 20ms or rmssd 19ms hrv was classified as low and in case of sdnn �20ms orrmssd �19ms as normal these cutoff values were based on cutoff values used in otherstudies showing an association between lowhrv as sdnn 20ms and rmssd 19ms andcolorectal cancer as there is no standardised definition of low and normal hrv endpoints and definitionsthe primary endpoints of this study were overall and cancer free survival overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer patients dying without cancerrecurrence were censored on day of death secondary endpoints were elevated cealevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the claviendindoclassificationstatistical analysisin this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex bmicomorbidities and asaclassification heart rate and tumour characteristics as tnmstagetumour localisation and tumour differentiation and their association with hrv and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerprognosis normal distribution of the continuous variables heart rate age and bmi as well assdnn and rmssd were tested with a kolmogorovsmirnov test because of normal distribution heart rate age and bmi were compared between hrvgroups using unpaired ttest allother variables were categorical and were compared between hrvgroups using chisquarestatistics as groups were all of sufficient powerdifferences in overall survival and cancer free survival in months according to sdnn andrmssd were visualized by means of kaplanmeier curves and statistically tested using the logrank test multivariate coxregression analyses were conducted to calculate the prognosticassociation between hrv and overall and cancer free survival while adjusting for other prognostic variables multivariate logistic regression was used to assess the independent effect ofsdnn and rmssd on cealevels and the occurrence and severity of postoperative complications variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including crohnsdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome differences in cealevel at baseline and one year checkup between and within groups of low hrv and normal hrv were assessed with a repeatedmeasures linear model and tested using the tukey test to test the implication of a longer timebetween ecg and treatment all analyses were repeated after excluding patients with an ecgolder than months a twotailed pvalue � was considered significant in all analysesdata were analysed using ibm spss statistics version ibm corp ny armonk usaresultsof colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study reasons for exclusion are presented in fig median sdnn andrmssd were 204ms interquartile range iqr 115ms351ms and 175ms iqr 99ms299ms respectively table shows descriptive data of the included patients by hrv groupsbaseline heart rate and age were negatively associated with hrv the group of patients withlow hrv contains more patients with a history of cardiac disease regardless of the hrv defining parameter when defining low hrv by rmssd 19ms more patients in this group havehypertension as comorbidity this group also contains more patients with an asa classification greater than oneduring a median followup of months iqr all causedeath occurred in patients cancer recurrence occurred in patients during a median followup of months iqr to rule out any distort in results caused by a delay between ecg and treatment all analyseswere repeated after exclusion of ecgs older than months this did not lead to any new significant results therefore these results were not displayed in detail in this papersurvivalin low hrv groups slightly more patients died compared to normal hrv groups sdnn20ms versus �20ms versus respectively rmssd 19ms versus�19ms versus respectively these observed differences between hrvgroups in overall survival were not significant fig a sdnn p b rmssd one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig flowchart of the study101371 pone0237244g001p after adjustment for some potential confounders these associations remained notsignificant sdnn 20ms versus �20ms hr p and rmssd19ms versus �19ms hr p tables and age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival age also differed at baseline and was identified as a confounder when defining low andnormal hrvgroups by sdnn cardiac disease was identified as confounder when conducting sensitivity analyses with sdnn and rmss as continuous variables results remained thesame there was no association between hrv and overall survival sdnn hr p and rmssd hr p in low hrv groups slightly more patients had recurrence of cancer compared to normalhrv groups sdnn 20ms versus �20ms versus respectivelyrmssd 19ms versus �19ms versus respectively these observed differences between hrv groups in cancer free survival were not significant fig a sdnnp b rmssd p as in overall survival after adjustment for some potentialconfounders these associations remained not significant sdnn 20ms versus �20mshr p and rmssd 19ms versus �19ms hr p tables and in sdnnbased groups baseline heart rate was identified asa confounding variable sensitivity analyses with sdnn and rmssd as continuous variables one 101371 pone0237244 august one 0ctable descriptive data of included patients according to prespecified hrv groupssdnn 20ms n sdnn �20ms n prmssd 19ms n rmssd �19ms n phrv and prognosis in colorectal cancerheart rate�age�age n year� yearsex nmalefemale [] [] [] [] [] [] [] [] mean bmi sd [] [] [] []comorbidities ncardiac diseasehypertensiondiabetes mellitusthyroid diseasepulmonary diseasevascular diseaseneurological diseaseotherasa nasa1asa2asa34localisation tumour nright colonleft colonrectumtumour stage niiiiii differentiation grade nwellmoderate poorundifferentiated � non normaldistributed data presented as median with interquartile range101371 pone0237244t001did not alter these results there was no association between hrv and cancer free survivalsdnn hr p and rmssd hr p cealevelcealevel at baseline and one year checkup was registered in patients and elevated in of these patients this was elevated at one year checkup in only patients low hrv was notsignificantly associated with elevated cealevels at one year checkup as shown in table sensitivity analyses with sdnn and rmssd as continuous variables did not alter these resultssdnn hr p and rmssd hr one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for overall survival in groups of low hrv and normal hrv presented as a sdnn andb rmssd101371 pone0237244g002 one 101371 pone0237244 august one 0ctable multivariate analyses of associations of sdnn and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancersdnn20ms�20msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t002p adjusting for covariates was not possible because of the small number of patientswith an elevated cealeveldifferences between cealevel at baseline and one year checkup were compared betweenand within hrvgroups results were displayed in fig there were no significant differencesin cealevel at baseline and one year checkup between the low hrv group and normal hrvgroup defined by sdnn and rmssd p and p respectively also there were nosignificant differences in cealevel at baseline and at one year checkup within the low hrvgroup and normal hrv group defined by sdnn and rmssd p and p respectivelypostoperative complicationsin patients one or more postoperative complications occurred within days after surgerythe occurrence of a postoperative complication was not significantly associated with lowhrv defined as sdnn 20ms or rmssd 19ms even after adjustment for some potentialconfounders table heart rate age cardiac disease and hypertension were identified asconfounding covariates when conducting sensitivity analyses with sdnn and rmss as continuous variables the association between occurrence of a postoperative complication with one 101371 pone0237244 august one 0ctable multivariate analyses of associations of rmssd and covariates with overall and cancer free survivalhrv and prognosis in colorectal cancerrmssd19ms�19msheart rateagebmicardiac diseasenoyeshypertensionnoyesasaclassificationasa1asa2asa34localisationright colonleft colonrectumtumour stageiiiiiioverall survivalhr cipcancer free survivalhr ci reference reference p reference reference reference reference reference reference reference reference reference reference postoperative complicationnoyes reference reference 101371 pone0237244t003baseline hrv remained not significant sdnn hr p andrmssd p the same applied when postoperative complicationswere graded according to the claviendindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationstable discussionin this observational cohortstudy we determined the heart rate variability in preoperativeecgs of patients with primary colorectal cancer who underwent curative surgical treatment hrv refers to physiological variations in beattobeat intervals it was presented intimedomain parameters sdnn and rmssd hrv was not significantly associated with overall survival or cancer free survival independent of some risk factors also this study showedno significant differences in cealevels at one year checkup between patients with low hrvand patients with normal hrv patients with low hrv did not have more or more severepostoperative complications compared to patients with normal hrvtumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing dnadamage and one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig kaplanmeier curves for cancer free survival in groups of low hrv and normal hrv presented as a sdnnand b rmssd101371 pone0237244g003 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable univariate analyses of low hrv and risk of elevated cealevel at one year checkupcea μglor cipcea μglor ciindependent of baseline ceaindependent of baseline ceasdnn 20ms n rmssd 19ms n sdnn �20ms n normal baseline cea � μglsdnn 20ms n sdnn �20ms n elevated baseline cea μglsdnn 20ms n reference reference rmssd �19ms n normal baseline cea � μglrmssd 19ms n rmssd �19ms n elevated baseline cea μglrmssd 19ms n reference reference sdnn �20ms n referencermssd �19ms n referencep101371 pone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression it has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation this is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway malfunctioning of this route may play part in the onset of cancer this theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [] the vagus nerve is also believed to modulate tumour progression an active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [] absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls epidemiologically low hrv has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [] however the results of the present study do not support thesefindingsto our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment de couck studied the relationship between hrv and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer independent of confounders the hypothesised inverse relationship of hrv and the tumour markerpsa at and months after diagnosis was only significant in patients with stage iv prostatecancer not stage ii and iii in colorectal cancer mouton found that low hrv definedas sdnn ms predicted significantly higher levels of the tumour marker cea at months after diagnosis again these results were only found in patients receiving palliativetreatment not curative only one previous study showed a significant inverse relationshipbetween hrv and mortality in cancer in general independent of stage this study of guo had a large study population of patients with various types of cancer low hrv wasdefined as sdnn 70ms and was significantly associated with shorter survival times thissuggests that hrv is a predictive indicator of survival time not only in palliative but also incurative patients however results were not controlled for cancer type which could affect bothhrv and survival and should therefore be interpreted with caution the fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in hrv in this sample one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancerfig bar chart for cealevel at baseline and one year checkup in groups of low hrv and normal hrv presented as asdnn and b rmssd101371 pone0237244g004 one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and risk of occurrence of a postoperative complication within dayspostoperative complicationor ciunadjustedsdnn 20ms n sdnn �20ms n referenceadjusted�sdnn 20ms n sdnn �20ms n referenceunadjustedrmssd 19ms n rmssd �19ms n referenceadjusted�rmssd 19ms n rmssd �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371 pone0237244t005psome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer however based on current evidence on this subject we cannot supportthis hypothesis the positive association between low hrv and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low hrv midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased hrv a study of de couck showed that cancerpatients in general have a significantly lower hrv than healthy people the same resultswere found in studies of bijoor where rmssd was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group when comparing patients with advanced stage cancer tnm iii and iv to patients with anearly stage of cancer tnm i and ii rmssd was found to be significantly lower in patientswith advanced stages of cancer thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased hrv besides the proposed influence of low hrv on survival of colorectal cancer patientsthrough development and increased progression of cancer reimer suggested that lowhrv could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications however the results found in this study were notconcurrent with those of reimer who included patients with asa undergoingelective surgery their analysis of hrv was through powerspectrum parameters based on longer ecgrecordings instead of the timedomain parameters based on 10s ecgs used in thisstudy but the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatrmssd and sdnn based on ecg recordings of 10s are in substantial agreement with those of45min and a 10s ecg therefore suffices to determine time domain hrv parameters howeverreimer did not correct for possible confounders in their study patients with low hrvwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low hrv group correcting for this comorbidity may change the significance of their findings a study of one 101371 pone0237244 august one 0chrv and prognosis in colorectal cancertable low hrv and severity of postoperative complications according to claviendindo classificationunadjustedminor grade i and iior cipmajor grade iii iv vor cisdnn 20msn n sdnn �20msn referencen referenceadjusted�sdnn 20msn sdnn �20msn referenceunadjustedrmssd 19msn rmssd �19msn referenceadjusted�n n referencen n referencermssd 19msn n rmssd �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371 pone0237244t006scheffler did not show any significant preoperative differences in hrv between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels thus also in relation to predicting postoperative complications results are not uniformthis study was subject to a number of limitations due to its retrospective character confounding may have occurred we attempted to correct for all possible confounders withindatabase availability with regression analyses also not all possible confounding factors werewithin database availability reasons for diagnostic procedure smoking alcohol abuse are presumed to have an effect on hrv and outcomes but were not documented in enough patientsto be taken into account when conducting multivariate analyses analysed ecgs were thoseperformed before or at time of preoperative screening in this cohort ecgs were madewithin the year before surgery only ecgs were older than five years at time of surgery theprecise effects of these differences in time from ecg to cancer treatment on hrv areunknown to test the implication o | 0 |
"assess the antioxidative activity of seleniumenriched ChrysomyiaMegacephala Fabricius C megacephala larvae powder SCML and its impact on the diversity and structure ofintestinal microflora in a mouse model of Dgalactose Dgalinduced oxidative damageMethods Sixty male ICR mice were equally randomized to a normal control NC group a model group a positivegroup a lowdose SCML LSCML group a middose SCML MSCML group and a highdose SCML HSCMLgroup Animals in NC and model groups received water animals in the positive group received mgKg vitamin EVE and those in the three SCML groups received SCML which include and μgKg selenium Serespectively An oxidative damage model induced by subcutaneous injection of Dgal for weeks via the neck wasestablished Serum oxidative stress levels and tissue appearance were evaluated Tissues oxidative stress levels weredetected by commercially available kit Nuclear erythroid 2related factor Nrf2 and gut microbiota weredetermined by western blot and high throughput sequencing 16S rRNA gene respectivelyResults An oxidative damage model was established successfully as represented by a significant elevation ofmalondialdehyde MDA and protein carbonylation and inhibition of the antioxidants including superoxide dismutaseSOD glutathione peroxidase GSHPx total antioxidant capacity TAOC and glutathione GSH It was found thatoxidative damage and histological alterations were attenuated the expression of Kelchlike ECHassociated proteinKeap1 was decreased and the expression of Nrf2 and hemeoxygenase1 HO1 was increased after SCML treatmentIn addition significant changes were observed in the gut microbiota including Proteobacteria and the ratio ofBacteroidetes to Firmicutes at the phylum level as well as Helicobacter Clostridium and Lactobacillus at the genus levelContinued on next page Correspondence jiangzcmueducn Dandan Xie and Liqin Jiang contributed equally to this work1College of Pharmaceutical Science Zhejiang Chinese Medical University Binwen Road Hangzhou ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cXie BMC Complementary Medicine and Therapies Page of Continued from previous pageConclusion SCML exerted an antioxidative effect in vivo probably by increasing the antioxidant activity and reducingthe production of oxidation products via the Nrf2 signaling pathway SCML could also redress the intestinal floraimbalance induced by oxidative stress All these findings suggest that SCML could serve as a functional food andnatural drug additive to protect the human body against oxidative damageKeywords Seleniumenriched Chrysomyia Megacephala Fabricius larvae powder SCML Antioxidant activity Nrf2Intestinal microbiota In vivoBackgroundAging is a natural process that involves the gradual loss ofphysiological functions causing enhanced morbidity andmortality due to various diseases This process is closelyrelated to oxidative stress [] One prevalent theory toexplain aging is the theory of the oxygen free radical []This theory posits that the macromolecules such as nucleic acids lipids sugars and proteins that make up cellsand tissues are subjected to oxidative stress induced bysuperoxide and other free radicals These macromoleculesthen undergo different degrees of oxidation which initiates oxidative damages and ultimately leads to an function impairment and aging [ ] Changes in the level ofoxidative stress affect the microbial environment in the intestine and lead to intestinal flora disorder [] Disorderedintestinal flora may affect the antioxidant activity and lipidmetabolism [] Hence it may be possible to inhibit oxidative stress by regulating the composition and structure ofthe gut flora To prevent oxidative stressassociated cellular damage it is therefore important to keep prooxidantantioxidant balance by supplementation or induction ofcellular antioxidants A high dose of dgalactose is converted to aldose and hydrogen peroxide by dgalactoseoxidase The products then generate reactive oxygen species through oxidative metabolism and glycosylation leading to oxidative stress The accumulation of oxidationproducts further exacerbates the oxidative damage to tissues and cells which then accelerates the aging process[] Therefore dgalactose overload has been used to establish animal models used to conduct aging related metabolic dysfunction and oxidative stress [ ]Selenium Se is an essential trace element for humanbody and other animals The role of Se is reported to beclosely associated with antioxidant activityimmune response and chemoprevention [] Se is mainly presentin the active site of enzymes in the form of selenocysteineMultiple Secontaining proteins such as GSHPx and thioredoxin reductase play important roles in preventing oxidativeimportance of Sesupplementation in boosting up the internal antioxidativedefense has been highlighted in recent years Studies haveshown that anic Se supplements can improve tissue Sedeposition antioxidant level and gene expression whereasSe deficiency may result in cardiac muscular osseous and[] Thereforeinjurytheimmune disturbances [ ] Therefore the healthrelatedbenefits of Se including the type of selenium supplementsand optimal dosage remain to be exploredThe importance of Se has inspired researchers to usebioenrichment to prepare high Se compounds [ ] Cmegacephala larvae is a traditional Chinese medicine witha wide range of pharmacological actions including antioxidant antibacterial and antiinflammatory activitieswhich has been widely applied in agriculture and medicine[] Seenriched C megacephala larvae SCML isgenerated from C megacephala larvae by biological transformation and enrichment of Se Our previous workshowed that SCML was an effective anic Se sourcewith low toxicity and high Se content [] Yet no studyhas reported the antioxidant activity of SCML in vivo andits impact on the gut microbiota which is susceptible toundergo alterations under oxidative stressThe objective of the present study was to evaluate theantioxidant activity of SCML in vivo explore the underlying mechanism as well as evaluate its impact on thegut microbial diversity and structure hoping that the results could provide a scientific basis for a comprehensiveutilization of SCMLMethodsMaterials and chemicalsSCML was provided by Beijing Ershang Biological Technology Co Ltd Beijing China Vitamin E was purchasedfrom Archer Daniels Midland Dictor USA DgalactoseDgal of ¥ purity was purchased from Aladdin Industrial Corporation Shanghai China GSHPx SOD TAOC GSH MDA and protein carbonyl assay kits werepurchased from Nanjin Jiancheng Bioengineering InstituteNanjin China RNA trizol reagent and FastStart Universal SYBR Green Master Rox were purchased from Servicebio Wuhan China The primers for Nrf2 SOD1GSHPx and GAPDH were synthesized and purified byWuhan Servicebio Technology Co LTD Wuhan ChinaThe kits for Revert Aid First Strand cDNA synthesis andHyPure¢Molecular Biology Grade Water were purchasedfrom Thermo Waltham USA and HyClone LoganUSA respectively Keap1 Nrf2 and HO1 polyclonal antibodies were obtained from Proteintech Chicago USARIPA Actin bicinchoninic acid BCA assay kit 0cXie BMC Complementary Medicine and Therapies Page of Western Lightening¢ PlusECL Enhanced chemiluminescence substrate assay kit and the secondary goat antimouse horseradish peroxides HRP were from ServicebioWuhan China All other chemicals and reagents used inthe study were of analytical grade Water used in the experiments was ultrapureDetermination of the compositions of SCMLCompositions of SCML including protein crude fat andmoisture content were analyzed according to methodGB5009 of China National Food Safety StandardSe content was detected by Inductively Coupled PlasmaICP according to Vu with minor modifications[] The results are shown in Table Animal experimentsSixty ICR male mice aged weeks and weighing ± gwere purchased from SinoBritish SIPPRBK Lab Animal Ltd Approval No SCXK HU Theanimal experiments were performed in accordance withthe guidelines of the Laboratory Animal Center of Zhejiang Chinese Medical University Permit No SYSKZHE Allthe experimental procedureswere strictly conducted according to the internationalstandards and nationallegislation on animal care anduse The mice were kept under controlled light conditions h lightdark cycle with free access to food andwater normal light circadian rhythm and 7day adaptivefeeding in a quiet environmentAfter oneweek acclimatization mice were equallyrandomized to six groups normal controlNCgroup model group positive group receiving mgKg·d vitamin E VE group lowdose SCML LSCML group receiving SCML μgKg·d Se middose SCML MSCML group receiving SCML μgKg·d Se and highdose SCML HSCML group receiving SCML μgKg·d Se Except for the mice inNC group animals in the other five groups were givensubcutaneous injection of mgKg·d Dgal for weeksinto the neck to prepare oxidative stress model Animalsin NC and model groups received water and animals inthe other groups as previously described received VE orSCML by intragastric gavage for weeks The experiments were conducted at A certain amountof SCML and gellan gum were weighed precisely anddissolved in purified water heated slightly to a suspension There were three different concentrations and μgmL Se Meanwhile VE was dissolved in purified water containing gellan gum which became aTable Compositions of SCMLsuspension mgmL Dgal was dissolved in physiological saline mgmLThe mice were weighed throughout the experimentThe appearance appetite mental condition and behavioral activity of the mice during the experiment werealso observed and recorded Stool samples were collected at weeks after treatment Blood samples wereobtained from the retrobulbar venous plexus at weeksafter treatment The mice were sacrificed by cervical dislocation and the liver kidney heart brain and caecumwere stripped The dissected ans were divided twoparts one for histological analysis and the other for biochemistry analysis Samples for analysis were thawed onice homogenized with mL cold buffer mM potassium phosphate with mM EDTA pH per gram oftissue and centrifuged at Ãg for min at °CThe supernatants were collected for analysisAnalysis of serum oxidative stress indexesSerum oxidative stress indexes GSHPx SOD and MDAwere determined by using the respective commercial kitsaccording to the manufacturers instructionsAnalysis of tissue oxidative stress indexesThe oxidative stress indexes were determined by measuring GSHPx SOD TAOC GSH MDA and proteincarbonylation ofthe tissue homogenate supernatantusing the commercial kits according to the manufacturers instructionsHistological analysisFor histological analysis the animal tissues were fixed in paraformaldehyde for h dehydrated in alcoholparaffin embedded sliced into μm thick sectionsstained with hematoxylineosin HE and finally photographed under a microscope à objective lensRNA extraction and realtime quantitative PCRexperimentsTotal RNA was extracted from the liver and kidney tissues using Trizol reagent RNA was reverse transcribedinto cDNA using RevertAid First Strand cDNA SynthesisKit Realtime quantitative PCR qRTPCR was performed using FastStart Universal SYBR Green MasterRox and the ABI7900Faxt Sequence Detection systemThe thermal cycle condition was cycle at °C for min followed by cycles of amplification at °C for s and then °C for 30s And the dissolution curvestarted from °C then ascending to °C at °C15ContentProtein g100 gCrude Fat g100 gMoisture g100 gSe μggSCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of s All samples were run in triplicate in each experimentValues were normalized to that for GAPDH The sequences of the primers used are shown in Table Theresults were calculated by using the 2ÎÎCT methodliverandtissueskidneyWestern blot analysisTotal protein and nuclear protein were extracted from mgusing RadioImmunoprecipitation Assay RIPA lysis solution and anuclearcytoplasm protein extraction kit The concentrations of protein lysates were quantified using a BCA protein kit Samples containing an equal amount of protein μg were mixed with the loading buffer containing 2mercaptoethanol heated for min at °C andloaded onto a SDSPAGE gel The proteins fromthe electrophoresing gel were then transferred ontopolyvinylidenethenblocked with milk and Tween in Trisbuffered saline and incubated overnight at °C withantiKeap1 antiNrf2 antiHO1 and actin Then the appropriate horseradish peroxideconjugated secondary antibody wasadded to the membranes at room temperature Finallythe proteins were detected with chemiluminescent substrate Gray semiquantitative analysis was performed byImage J The protein bands were quantified using densitometry Values are expressed as the fold change withrespect to betaactindifluoride membranes whichIntestinal microbiota analysisThe stool samples were sent to BGI Co Ltd WuhanChina for sequencing of the 16S rRNA gene Total genomic DNA of the gut microbiome was extracted and theV3V4 region of the 16S rRNA gene from the sample wassubjected to PCR amplification After normalization of thegenome DNA to ng per PCR reaction V3V4 dualindex fusion PCR primer cocktail and PCR master mixwere added and then a PCR was performed The PCRproducts were purified with Agencourt AMPure XP beadsto remove the unspecific products Pairedend sequencingTable Primers for realtime PCR analysesAccession NoGeneNrf2NM_0109023SOD1GSHPxGAPDHNM_0114341NM_0081606NM_0080842was performed on the Illumina Hiseq platform and theobtained data were subjected to bioinformatics analysisTo obtain clean reads the clean pairedend reads withoverlap were merged to tags using FLASH fast lengthadjustment of short reads v1211 Then the tags wereclustered to operational taxonomic units OTUs at sequence similarity by scripts of software USEARCHv701090 The RDP classifier v22 was used to compare OTUs with the database to comment on the OTUsspecies Finally intestinal microbial diversity and structure were analyzed based on OTUs and taxonomic ranksusing software R v311Statistical analysisAll data are expressed as the means ± SD or means ± SEand analyzed using Statistical Analysis Software SPSS The experimental values were analyzed by oneway ANOVA followed by the Duncans multiplerangetests and Pvalue were considered to be statistically significantResultsEffects of SCML on daily behavior and weight gain inmiceUsual performance of the mice was observed and recorded and no abnormal phenomenon found duringthe experimentincluding antifeeding and vomitingSymptoms such as slow movement and listlessnesswere obviously observed in model groupindicatingthat the oxidative stress model induced by subcutaneous injection of Dgal was successfully establishedHowever the above symptoms receded in varying degrees in VE and SCML groups The weight gain ofthe mice is exhibited in Fig Compared with NCgroup body weight of the mice in model group significantly increased slowly P and increasedsteadily in drug treatment groups MSCML μgKg Se group showed a significant difference compared to the model group P Primer Sequences CTGGCTGATACTACCGCTGTTCAGGTGGGATTTGAGTCTAAGGAGATGTGACTGCTGGAAAGGACGCGCAATCCCAATCACTCCACCCAGGAGAATGGCAAGAATGAGGAAGGTAAAGAGCGGGTGACCTCGTCCCGTAGACAAAATGTGAGGTCAATGAAGGGGTCGTProduct Sizebp bp bp bp bp 0cXie BMC Complementary Medicine and Therapies Page of tissues were decreased significantly compared to NCgroup P except for SOD in the heart as well asGSHPx in the liver and brain After administration ofVE or SCML the activity of GSHPx and SOD as wellas the content of TAOC and GSH were increased gradually As shown in Fig 3a the activity of GSHPx in thekidney and heart was increased significantly comparedto model group P except for the heart in VEgroup and LSCML μgKg Se group The activityof GSHPx in the liver and brain remained unchangedsignificantly compared to model group except for VEgroup in the liver As shown in Fig 3b the activity ofSOD in the kidney and brain was increased significantlycompared to model group P except for the brainin LSCML μgKg Se group However the activityof SOD in the liver and heart remained unchanged significantly compared to model group except for the liverin VE group As seen in Fig 3c the content of TAOCin the liver kidney and heart was increased significantlycompared to the model group P except for theliver in LSCML μgKg Se group The content ofTAOC in the brain was not significantly altered compared to model group except for VE group As shownin Fig 3d the content of GSH in liver kidney and brainof VE group and in the kidney of HSCML μgKgSe and MSCML μgKg Se groups was increasedsignificantly compared to model group P As shown in Fig 3e the MDA level in model group wasincreased significantly compared to NC group P and decreased significantly after VE or SCML treatmentcompared to model group P except for LSCML μgKg Se group in the kidney In Fig 3f the proteincarbonylation level in the liver kidney and brain of modelgroup was increased significantly compared to NC groupP However the level decreased significantly inthe mice treated with SCML or VE except for in the liverand brain of LSCML μgKg Se group compared tomodel group P And compared with model groupFig Percentage of weight gain in mice at weeks Valuesrepresent means ± SD n and evaluated by oneway ANOVAfollowed by the Duncans multiplerange tests Compared with NCP Compared with Model P SCML SeleniumenrichedC megacephala larvae powderEffects of SCML on serum oxidative stress indexes in miceAs shown in Fig the serum antioxidative enzyme activities in model group were decreased significantly andthe MDA content was increased significantly comparedto NC group P As shown in Fig 2a the GSHPxactivities in animals treated with SCML or VE were increased significantly P As shown in Fig 2b theactivities of SOD in MSCML μgKg Se and VEgroups were significantly increased compared to modelgroup P As shown in Fig 2c the MDA levels inanimals treated with SCML or VE were decreased significantly P Effects of SCML on tissue oxidative stress indexes in miceAs illustrated in Fig after 6week subcutaneous injection of Dgal the activity of the antioxidative enzymesand the content of the antioxidants in different miceFig Oxidative stress level indexes of the mice serum a GSHPx activity in the mice serum b SOD activity in the mice serum c MDA content inthe mice serum Values represent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by theDuncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig Oxidative stress indexes of the mice tissue a GSHPx activity in the mice tissue b SOD activity in the mice tissue c TAOC content in themice tissue d GSH content in the mice tissue e MDA content in the mice tissue f protein carbonylation content in the mice tissue Valuesrepresent means ± SD from three independent replicates n and evaluated by oneway ANOVA followed by the Duncans multiplerangetests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powderFig Optical micrographs of mice tissue sections HE staining à Black arrow derangement of hepatic cord cells Red arrow infiltration ofinflammatory cells White arrow pyknosis Blue arrow cavitation and deformation Orange arrow atrophy and breakage of the villus Yellow arrowthinning of the intestinal wall Scale bar50 μm SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of HSCML μgKg Se group in heart also significantlydecreased in protein carbonylation levelEffects of SCML on histopathological changes in miceThe histopathological results are shown in Fig Normal histological architectures were observed in the tissuesections in NC group However the liver tissue sectionsin model group showed that the number of double nuclei was increased the hepatic cords were disarrangedliver cells expanded widely and infiltration oflargenumbers of inflammatory cells was observed Comparedto NC group kidney histopathology in model groupshowed that the glomeruli became atrophic or even disappeared the number of epithelial cells was reduced therenal proximal tubules were dilated Histologically theheart tissue was seen abnormally structured in modelgroupincluding cavitation and deformation in somemyocardial cells nuclear pyknosis and inflammatory cellinfiltration In model group the brain tissue was alsoseen abnormally structured including nuclear pyknosisand incomplete dissolution of nerve fibers The caecallesions including atrophy and breakage of the villus irregular cell arrangement and thinning of the intestinalwall were observed in model group SCML or VE treatment significantly attenuated these abnormal histologicalchanges of the tissues induced by DgalEffects of SCML on oxidative stress gene expression inmiceThe Nrf2 pathway maintains the redox homeostasis exerts antioxidant activity by regulating its multiple downstream cytoprotective genes thereby plays a vital role incell survival The effect of SCML on oxidative stressgene expression is shown in Fig As shown in Fig 5athe Nrf2 expression in model group in liver was lowerthan that of NC group P Except for LSCML μgKg Se group in the liver the Nrf2 expression in liverwas increased all other drug treatment groups comparedwith model group P The Nrf2 expression was increased in the kidney of in HSCML μgKg Se groupcompared to model group P As shown in Fig 5bthe expression of GSHPx mRNA in the liver of modelgroup was decreased P After SCML treatment theGSHPx mRNA expression in the liver was significantly increased compare to model group P and MSCML μgKg group and HSCML μgKg Se group inkidney was increased significantly compared to model groupP As shown in Fig 5c the expression of SOD1mRNA was decreased in model group especially in the kidney compared to NC group P However the expression was obviously increased in the liver of HSCML group μgKg Se and MSCML group μgKg Se compared to model group P Significant change was alsoobserved in SOD1 mRNA expression in the kidney of HSCML group μgKg Se and MSCML μgKgSe group compared to model group P Effects of SCML on oxidative stress protein expression inmiceTo determine whether Nrf2 activation played a role inSCML protection against Dgal induced oxidative stressthe expression of Keap1 Nrf2 and HO1 in the mouseliver and kidney was detected As shown in Fig compared with NC group the western blot results showedthat the Nrf2 and HO1 protein expression in modelgroup was significantly decreased P while theKeap1 protein expression was increased in model groupAfter SCML or VE treatment the Keap1 expression inthe treatment groups was decreased though the difference was not statistically significant Compared withmodel groupthe Nrf2 expression in the treatmentgroups was increased significantly P except forLSCML μgKg Se group in liver Compared withmodel group the HO1 expression in SCML groups wasincreased especially in the liver of HSCML μgKgSe group P Sequencing depth and diversityA total of sequences from all intestinal microbiota samples were produced averaging sequencesFig The effect of SCML on the expression of Nrf2 SOD1 and GSHPx mRNA in the liver and kidney of the mice a Nrf2 mRNA relativeexpression in the liver and kidney b GSHPx mRNA relative expression in the liver and kidney c SOD1 mRNA relative expression in the liver andkidney Values represent means ± SD from three independent replicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with Model P SCML Seleniumenriched C megacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of Fig The effect of SCML on the protein expression of Keap1 Nrf2 and HO1 in the liver and kidney tissue of the mice a Protein strip b Keap1actin relative density c Nrf2actin relative density d HO1actin relative density Values represent means ± SD from three independentreplicates and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Compared with ModelP SCML Seleniumenriched C megacephala larvae powderclusterper sample These sequences resulted in a mean sequencelength of approximately bp Based on the Clean Tagstheanalysis was processed by USEARCHv701090 The sequences were delineated into operational taxonomic units OTUs at similarity Thevalue of coverage for the observed OTUs was above The species accumulation curves showed clear asymptotes and the curve tended to be flat or reached theplateau stage Fig 7a indicating a nearcomplete sampling of intestinal microbial communities of mice Theboxplot of Shannon index showed that the diversity of theintestinal microbiota was decreased in model group compared to NC group and the diversity of VE group and HSCML μgKg Se group was increased compared tomodel group Fig 7b As shown in Fig 7c the contribution value of PC1 and PC2 for the sample difference was and respectively All intestinal microbiotasamples were presented as three distinct groups Thesefindings indicate that the main components of the intestinal microbiota in model group were different from thoseFig Alpha diversity of the gut microbiota and principal component analysis PCA plots based on abundance of operational taxonomic unitsOTUs a Species accumulation curves b Bacterial diversity estimated by the Shannon index c PCA plots SCML Seleniumenriched Cmegacephala larvae powder 0cXie BMC Complementary Medicine and Therapies Page of in NC group After VE and SCML treatment the components of the intestinal microbiota were different fromthose in model group while there was an insignificant difference between HSCML μgKg Se group and NCgroup BacteroidetesEffects of SCML on species structures in miceThe species profiling histogram was obtained to knowthe community structural composition of differentgroups at phylum and genus levels Fig As shown inFig 8a the most prevalent phyla in all samples were Firmicutesand Proteobacteria There were otherphyla level bacteria with low abundance in the intestinaltract of mice As shown in Fig 8b species were usedto describe the relative abundance ofthe intestinalmicrobiota at the genus level showing that Prevotella Helicobacter and Clostridium were the most abundant followed byOscillospira Bacteroides andLactobacillus Effects of SCML on intestinal bacteria of differentclassification levels in miceAs shown in Table Proteobacteria were increased significantly at the phylum level in model group comparedto NC group P Proteobacteria were decreasedsignificantly and Bacteroidetes were increased significantly in VE and SCML groups compared to modelgroup P In addition VE group MSCML μgKg Se group and HSCML μgKg Segroup showed significant differences in Firmicutes compared to model group P and MSCML μgKg Se group showed a significant difference in Actinobacteria compared to model group P As shown in Table there was not significant alteration in Bacteroides Lactobacillus Oscillospira Prevotella and Sutterella at the genus level in model groupcompared to NC group Compared with NC group Helicobacter and Clostridium were increased significantlyand Ruminococcus was decreased significantly in modelgroup P Clostridium Helicobacter and Oscillospira were decreased significantly in VE and SCMLgroups compared to model group P while VEgroup and LSCML μgKg Se group showed a significant difference in Bacteroides P In additionLactobacillus in MSCML μgKg Se group and HSCML μgKg Se group Prevotella in VE groupand Sutterella in MSCML μgKg Se group wereall increased significantly compared to model group P There were not significant alterations in Ruminococcus in VE and all SCML groups compared to modelgroupCorrelation analysis of changes in flora abundance andserum biochemical indexesIn order to explain the relationship between the intestinal flora abundance changes of mice and serum biochemicalindexes Spearman correlation analysis wasperformed to analyze correlation between serum biochemical indexes and Clostridium and Helicobacter theabundance of which were significant difference in eachgroup The change of Clostridium abundance was foundto be negatively correlated with GSHPx and SOD andpositively correlated with MDA There was not significant correlation in Helicobacter and serum biochemicalindexes The specific correlation analysis results wereshown in the Table DiscussionThe results of the present study showed that the dailybehaviors of the mice in model group were differentfrom those of the mice in NC group In addition the tissues of the modeled mice underwent significant pathological changes The antioxidant system parametersincluding GSHPx SOD TAOC and GSH in the antissues or serum were decreased while the MDA andcarbonylated protein levels were increased All these results indicated that the Dgalinduced oxidation mousemodel was successfully established in the present studyVE the monomer of which is often used as the positivecontrol for the studies of aging in mice induced by DgalFig Taxonomic composition of the gut microbiome in the mice a Phylumlevel b Specieslevel SCML Seleniumenriched C megacephalalarvae powder 0cXie BMC Complementary Medicine and Therapies Page of Table Oneway ANOVA test of species differences at the phylum and species level BacteriaNCModelVEPhylumBacteroidetes ± Proteobacteria ± Firmicutes ± GenusActinobacteriaTenericutesBacteroidesClostridiumHelicobacterLactobacillusOscillospiraPrevotellaRuminococcus ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± SCML μgKg Se ± ± ± ± ± ± ± ± ± ± ± ± Sutterella ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Values represent means ± SE n and evaluated by oneway ANOVA followed by the Duncans multiplerange tests Compared with NC P Comparedwith Model P SCML Seleniumenriched C megacephala larvae powder ± ± ± ± [ ] Studiesshowed that mice subcutaneouslyinjected with Dgal in the neck exhibited a significantbody weight declined [] In this study Dgal was foundto significantly inhibit weight gain in mice howeverSCML and VE could increase the body mass in varyingdegrees indicating that SCML and VE could effectivelyenhance the constitution of aging mice Oxidative damage appears in body ans to a large extent Our resultsshowed that Dgalinjection for weeks for mice resulted in severe histopathological changes in the antissues However SCML and VE could alleviate these Dgalinduced pathological damages in an tissues ofmice Recent research work has demonstrated that senescent cells accumulated in various tissues of age anddisease [] Cellular senescence is associated with agerelated phenotypes causally and decreasing senescentcells can retard tissue dysfunction and extend healthspan[] The results suggested SCML c | 2 |
complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen Peoples Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [] Following these discoveries The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions []The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [] and a Cterminalregulatory tail [] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ ] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ ] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins []It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions []its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies []Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as undruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain undruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ ] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ ] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPKssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPKs regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ ] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous []YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucellslatorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic | 2 |
" mutations in the exonuclease domain of pole a dna polymerase associated with dna replicationand repair lead to cancers with ultrahigh mutation rates most studies focus on intestinal and uterine cancers withpole mutations these cancers exhibit a significant immune cell infiltrate and favorable prognosis we questionedwhether loss of function of other dna polymerases can cooperate to pole to generate the ultramutatorphenotypemethods we used cases and data from cancer types in the cancer genome atlas to investigate mutationfrequencies of different dna polymerases we tested whether tumor mutation burden patient outcomediseasefree survival and immune cell infiltration measured by estimate can be attributed to mutations in polqand polzrev3lresults thirty six percent of colorectal stomach and endometrial cancers with pole mutations carried additionalmutations in polq eq polzrev3l ez or both dna polymerases ezq the mutation burden in thesetumors was significantly greater compared to poleonly e mutant tumors p in addition eq ez and eqz mutant tumors possessed an increased frequency of mutations in the pole exonuclease domain p colorectal stomach and endometrial eq ez and eqz mutant tumors within tcga demonstrated diseasefree survival even if the pole mutations occurred outside the exonuclease domain p however immunescores in these tumors were related to microsatellite instability msi and not pole mutation status this suggeststhat the host immune response may not be the sole mechanism for prolonged diseasefree survival ofultramutated tumors in this cohort results in this study demonstrate that mutations in polq and rev3l in pole mutant tumors shouldundergo further investigation to determine whether polq and rev3l mutations contribute to the ultramutatorphenotype and favorable outcome of patients with pole mutant tumors correspondence beatriceknudsenpathutahedu1department of biomedical sciences cedarssinai medical center losangeles ca usa2samuel oschin cancer research institute socci cedarssinai medicalcenter los angeles ca usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chuang bmc medical genetics page of the analysis of thousands of cancers by the cancergenome atlas tcga consortium and academic institutions revealed a small group of cancers with mutationsin pole and an ultramutator phenotype [ ] multiplestudies have found significantly improved survival in patients with pole mutated endometrial cancers [ ]while the survival benefit was not as profound in polemutated colorectal carcinoma the pole geneencodes the catalytic subunit of dna polymerase epsilon which catalyzes the leading strand synthesis duringdna replication pole possesses highfidelity dnapolymerization proofreading and ²² exonuclease activities which promote accurate dna synthesis firstidentified and reported in of colorectal carcinomas[ ] pole mutations were also noted at frequencies of amongst uterine corpus endometrialcancers [ ] and in gastric adenocarcinoma mutations can be found across the entire pole genebut those in the pole exonuclease domain are mostprevalent in cancers with ultrahigh mutation rates mutmb these cancers exhibit higher mutationrates than microsatellite instable msi tumors associated with mismatch repair abnormalities in additionpole ultramutant cancers also possess a high frequency of ctoa transversions [ ] a tumor associated inflammatory response similar to thatin msitumors has been reported to occur early on duringdevelopment of pole mutated endometrial and colorectal cancers it is thought to be caused by neoantigens that are generated as a result of the high mutation burden [ ] and render pole mutant cancersresponsive to immunotherapy polymerase theta polq is a lowfidelity dna polymerase lacking a ² to ² exonuclease function theenzyme is involved in the alternative nonhomologousendjoining pathway altnhej which is a backupmechanism of double stranded dna break repair thispathway predominates in cancer cells when other dnarepair pathways are missing or when telomere ends aredeprotected [ ] the loss of polq sensitizes cellsto ionizing radiation and polqdeficient mice exhibit increased dna instability and genomic rearrangementssuggesting a role for polq as a guardian of the genome both overexpression and loss of polq increasemutation frequencies [ ] multiple structuralmotives in polq can interact with dna rad51 andbrca1 in addition polq forms a complex withparp1 in a pathway of synthetic lethality with brca1and is thus considered a therapeutic target [ ]however which domains in polq should be targetedremains to be determined rev3l rev3 like dna directed polymerase zetapolz catalytic subunit is involved in dna synthesisthat reads through damaged dna translesion dnasynthesis tls the high efficiency of polz bypassing a broad spectrum of dna lesions led to its recognition as a master tls polymerase polzrev3l has been linked to carcinogenesis in breastlung gliomas and gastric cancers and modulatescisplatin sensitivity []because studies describe overlapping functions andsynergy among the over dna polymerases [ ] we undertook an unbiased approach to identifymutations in polymerases within the tcga tumorcompendium this analysis revealed a greater frequencyof mutations in polq and polzrev3l compared toother polymerases of similar gene length therefore wepropose that polq and polzrev3l may cooperatewith pole in generating ultrahigh mutation ratesmethodsdata acquisitionthe data used in this study are based upon the wholeexome sequence data sets generated by the tcgaresearch network httpcancergenomenihgov thelocations and frequencies of somatic mutations msistatus and clinical stage and follow up information intcga provisional datasets were obtained from cbioportal httpwwwcbioportal [ ] up to supplementary table mutation data were obtainedby first selecting the query tab from cbioportal thecancer type specific data were chosen from the tcgapancancer atlas studies we entered the genes ofinterest pole polq and rev3l to retrieve mutationdata from the genes when the result page wasdisplayed we accessed the information underneath themutations tab we downloaded the number of mutations in each sample the annotation of protein aminoacid change and the type of mutation the survivalinformation for each case months after diagnosis wasobtained through the comparisonsurvival link functional domains of the proteins were provided by pfamdatabase data visualization for mutations wasperformed with mutationmapper in cbioportalcase selection criteriawe searched all cancer types in tcga for those thatpossess or more cases with pole mutations thisyielded tumor types that we named the pancandata set in this study we then determined the frequencyof mutations within additional polymerase within pancan we selected the three adenocarcinomas uterinecorpus endometrial adenocarcinoma colorectal adenocarcinoma and stomach adenocarcinoma with the highesttriple mutated dnapolymerase status for more detailed analysisfrequency of double or 0chuang bmc medical genetics page of studies performedkaplan meier survival plots were generated using clinicalfollowup data available within the tcga database todetermine the global mutational spectrum we classified types of nucleotide transitions or transversions thefrequency of each mutation type was calculateddigital images of all eq mutant tumors and representative cases of tumors with neither pole nor polqmutations and of msi tumors were assessed by one author jr for the amount of tumor immune infiltratethe combination of tumor infiltrating lymphocytes andthe peritumorallymphoid infiltrate was graded on ascale between and tumor associated lymphocyteswere graded as none minimal rare to per highpowered field hpf to per hpf and perhpf peritumoral lymphocytes were assessed at thedeepest advancing tumor front graded at low powernone minimal mild moderate andmarked these visual semiquantitative scores werecompared with the immune scores evaluated using estimate estimation of stromal and immune cells inmalignant tumor tissues using expression data estimate score were obtained from the website ofestimate at md anderson httpsbioinformaticsmdandersonestimatediseasehtml theplatformtype selected was rnaseqv2 data for colorectalstomach and endometrial cancer types were downloaded next we separated the colorectal stomach andendometrial cancer cases into quartiles defined by thehighest intermediate and lowest of mutational counts or msi status and calculated the immunescores for each groupstatistical data analysisall statistical analyses were conducted in r v313 plotswere generated using ggplot2 package in r datavisualization methods were described previously the horizontal lines in the boxplots represent the 1st2nd and 3rd quartiles and whiskers outside the boxshow the interquartile range the significance of thedifferences of data illustrated in the boxplots wascalculated using the wilcoxon ranksum tests the chisquare test was performed to test the significance ofdifferences in frequencies of all tables the significancein the kaplanmeier survival plot was calculated usingthe log rank test statistical significance was accepted atp resultsof the cancer types in the tcga database weidentified cancer types pancan with or morecases that possessed mutations in the pole proteincoding region fig 1a and supplementary table these cancer types contained cases with polemutations anywhere in the exome of these polemutant tumors carried mutations in one or more of the other dna polymerases we observed dna polymerases polq and polzrev3l to be most frequentlymutated fig 1b these two polymerases were mutatedin of tumors with pole mutations in fact thesetwo polymerases were even more commonly mutatedthan pole in our pancan cohort supplementary figure altogether cases with pole and polq mutations eq cases with pole and polzrev3lmutations ez and cases with pole polq andpolzrev3l eqz mutations were identified in thepancan cohort fig 1c mutations in the exonucleasedomain of pole are responsible for causing the ultramutator phenotype in colorectal and uterine corpuscancers [ ] in order to determine the contribution of polq and rev3l to the ultramutator phenotype we compared the mutation frequencies of tumorswith mutations in only pole to eq ez and eqzmutant tumors mutation frequencies in the cellulargenome increased in the following order no polemutations poleonly mutations anywhere with thepole exome eq ez eqz mutations fig 1dthe median mutation count of eqz tumors was morethan 10fold higher p than that of tumors withonly pole mutations e q and e z mutant tumorsalso displayed significantly higher mutation countscompared to eonly mutant tumors suggesting a contribution of mutationally altered polq or polzrev3l tothe overall cancer mutation rates next we determinedthe number of mutations in the exonuclease and polymerase domains of pole in the cancer types withinour pancan compendium fig 1e the percentage ofpole mutations in the exonuclease domain was greaterin eq ez and eqz mutant tumors compared topoleonly mutant tumors p in contrast thepercentage of mutations in the dna polymerase domainwas similar thus our data confirm the notion thatmutations in the exonuclease domain of pole areresponsible for ultrahigh mutation rates in additionmutations in polq and rev3l may further increasethe mutation burden however why mutations in polqand rev3l preferentially increase tumor mutationfrequencies remains elusive endometrialto further investigate a potential role of these mutantdna polymerases in the ultramutator phenotype wefocused on colorectalucec andstomach stad cancers these cancer types containthe highest numbers of tumors with eq ez and eqz amongst the cancer types included in pancanfig 1a supplementary table among these cancertypes we identified cases with eq cases with ezand cases with eqz mutations fig 2a in thesecancers the mutation burden in pole eq ez and e 0chuang bmc medical genetics page of fig cancer types pancan with poleqz mutations in tcga a number of cases with pole only eq ez and eqz mutations in cancertypes cohort referred to as pancan within tcga the xaxis shows the actual number of cases with pole green eq orange ez pink and eqz blue mutations the yaxis displays the cancer types uterine corpus endometrial carcinoma ucec stomach adenocarcinoma stadcolon and rectum adenocarcinoma skin cutaneous melanoma skcm lymphoid neoplasm diffuse large bcell lymphoma dlbc lungadenocarcinoma luad breast invasive carcinoma brca sarcoma sarc cervical squamous cell carcinoma and endocervical adenocarcinomacesc pancreatic adenocarcinoma paad lung squamous cell carcinoma lusc head and neck squamous cell carcinoma hnsc bladderurothelial carcinoma blca kidney renal clear cell carcinoma kirc and liver hepatocellular carcinoma lihc b case numbers with mutationsin polymerase genes the number of cases in pancan with mutations in the following polymerases is displayed on the yaxis dntt pola1polb pold1 polg polh poli polk poll polm poln polq rev1 rev3l c venn diagram displaying the number of cases in pancan withmutations in or pol genes d mutations per mb yaxis of pancan cases without pole mutations other or with pole eq ez and eqzxaxis mutations number of cases in each group are listed in parenthesis e mutation frequencies in pole exonuclease and polymerase domainsas a percentage of total number of mutations in the pole exome other refers mutations in the entire exome outside the exonuclease orpolymerase domains the cases are grouped by their polymerase mutation status on the yaxis and the number in parenthesis represents thetotal number of pole mutations within each group 0chuang bmc medical genetics page of fig poleqz mutations in colorectal endometrial ucec and stomach stad cancers a venn diagram displaying the number of caseswith mutations in or pol genes b mutation groups of cases without polymerase mutations other or with mutations in pole only eq ez and eqz the number of cases in each group is listed in parenthesis the pvalue for comparison of pole and eq groups is not significantp c number of cases with mutations in pole exonuclease domain in various mutation groups d percentages the ratio of transitions tiand transversions tv are shown on the yaxis for core stad and ucec the xaxis shows the mutation groupsqz mutant tumors paralleled the mutation burden inthe whole pancan cohort compare fig 2b and fig1c supplementary figure 2ad eqz mutant tumors demonstrated on average an 8fold increase inmutation frequencies compared to tumors with onlypole mutations amongst pole mutant tumors tumors carried mutations outside the pole exonuclease domain while tumors carried mutations withinthe exonuclease domain the frequency of poleexonuclease mutationsin fig 2cprovides a valid explanation forthe difference inmutation rates and potential association of poleexonuclease domain mutations with mutationsinpolq and polzrev3l which are the other twomost frequently mutated dna polymerases casesoverall exonuclease domain mutations were identifiedin cases of pole only mutant tumors eqcases ez cases and of eqz cases fig 2cstratified by cancer types pole exonuclease domainmutations occurred in colorectal endometrial and stomach tumors demonstrating cancertype specific frequencies supplementary figure 3a incontrast to the pole gene that demonstrates mutationalhotspots in the exonuclease domain mutational hotspotsin the polq gene are not associated with a functionalprotein domain supplementary figure 3b c dwhile rev3l does not reveal mutational hotspotsapproximately of mutations lead to truncated protein expression supplementary figure 3e f anothercharacteristic of pole mutant tumors are c to a and g 0chuang bmc medical genetics page of to t transversions we observed the greatest increaseof nucleotide transversions in cancers with eqz mutafig 2d consistent with the loss of poletionsexonuclease activity in these tumorssince mutations in pole confer increased disease freesurvival dfs in patients with uterine cancer even inthose patients with highgrade tumors [ ] we investigated the prognostic role of polq and rev3l mutations in pole mutanttumors kaplanmeier curveswere constructed for colorectal endometrial and stomach cancer cases with followup data fig 3a using thetcga annotations of dfs in individual patients nocancer recurrences were observed in the eq ez andeqz mutant groups pole exonuclease domain mutations were observed in cases in the good survivalgroup and case in the poor survival group consistentwith the expected long dfs periods of patients withpole exonuclease domain positive tumors in additionanalysisin the pancan cohort cases with mutations in pole outside the exonuclease domain were in the good survival group of those had concurrent mutations in polq or rev3l orin both polymerases fig 3b furthermore a kaplanmeierrevealedimproved dfs associated with mutations in polq andrev3l the favorable survival outcome was observed incolorectal endometrial and stomach cancers howeverno favorable outcome was observed in diffuse bcelllymphoma p these data provide preliminaryevidence of cancertype specificfavorable survivaloutcomes in tumors with pole mutations that arelocated outside the pole exonuclease domain if concurrent mutations in polq rev3l or in both polymerasesare presentcompared to microsatellite stable tumorsmssmicrosatellite instability msiin colorectal cancerconfers a better prognosis to determine whetherfig survival and clinical characteristics of patients with polymerase mutations colorectal endometrial ucec and stomach stad cancers akaplanmeier curves of diseasefree survival dfs for groups of patients pole only n median followup months green line eqn median followup months orange line ez n median followup months pink line and eqz n median followup months blue line tumors without mutations in pole polq or rev3l exomes grey line the overall pvalue is p individual pvalues eqz vrs pole p eqz vrs none p eq or ez or eqz vrs pole p b polymerase mutation analysis ofcases in the good survival group in panel a the red bar indicates cases with pole exonuclease mutations c cancer typespecific illustration ofmutation count pole polq and rev3l mutations microsatellite instability msi and tumor stage 0chuang bmc medical genetics page of the favorable outcome of eq ez and eqz mutantcancers can be explained by msi or tmn stage we examined the relationship between msi statustumorstage and polymerase mutations in colorectal endometrial and stomach cancers fig 3c and supplementaryfigure and supplementary table despite improveddfs rates the full range of tumor stages was observedamongst eq ez and eqz tumors p supplementary table 2a comparing the poleonly and eqz mutant cancers did not reveal a significant difference in tumor stage but differed in the frequency ofmsi cases p pole mutant tumors were morefrequent in the msi group than in themss group in addition the frequency ofmsi cases in pole mutant tumors differed between the cancer types p supplementary table 2b c although msi is enriched in samples with highmutation levels supplementary table 2d as expectedp were10fold higher mutation countsobserved in cancers with eqz mutations compared tomsi without eqz mutations supplementary figure these results suggest that mutations in eq ez and eqz confer a better prognosis independent of msi statusand tmn stage in colorectal endometrial and stomachadenocarcinomaswe next examined the amount ofthe cancerassociated immune infiltrate the immune scoreobtained through estimate corresponded tothe categorical score of the immune infiltrate derivedfrom digital he images supplementary fiure therefore we used the estimate immune scoresfor further analysis of colorectal endometrial andstomach cancers as shown in fig 4a a significantdifference was observed in the median immunescores between groups with lowintermediate andhigh mutation burden grouped based on mutationburden and not on e q z mutantseemethods and as expectedthe median immunescores increased with total mutation levels surprisingly the immune scores in eqz mutant tumorsdid not differ significantly from tumors with a lowlevel of mutationsfig 4b as expected msitumors possessed higher immune scores than msstumors p immunescores of msi and eqz mutation tumors weresimilar to those in the msi group and higher thanmss and eqz mutation tumors fig 4d withinthe group of tumors with pole exonuclease domainmutations mss tumors possessed lowerimmunescores than msi tumors but the difference was notsignificanttogether results in this tcga cohort demonstratethat the immune response is driven by msi ratherthan pole exonuclease domain mutationssupplementary fig 4c finallystatusp figurediscussionan analysis of dna polymerases in tumors withmutations in the pole revealed additional mutations inspecific polymerases most commonly in polq andpolzrev3l among the cancer types colorectaluterine and stomach cancer were mostfrequentlyafflicted by these mutations cancers with mutations inpole and polq eq pole and polzrev3l ezand in all polymerases eqz were associated withthe highest mutation burden and an excellent prognosisindependent of msi status and tumor stage mutationsin the exonuclease domain were observed in of eqz mutant tumors but only in of poleonly mutant tumors or in of eq ez tumorshowever despite harboring 10fold more mutationsthan msi tumors and 8fold more mutations than themutation frequencies associated with poleonly mutanttumors eqz mutant tumors did not display significantly more inflammationthe main result from that analysis is that patients withcolorectal stomach and endometrial cancers bearing eq ez and eqz mutations have disease free survival dfs at a median follow up time of months incontrast patients with tumors bearing mutations inpole only most of which outside the pole exonucleasedomain had a dfs of at follow up of monthsthe favorable dfs in eq ez and eqz mutated tumors occurred even in tumors with mutations in polethat are located outside the pole exonuclease domainthe contribution of mutations in pole to tmb casubstitutions and cancer type associations are describedin table of raynor using a larger resourceof cases and should be used to interpret the mutationsin the current study listed in supplementary figure as a whole the current study expands the spectrum ofpole mutant tumors with an excellent prognosis thefavorable prognosis included patients with high tumorstage which echoes prior studies demonstrating a favorable outcome of uterine tumors with pole exonucleasemutations despite adverse standard clinicopathologicindicators including high grade high stage and lymphovascular invasion [ ] while the high mutationfrequencies may cause an early growth advantage as tumors evolve they may succumb to high mutationburden as new mutations can no longer be tolerated andcause tumor cell death [ ] or increased sensitivityto therapeutic agentsthe prevailing hypothesis for the favorable prognosisof cancers displaying the hypermutator phenotype is theincreased attack by the immune system evidence insupport of this theory is the observation that tumorinfiltrating and peritumorallymphocytes are increased and that cytotoxic activities in cd8 and cd4are heightened in polelymphocyte populations 0chuang bmc medical genetics page of fig estimate immune scores by mutation frequency quartiles eqz mutation groups and msi in colorectal endometrial ucec and stomachstad cancers a estimate immune scores in cancers within high intermediate and low overall mutation quartiles b immune scores of sampleswith eq ez and eqz mutations compared to the low mutation quartile from panel a c immune scores in groups of cancers separated by msistatus d immune scores in msi and mss eqz cases compared to all other msi cases for each panel the number of cases within each group isincluded in parentheses on the xaxismutated endometrial cancers [] similar to hypermutated msi tumors this observation has led tothe hypothesis that immune checkpoint inhibitors maybe efficacious in pole ultramutated tumors ourresults question a direct relationship between mutationburden tumor immune response and pdl1 expressionalso raised in a larger study across cases from cancer types in tcga while we observed a concordance between the computational and histological assessments of the immune infiltrate the immune score intumors with eqz mutations depended on msi statusthis result suggests that the immune infiltrate attributable to mutations in eqz mutant tumors may be lessor that its composition may involve immune cells otherthan lymphocytes lesser cd8 and gammainterferongene expression signatures have also been observed ingastrointestinal tumors with a large single nucleotidevariantsnv burden that was attributed largely topole exonuclease mutations perhaps eqz mutations occur at a later point in tumor evolution whenimmunosuppressive factors already dominate we alsocannot rule out the possibility of increased numbers ofcytotoxic lymphocytes intermixed with eqz mutanttumor cells because computational methods and inspection of he images are not sensitive enough to detectsmall differencesinfiltrating lymphocytestils that may have large antitumoral effectsin tumora significant limitation of the study lies in the relatively small number of tumors this limitation cautionsthe generalization of results and seemingly novel insights 0chuang bmc medical genetics page of into the hypermutator phenotype studies by othergroups attributed hypermutator phenotype to specificmutations primarily within the pole exonucleasedomain given the proofreading function of the exonuclease domain it makes sense that mutations outsidethe domain have a lesser effect on tmb in agreementwith this concept our study reveals that compared totumors harboring only a mutation in pole polesinglemutant tumors of cases pole exonucleasedomain mutations are more common in tumors withboth double ez and eq and triple eqz dnapolymerase mutations of cases and doubleand triple mutant tumors have higher mutation countsthan pole single mutant tumors while it cannot befully excluded that polq and polzrev3l mutationsare bystander events in pole mutanttumors weobserve a higher tmb in cases with mutations in allthree polymerases supplementary figure 2a and bmechanistically polq and polz are thought to function in different repair processes polq in alternativemicrohomologymediated nonhomologous dna repair pathway and polz in translesion dna synthesishow these dna repair processes cooperate with thereplicative dna polymerase pole to prevent genomeinstability remains unknown this will be an importantsubjectthe mechanismfor further understanding ofunderlying the hypermutator phenotypesif validated in additional cohorts our findings may haveimportant clinicalimplications they build upon andexpand the previously well documented good prognosticimpact of pole exonuclease mutationsin uterinecancer that have generated intense interest in part dueto the paradox of a favorable prognosis in tumors withpathologic indicators of poor prognosis while in thisstudy prolonged dfs is observed in colorectal endometrial and stomach cancers with eqz mutations thisis not the case in other noncarcinoma cancer typeswithin tcga thus we find that the positive outcomeprediction is cancer type specific altogether resultsfrom this study provide a rationale for including polqandor polzrev3l mutations in clinical outcomestudies of tumors with pole mutations however future validation is required to confirm the concept that isrevealed in the current studycolorectal core stomach stad and endometrial ucec cancers byspecific polymerase mutated groups in tcga data sets supplementaryfigure locations of mutations in pole polq and rev3l exomes inindividual colorectal core stomach stad and uterine cancers ucecsupplementary figure relationships between mutation spectrumand mutation counts pole polq and rev3l exome mutations msi andtumor stage of individual cases supplementary figure mutationrates per mb yaxis of core stad and ucec cases with msi and eq ez and eqz xaxis mutations supplementary figure relationshipbetween pathology inflammation score and estimate immune scores forcore stad and ucec supplementary figure estimate immunescores in colorectal core endometrial ucec and stomach stadcancers supplementary table number of cases with pole polq zrev3l or multiple exome mutations in the pancan cohortsupplementary table contingency tables showing number of casesof colorectal endometrial and stomach cancers in each categoryabbreviationspole polymerase epsilon polq polymerase theta polzrev3l rev3 likedna directed polymerase zeta polz catalytic subunit tcga the cancergenome atlas msi micro satellite instability mss micro satellite stabilitytmb tumor mutation burden tnm tumor lymph node metastasis estimate estimation of stromal and immune cells in malignant tumor tissuesusing expression dataacknowledgementsnot applicableauthors contributionsall authors have read and approved the content of this manuscript fhstudy design and data analysis ht data interpretation jr data analysis andmanuscript writing bsk data interpretation and manuscript writingfundingthe prostate cancer foundation funded salaries of fh and bsk forcomputational analysis steven spielberg team science award andr01ca131255 bsk funded salaries for data analysis and paper writing of fhand bsk we also acknowledge the institutional support of salaries throughthe nih g20 rr030860 to the cedarssinai biobank and translational research core for salaries of bsk and fh the content of this publication doesnot necessarily reflect the views or policies of the department of health andhuman services nor does any mention of trade names commercial products or anizations imply any endorsement by the us governmentavailability of data and materialssequencing data can be obtained from national cancer institue gdc dataportal and are published in raw genomic and clinical data can befound at the nci genomic data commons httpsportalgdccancergovlegacyarchive the mc3 mutation annotation file can be accessed at httpsgdccancergovaboutdatapublicationsmc32017 and the processed datafiles can be viewed at httpsgdccancergovaboutdatapublicationspancanatlasethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors do not declare any competing interestssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12881020010899additional file supplementary figure number of cases polemutations n and mutations in the exomes of dna polymerasegenes in pancan supplementary figure mutation counts forauthor details1department of biomedical sciences cedarssinai medical center losangeles ca usa 2samuel oschin cancer research institute soccicedarssinai medical center los angeles ca usa 3surgerycedarssinai medical center los angeles ca usa 4pathology andlaboratory medicine cedarssinai medical center los angeles ca usa 5department of pathology university of utah salt lake city ut usa 0chuang bmc medical genetics page of received january accepted july referenceskandoth c schultz n cherniack ad akbani r liu y | 0 |
environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at Ëc under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbecos modified eagles medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ ]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hamss f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at Ëc for sannealing at Ëc for s and extension at Ëc for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at Ëc with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukeys or dunnetts posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukeysposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [] and cancer cells [] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when | 0 |
" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didnt show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision¢ flex mouselinker and then envision¢ flex hrp reagent finally the ihc was visualized by envision¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplanmeier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didnt reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of heà and pdl1à ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range bmnegativepositiveihc hans algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueÏ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didnthave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci p_valueoshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci p_value pfshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s aswerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncollesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" lymph node staging of ductal adenocarcinoma of the pancreatic head pdac by crosssectionalimaging is limited the aim of this study was to determine the diagnostic accuracy of expanded criteria in nodalstaging in pdac patientsmethods sixtysix patients with histologically confirmed pdac that underwent primary surgery were included inthis retrospective irbapproved study crosssectional imaging studies ct andor mri were evaluated by aradiologist blinded to histopathology number and size of lymph nodes were measured shortaxis diameter andcharacterized in terms of expanded morphological criteria of border contour spiculated lobulated and indistinctand texture homogeneous or inhomogeneous sensitivities and specificities were calculated with histopathologyas a reference standardresults fortyeight of patients had histologically confirmed lymph node metastases pn sensitivityspecificity and youdens index for the criterion size were and for inhomogeneous signal intensity and and for border contour and respectively there was a significant associationbetween the number of visible lymph nodes on preoperative ct and lymph node involvement pn p lymph node staging in pdac is mainly limited due to low sensitivity for detection of metastatic diseaseusing expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivityremaining low combining specific criteria yields improved sensitivity with specificity and ppv remaining highkeywords ductal adenocarcinoma of the pancreatic head staging lymph nodes computed tomography magneticresonance imaging crosssectional imaging neoadjuvant therapy correspondence florianlochcharitede1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germanyfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cloch world of surgical oncology page of pancreatic cancer remains one of the most lethal malignancies being the fourth leading cause of cancerdeath in the usa and predicted to be the secondleading cause of cancer death by the overall5year survival after diagnosis is and at thetime of diagnosis the main proportion of patients hasadvancedstage disease leaving only qualifiedfor resective surgery pancreatic cancer is locatedin the head of the pancreas in of the cases even after successful resective surgery in patients withcancer of the pancreatic head the 5year survival remains as low as these data underline theimportance of establishing multimodaltherapeuticconcepts for patients with pancreatic cancer as perother entities of abdominal cancerapart from the potential to increase the resectabilityrate of pancreatic cancer by neoadjuvant therapy [ ]there is evidence that patients which are successfullydownstaged from nodepositive disease cn1 to nodenegative disease ypn0 prior to surgery benefit in termsof higher 5year survival rate this would qualifynodalinvolvement as a sufficient basis for indicatingneoadjuvant therapy yet even given advanced imagingtechnologies identifying lymph node metastasis remainschallenging consequently the indication of a potentiallyeffective neoadjuvant therapy cn with side effects inlymph nodepositive patients cn is mainly based onunreliable clinical stagingthe established criterion for lymph node involvement in pancreatic cancer is size using the size underlies the assumption that tumor spread to regionallymph nodes leads to an enlargement of the respective lymph node the usual cutoff value is a shortaxis diameter of mm [ ] it has been shownthough that lymph nodes of ¥ mm are not seenmore frequently in patients with histopathologicallymph node involvement pn in various othertumor entities expanded morphological criteria suchas texture and border contour oflymph nodes areused for the assessment oflymph node malignancyon both computed tomography ct and magneticresonance mr imaging this is utilized in order toimprove the accuracy of lymph node staging []by applying morphological criteria instead of brown size criterion alone the sensitivity was improvedfrom to and the specificity from to inlymph node staging of rectal cancer thus the aim of this study was to determine thelymph node staging in patients withaccuracy ofductal adenocarcinoma ofthe pancreatic head byboth computed tomography and magnetic resonanceimaging using sizeand expanded morphologicalcriteriamaterial and methodspatientsin this retrospective singlecenter study approved by thelocal ethics committee consecutive patients with histologically proven ductal adenocarcinoma of the pancreatichead that underwent primary surgery between february and november at the department of surgerycampus benjamin franklin charituniversity medicine berlin germany were included patients were retrieved from the database of our pancreatic cancercenter certified by the german cancer society n inclusion criteria were primary oncologic tumor resection and the presence of preoperative crosssectional imaging of sufficient quality see below exclusion criteriawere neoadjuvant therapy presence of a potential simultaneous cause of lymphadenopathy of the upper abdominal region eg abdominal lymphoma neuroendocrinetumor and main tumor mass located outside the pancreatic head on histopathology the process of patientselection with the respective reasons for inclusion andexclusion is shown in fig crosssectional imagingall images were retrospectively analyzed for the purposeof this study by a single abdominal radiologist with morethan years of experience in staging of tumors of the visceral ans blinded to the results of histopathologyall crosssectional imaging studies were assessed forsufficient image quality by the radiologist prior to commencement for ct imaging the minimum quality wasdefined as either thinsection ct mm reconstructedslice thickness or contrastenhanced ct with a slicethickness of mm for mri minimum quality was defined as availability of an axial t2weighted sequencewith fat suppression slice thickness mm in combination with a venous phase postcontrast 3d gradientecho sequence slice thickness mmfor lymph node assessment all visible regional lymphnodes in the field of view were recorded on a score chartand the total number of visible lymph nodes per patientwas calculated then for each patient all lymph nodeswere characterized in terms of size long and shortaxisdiameter in millimeters and the expanded morphological criteria border contour lobulated spiculated indistinct or unaltered and texture homogeneous orinhomogeneous fig based on kim regional lymph nodes of the pancreas are defined asthe following lymph node station numbers 8a8p 11p 11d 12a 12b 12p13a 13b14p14d 17a17b and in all cases in which a lymph node wasnot definitively regional correlation with postoperativecrosssectional imaging was performed to assess whetherthe lymph node was resected or not only resectedlymph nodes were analyzed in this study 0cloch world of surgical oncology page of fig flowchart of patient recruitment the process of patient selection with the respective reasons for inclusion and exclusion is showna second radiologist with more than years of experience in staging of tumors of the visceral ans alsoblinded to the results of histopathology evaluated thect examinations of a representative subgroup of patients for evaluation of interobserver agreementsurgeryall patients underwent primary oncologic pyloruspreserving pancreaticoduodenectomy or whipple procedure with complete lymphadenectomy of the regionallymph nodes mentioned aboveformalinembedded surgicalhistopathologythe original histopathological reportsfor the studyspecimens wereusingreviewed cancer of the pancreatic head was defined as amalignant tumor located within the pancreas to the rightof the superior mesenteric vein and portal vein each patient with histologically proven lymph node metastaseswas classified as nodepositive pn regardless of thenumber of metastatic lymph nodes patients without anymetastatic lymph nodes were classified as nodenegativepn the ratio of metastatic lymph nodes vs the totalnumber of retrieved lymph nodes was documented infig morphological characterization of lymph nodes based on kim the morphological criteria for lymph node assessment used inthis study are shown smooth and homogeneous lymph nodes were considered normal 0cloch world of surgical oncology page of the histopathological report eg or tumorswere classified according to their respective tnm stageusing the 8th edition of tnm classification of malignant tumors table demographic data of patients with ductal adenocarcinomaof the pancreatic head undergoing primary tumor resectionpatientsagen comparison of crosssectional imaging andhistopathologysensitivity specificity and positive predictive value ofthe nodal status using ct and mri with histopathologyas a reference standard were calculated for lymph nodeinvolvement using size and morphological criteria specifically nodal involvement criteria were based on eithersize shortaxis diameter altered border contour lobulated spiculated or indistinct and inhomogeneous signal intensity fig ct and mri examinations wereconsidered nodepositive cnif at least one lymphnode met one of the respective criteria used for involvement if no lymph node with the respective criteria wasseen on ct or mri then the examination was considered nodenegative cnstatistical analysissensitivities specificities and positive predictive valueppv for the size criterion and all morphological criteria were calculated for their respective cutoff valuesan index summarizing the sensitivity and specificity foryoudens index was calculated sensitivity specificity the number of lymph nodes visible on ctand mr images in the group with pn and withoutpn nodal metastases was compared using the mannwhitney u test when calculating the association between ct and mri criteria and lymph node positivitythe Ï2test was used interobserver agreement was calculated using cohens kappa statistic a p value of was considered to indicate a statistically significantdifferenceresultspatientssixtysix patients were included in the study fig with the characteristics of the patients presented intable sixty of these patients were staged by preoperative ct twelve of which had additional stagingby mri and six patients were staged by only mri intwo patientsthe mri examinations were excludeddue to insufficient imaging quality both patients hadsufficient staging by ct and were therefore includedin the study of the patients patients receivedpreoperative biliary drainage eight ofthem werestaged by ct only and two by mri onlycomputed tomography ctlymph nodes were detected by ct in ofthe patients the median number of visible lymph nodesmedian age yearsage range yearssexfemalemalecrosssectional imagingct onlyct and mrimri onlyhistopathological stagingpnpnpt1pt2pt3 was range the smallest visible lymph node was mm of size whereas the largest measured mmshortaxis diameter the mean time between ct andsurgery was days with a median of days range the slice thickness in of the ct examinations was mm or less in five ct examinations slice thickness was mmsize criterion for lymph node involvement onpreoperative ctfigure shows the percentage of patients with pnand without pn lymph node metastases in which alymph node of the respective size was visible mmin table sensitivity specificity and youdens indexare presented for the respective cutoff values lymphnodes of small and medium size mm were visiblein patients with pn and withoutlymph node metastases pnineven frequency large lymph nodes mm wereseen more frequently in the lymph nodepositive group pnthan in the lymph nodenegative group pn the maximumvalue of youdens index for the size criterion was j ci when a cutoff value of mmwas applied yielding a sensitivity of and specificityof additionallylymph nodesgreater than mm on preoperative ct and the histopathological confirmation of a lymph node metastasispn showed a trend towards significance p the presence of 0cloch world of surgical oncology page of fig graph showing lymph node size and morphological criteria of lymph nodepositive and negative patients frequency of regional lymphnodes of the pancreatic head in percent xaxis with different shortaxis diameters and morphological features yaxis in patients with pn redbars or without histologically proven lymph node metastases pn blue bars on preoperative ct imagingexpanded morphological criteria for lymph node involvementon preoperative ctfigure shows the percentage of patients with pnand pn without lymph node metastases in which alymph node of the respective morphological criterionwas visible and table shows the sensitivity specificityand youdens index of the respective criterionlymph nodes of lobulated border contour were visiblewith a similar frequency in patients with pn and without lymph node metastases pn lymph nodes of spiculated or indistinct bordercontour were only occasionally detected in both groups vs in the lymph nodepositivegroup pn and vs in the lymphnodenegative group pnlymph nodes of inhomogeneous signal intensity were detected in only one patient of the lymph nodenegative group pn and more frequently in patients of the lymphnodepositive group pn resulting in the maximum value of youdens index for the morphological criteriaj ci consisting of a sensitivity of and a specificity of the ppv was comparison of size with expanded morphological criteriathe maximum value of the youdens index of the sizecriterion was j ci cutoff mmwhich is not inferior to the maximum value of the morphological criteria j ci inhomogeneoussignal intensity figure displays respective ct images ofpatients with and without lymph node metastasesfig ct images of patients with and without lymph node metastases a patient with enlarged suspicious lymph node adjacent to the portalvein and hepatic artery who had no lymph node metastases on pathology b patient with enlarged suspicious lymph node adjacent to thehepatic artery who had lymph node metastases on pathology c patient with multiple suspicious lymph nodes based on size and inhomogeneitywho had lymph node metastases on pathology 0cloch world of surgical oncology page of table sensitivity specificity ppv and youdens index for cutoff values and morphological criteria by ct and mrisensitivity specificity ppvyoudens indexctsize cutoff value mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctinhomogeneous mrisize cutoff value mm mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctnot visibleinhomogeneousnot visible number of visible lymph nodesthere was a significant association between number of visible lymph nodes seen on preoperativect and histopathologicallymph node involvementpn p seven or more lymph nodes were seen on preoperative ct in of patients with lymph nodemetastases pn and in of patients without lymph node metastasis pn p this resulted in a specificity of youdens index of and ppv of combination of number size and expanded morphologiccriteria on preoperative ctcombining the size criterion and the morphological criterion with the respective highest youdens index cutoff mm and inhomogeneous signalintensity andthe criterion visible lymph nodes n ¥ was significantly associated with nodal metastases pn p for this combined criterion specificity was sensitivity ppv and youdens index ci interobserver agreementinterobserver agreement was calculated for patientspn pn vs pnfor the criteria sizemorphology and number of visible lymph nodes withthe respective highest youdens indexinterobserveragreement was substantial for size mm cutoff κ p moderate for the presence of seven ormore lymph nodes κ p and fair forthe morphological criterion inhomogeneous signal intensity κ p magnetic resonance imaging mrilymph nodes were detected in of patientson preoperative mri the median number of visiblelymph nodes was range there was no significantassociation between number of visible lymph nodes seenon preoperative mri and histopathological lymph nodeinvolvement pn p the smallest visible lymph node was mm of sizewhereas the largest measured mm shortaxis diameter the mean time between mri and surgery was days with a median of days range the cutoff values of the highest diagnostic value were mm or mm for the size criterion sensitivity specificity youdens index the presence ofa lymph node of these sizes was not associated with lymphnode metastases pn p lobulated and spiculated lymph nodes were only seen in a few patients n and n and indistinct and inhomogeneous lymphnodes were not seen at all table discussionin this retrospective singlecenter study on lymph nodestaging by ct in ductal adenocarcinoma of the pancreatic head we could show that the morphologic criteriainhomogeneous signal intensity and size are specificfor regional nodal metastatic disease replacing the sizecriterion by morphologic criteria however did not improve diagnostic accuracy due to sensitivity remaininglow combining specific criteria yields improved sensitivity with specificity remaining highby ct lymph nodes of mm in shortaxis diameterwere seen just as often in patients with and without 0cloch world of surgical oncology page of lymph node metastases resulting in poor discriminationlarger lymph nodes mm had a higher prevalence inthe lymph nodepositive group leading to high specificity however these lymph nodes mm or mmwere seen infrequently resulting in a rather low sensitivity the maximum value of the youdens index for thesize criterion of was achieved when a cutoff valueof mm was applied consisting of a specificity of and sensitivity of yielding a ppv of as for morphologic criteria lymph nodes of lobulatedborder contour were seen in about half of the patients ofboth groups pn and pn and therefore isa criterion that is not suitable to differentiate betweenthe groups lymph nodes of spiculated and indistinctborder contour were seen in few cases in both patientgroups only pn and versus pn and making them poor diagnostic criteria however lymphnodes of inhomogeneous signal intensity were visible in of patients with lymph node metastases pn andonly in of the patients without lymph node metastases pn resulting in a youdens index of whichwas the maximum value for the morphological criteriaand a ppv of ideally a good discriminator for nodal metastases isnegative in patients without nodal involvement and positive for tumors with lymph node metastases in ourstudy each criterion ie size as well as different morphological features only met one of these prerequisitesthe size criterion mm as well as the presence of alymph node of inhomogeneous signal intensity as morphological criterion turned out to be negative in patientswithout nodal involvement pn and therefore highlyspecific yet lymph nodes of the respective characteristicwere not positive in a sufficiently high number of tumorswith lymph node metastases pn to reach high levelsof sensitivity and consequently did not have a significantdiagnostic valuethe maximum value of the youdens index for the sizecriterion was when a cutoff value of mm wasapplied and for the morphological criteria whenthe criterion inhomogeneous signal intensity was usedshowing that morphologic criteria do not yield in higherdiagnostic value than lymph node size in adenocarcinoma of the pancreatic head pdac patients this iscontrary to the findings of brown in rectal cancer one reason might be that brown used mri toassess morphologic criteria which has a higher soft tissuecontrast compared to ct which was used in most patients in our studyinterestingly we could show that with preoperativect the presence of seven or more lymph nodes wasseen more often in patients with lymph node metastasispn than in those without metastasis pn p when applying this as a sole criterion cn for lymphnode metastasis pn this led to a sensitivity of aspecificity of ppv of and youdens indexof in diagnostic test analysis criteria can be combinedin mainly two ways sensitive criteria can be taken together to improve specificity or specific criteria canbe accumulated to improve sensitivity when combining the highly specific criteria size cutoff value mm inhomogeneous signal intensity and number ofvisible lymph nodes n ¥ a highly significant association with nodal metastases pn p wasfound consequently the ct examination was considered nodepositive cn when at least one of thesecriteria was met the application of this criterion improved the sensitivity to with a remaining specificity of and ppv resulting in an alsoimproved youdens index of the results of the mri examinations must be viewedin a rather descriptive manner since the sample size waslimited n lymph nodes were detected in the majority of examinations generally allowing theevaluation of lymph nodes by mri as well upper abdominal mri generally has a lower spatial resolutionbut a higher soft tissue contrast compared to ct forthe size criterion a cutoff value of mm or mm ledto the best diagnostic results sensitivity specificity youdens index lymph nodes of abnormal morphological criteria were seen in only veryfew patients table the main limitation of this study is the retrospectivestudy design in which a nodebynode comparison ofcrosssectionalimaging with histopathology was notpossible this was of minor importance though sincelow sensitivity was the main factor that led to compromised diagnostic performance in our study we werealso able to correlate with postoperative crosssectionalimaging in all cases in which it was unclear whether alymph node had been resected during surgery or notalso in our cohort only patients who had subsequentsurgery were included presuming lower tumor stage ascompared to the average patient who undergoes imagingfor presurgical workupthe strength of our singlecenter study is reinforcedby a defined number of surgeons a high standardizationof the ct technique and an experienced radiologist whoperformed the analysisthe results of our study are consistent with recent andinitial data demonstrating that clinical staging by lowsensitivity underestimates histopathological lymph nodeinvolvement pn [ ] however by adding thecriterion inhomogeneous signal intensity and numberof visible lymph nodes to the size criterion we wereable to increase the sensitivity to in comparison toprevious findings roche nanashima 0cloch world of surgical oncology page of and cao with specificity remainingsufficientan additional imaging modality that has shown the potential to improve the sensitivity of detecting metastaticdisease is positron emission tomographycomputed tomography petct however a beneficial role ofpetct in locoregional nodal staging could not be established to date the majority of initial as well as recentstudies show very limited sensitivities for nodal status between and [] the petpanc study evaluated the incremental diagnostic accuracy and impact ofpetct in addition to multidetector ct in patients withsuspected pancreatic cancer in a prospective multicenterstudy that included patients in this study significantlymore patients with stage iib disease pn were correctlystaged by petct than by multidetector ct p but this only led to a moderate sensitivity of for petct versus for multidetector ct endoscopic ultrasonography eus is a wellestablisheddiagnostic procedure in pancreatic cancer with the benefitof a dynamic diagnostic examination that allows fineneedle aspiration for cytologic diagnosis two metaanalyses evaluating diagnostic accuracy of eus for locoregional nodal staging the pooled sensitivities and specificities were and li studies n patients and and nawaz studiesn patients advanced techniques such ascontrastenhanced eus cheus and eus elastographyare currently in evaluation to date ct remains the standard staging imaging modality recommended by nccn guidelines for locoregional staging of pancreatic cancer neither petctnor eus yields reliable diagnostic accuracy for nodalstagingan advantageous effect on resectability and overallsurvival os in unresectable cases including both borderline resectable and unresectable of pdac by multimodality therapy including neoadjuvanttherapy hasalready been described in several studies the benefit of neoadjuvant therapy in cases of primarily resectable disease at diagnosis is yet less revealedseveral phase ii trials showed that patients who completed neoadjuvant chemoradiation without progressivedisease at restaging had a higher chance of achieving r0resection and consequently higher median and oswhen compared to historical data as seen in othertumor entities a potential benefit of neoadjuvant therapyon the basis of positive nodal status cn is stronglyimplied cao found that the of patients thatwere successfully downstaged from nodepositive diseasecn1 to nodenegative disease ypn0 by neoadjuvanttherapy benefit in terms of higher rates of 5year survivalypn0 vs ypn1 p this is consistent with the findings of portuondo 5yearsurvival ypn0 vs ypn1 p the nccn guidelines for pancreatic adenocarcinomaappreciates these results by stating that considerationcan be given to neoadjuvant therapy for selected patientswith resectable tumor but poor prognostic features suchas large regional lymph nodes markedly elevated ca large primary tumors extreme pain and excessiveweight loss further clarification on this matter isexpected to come from the ongoing neonax trialnct02047513 a phase ii study comparing neoadjuvant plus adjuvant with only adjuvant nabpaclitaxelplus gemcitabine therapy forresectable pancreaticcancer theiii neopa trialphasenct01900327 compares neoadjuvant chemoradiotherapy with upfront surgery of resectable pancreatichead cancer a subgroup analysis in terms of nodalstatus would present reliable dataongoinggiven the suggested benefit of neoadjuvant therapybased on lymph node staging there is an urgent need tofind criteria and modalities to further improve the diagnostic value of lymph node staging by pretherapeuticcrosssectional imaging in patients with ductal adenocarcinoma of the pancreatic head to date none of theexisting modalities and criteria accomplishes reliablenodal staging larger prospective studies are ongoingand necessary to get a more precise idea of the prognostic advantage of neoadjuvant therapy in patients with regionalcn of pdac inpretherapeutic staginglymph node metastasisslymph node staging in pdac patients when using ctmorphological criteria such as border contour or homogeneity compared to diameter cutoff values does notlead to reliable diagnostic value diagnostic accuracy islimited due to low sensitivity for detection of metastaticdisease combining specific criteria yields improved sensitivity with specificity and ppv remaining high theseresults suggest an attentive interpretation of the resultsof pretherapeutic lymph node staging particularly incases in which lymph node metastases are absentabbreviationspdac adenocarcinoma of the pancreatic head ct computed tomographymri magnetic resonance imaging pn histopathologically involved lymphnodes pn histopathologically no involved lymph nodes cn clinicallyinvolved lymph nodes cn clinically no involved lymph nodes ppv positivepredictive value petct positron emission tomographycomputed tomography eus endoscopic ultrasonography cheus contrastenhancedendoscopic ultrasonography os overall survivalacknowledgementswe acknowledge support from the german research foundation dfg andthe open access publication funds of charit universittsmedizin berlinauthors contributionsall authors were involved in data acquisition and manuscript revision theconception of the design of the study and drafting of the manuscript was 0cloch world of surgical oncology page of done by fn loch c kamphues and p asbach image analysis was performedby p asbach and assisted by fn loch image analysis of the subgroup foranalysis of interobserver agreement was performed by m haas all authorshave approved the submitted version of the manuscript and account fortheir own contribution and the accuracy as well as the integrity of the workpresentedfundingthe study was not fundedavailability of data and materialsthe datasets used during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study was approved by the local ethics committee of the charituniversity medicine berlin informed consent of participation was waived bythe irb given the retrospective study design irb no ea411418consent for publicationconsent for publication is not applicable for this studycompeting intereststhe authors declare that they have no competing interestsauthor details1charit universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germany 2charit universittsmedizin berlin corporatemember of freie universitt berlin humboldtuniversitt zu berlin and berlininstitute of health department of radiology campus benjamin franklinhindenburgdamm berlin germany 3the johns hopkins universityschool of medicine department of surgery n wolfe street blalock baltimore md usareceived december accepted july zeman rk cooper c zeiberg as kladakis a silverman pm marshall jl tnm staging of pancreatic carcinoma using helical ct am jroentgenol m¼ller mf meyenberger c bertschinger p schaer r marincek b pancreatictumors evaluation with endoscopic us ct and mr imaging radiology midwinter mj beveridge cj wilsdon jb bennett mk baudouin cj charnleyrm correlation between spiral computed tomography endoscopicultrasonography and findings at operation in pancreatic and ampullarytumours br j surg r¶sch t braig c gain t feuerbach s siewert jr schusdziarra v staging of pancreatic and ampullary carcinoma by endoscopicultrasonography comparison with conventional sonography computedtomography and angiography gastroenterology prenzel kl h¶lscher ah vallb¶hmer d drebber u gutschow ca m¶nig sp lymph node size and metastatic infiltration in adenocarcinoma of thepancreatic head eur j surg oncol rollvn e blomqvist l ¶istm¶ e hjern f csanaky g abrahamnordling mmorphological predictors for lymph node metastases on computedtomography in colon cancer abdom radiol brown g richards cj bourne mw newcombe rg radcliffe ag dallimorens morphologic predictors of lymph node status in rectal cancer withuse of highspatialresolution mr imaging with histopathologic comparisonradiology kim jh beets gl kim mj kessels agh beetstan rgh highresolution mrimaging for nodal staging in rectal cancer are there any criteria in additionto the size eur j radiol isaji s murata y kishiwada m new japanese classification of pancreaticcancer in neoptolemos j urrutia r abbruzzese j b¼chler mw editorspancreatic cancer new york springer p brierley jd gospo | 0 |
Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group ARED PBM Group BINFRARED PBM Group CPLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring º Analyses of body contouring will be performed by non invasive methods º The waist circumference measurement will not discriminate the factors underlying the volume modifications º The habits of the participants such as diet and exercise routines may affect the results º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical TrialsReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individuals self esteem4 Studies have shown that aesthetic treatments significantly increase a patients quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6 There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10 The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18 Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions their frequency times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25 It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described below biggersmaller Ïn2 accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participants abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group ARED ± nm Group BINFRARED ± nm Group CPLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant powerone LED mWAperture diameterone LED cmPower density at aperture mWcm2Beam spot size at targetone LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContactweek for a month a total of sessionsInfrared LEDContinuousContactweek for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire The WHO Quality of Life WHOQOL BREF as well as the Body Shape Questionnaire BSQ34 Self Image Scale will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on a protected by password computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths nm and near the infrared regions nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18 Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39 If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individuals self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on a protected by password computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients Landecho MF Tuero C Valent V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts Kouris A Platsidaki E Christodoulou C et a0al Patients self esteem before and after chemical peeling procedure J Cosmet Laser Ther Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci Thornfeldt CR Thaxton PM Horn | 2 |
"Because the meQTL SNPs affecting CpG sites in gene body/non-CGI regions were mostly enriched for SQ risk (Fig. 5d) we performed further analysis in this category by integrating the ENCODE SAEC data. We chose SAEC data because this cell type may be involved in SQ development. We restricted enrichment analysis to the regulatory meQTL SNPs which localized in the CTCF binding regions DNaseI hypersensitive sites or histone marks (H3K27me3 H3K4me3 and H3K36me3) or had at least one LD SNP (r2? 0.95) residing in these regions. The strong enrichment in SQ was driven by SNPs overlapping with CTCF binding sites (P<10?4 based on 10000 permutations) or the repressive mark H3K27me3 (P<10?4 based on 10000 permutations) (Fig. 5e). The enrichment test was not significant after excluding the SNPs overlapping with these regulatory regions (P=0.14 based on 10000 permutations). Replication of meQTLs in TCGA breast and kidney tissues To explore the tissue-specificity of the genetic effects on DNA methylation we examined whether the meQTLs detected in EAGLE lung tissue data could be replicated in TCGA breast (n=87) or kidney (n=142) histologically normal tissue samples the only two ans to date with data available for a large number of normal tissues of European ancestry. Results are in and Supplementary Fig. 8. For both cis- and trans- meQTLs a large proportion of associations had the same direction of EAGLE meQTLs in both breast and kidney samples. For cis-associations 54.7% and 70.0% were replicated with FDR=5% based on single-sided P-values in two data sets respectively. For the strong cis associations with P<10?10 in EAGLE the replication rates increased to 82.7% and 89.2% in the two data sets. For trans- associations 83.4% and 86.4% were replicated in breast and kidney samples respectively. The detected master regulator (Fig 2a) was strongly replicated in both data sets (Supplementary ). Interestingly some cis-meQTLs but not trans-meQTLs had an opposite but very strong association (P<10?6) in breast (n=7) or kidney (n=58) compared with the EAGLE lung data a phenomenon previously reported in a cell-type specific eQTL study40. Discussion We found that inherited genetic variation profoundly and extensively impacts DNA methylation in target ans. Based on high-density methylation arrays in a large sample size we identified 34304 cis-meQTLs and 585 trans-meQTLs one to two orders of magnitude larger compared to previous studies357. meQTLs involved nearly half of the autosomal genes of which 9330 in cis and 373 in trans with 9525 unique genes in total. We show that approximately 10% of the cis-meQTLs were affected by at least two SNPs independently. Moreover we detected a master regulator SNP associated with the methylation levels of five CpG probes on different chromosomes demonstrating the existence of regulatory hotspots for DNA methylation as previously shown for eQTL4142. Most meQTLs were replicated in independent histologically normal lung tissue samples from TCGA. We also showed a high similarity of genetic control on DNA methylation across different tissues. Our findings show that genetic effects on DNA methylation are extensive in scale and complex in structure across the whole genome and suggest a series of important biological implications. First our results show that the genomic architecture surrounding cis- and trans-meQTLs is distinct. cis-meQTLs are very large in number impact predominantly the CpG sites mapping to non-gene regions and when they occur in genes are mostly in non-promoter and non-CpG island regions. In contrast trans-meQTLs are rarer mainly affect promoter CGI regions and may be associated with distal CpG sites through the mediation effect of proximal CpG sites. We found preliminary evidence that the cis-CpG sites mediating the trans-meQTL associations were enriched for genes involved in methylation regulation such as genes encoding for GTPase or proteins involved in genetic imprinting. GTPase-related gene pathways appear to modulate expression of DNA methyltransferases43. Methylation-induced expression changes of these genes may result in further methylation changes of other genes (i.e. in trans). Moreover a noncoding RNA within the intron of KCNQ1 a key gene regulating genetic imprinting can influence chromatin 3-D structure via a protein complex including DNA methyltransferase proteins4445. These findings suggest intricate mechanisms for trans-regulating effects through proximal methylation. cis-meQTLs may affect cancer risk. To understand the functional consequences of GWAS loci is challenging and multiple principles for post-GWAS functional characterization of genetic loci have been proposed including the exploration of epigenetic mechanisms46. In our study the top GWAS lung cancer loci were strongly associated with methylation levels of CpG sites in nearby gene bodies through cis-regulation and adjusting for smoking status or intensity did not change the results. Furthermore SNPs affecting the DNA methylation of gene bodies (which are typically methylated) were also collectively associated with risk for squamous cell carcinoma after excluding the established GWAS loci and were enriched for genes in cancer pathways. In contrast no enrichment was observed for SNPs affecting the methylation of gene promoters or CGI regions which are typically not methylated in normal tissues. This suggests a potential novel mechanism for genetic effects on cancer risk. In fact gene body-enriched cis-meQTLs outside CGI regions may increase the risk for germline and somatic mutations due to their increased propensity to become mutated1112. Upon spontaneous hydrolytic deamination methylated cytosine residues turn into thymine which are less likely to be efficiently repaired than the uracils that result from deamination of unmethylated cytosine residues. For example about 25% of mutations in TP53 in cancers are thought to be due to epigenetic effects47. Indeed analyses of comprehensive human catalogues of lung tumors have identified frequent G>T mutations enriched for CpG dinucleotides outside CGI regions suggesting a role for methylated cytosine since CGI as we confirmed are usually unmethylated48. A similar signature was recently observed in other tumors14. Thus inherited genetic variation may have a profound impact on carcinogenesis by regulating the human methylome. We observed a high similarity of genetic control on DNA methylation across tissues. Since tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns49 a natural question is whether the genetic regulation of methylation is tissue specific. While the tissue-specificity of eQTLs has been investigated for a few tissues50 for cis-meQTL only a recent investigation was conducted6 showing that 35.7% of 88751 cis meQTLs detected in 662 adipose samples were replicated in ~200 whole blood samples. We found that a large proportion of meQTLs in EAGLE lung samples particularly those with large effect sizes were robustly replicated in breast and kidney tissue samples from TCGA suggesting a high similarity of genetic regulation of methylation across these tissues and related impact on somatic mutation rates1448. " | 1 |
High burden of depression among cancerpatients on chemotherapy in University ofGondar comprehensive hospital and FelegeHiwot referral hospital Northwest EthiopiaAdhanom Gebreegziabher BarakiID1 Getahun Mengistu Tessema2 EyayawAdisu Demeke3 Department of Epidemiology and Biostatistics College of Medicine and Health Sciences Institute of PublicHealth University of Gondar Gondar Ethiopia Department of Internal Medicine College of Medicine andHealth Sciences School of Medicine University of Gondar Gondar Ethiopia Department ofPhysiotherapy Bahirdar University Bahirdar Ethiopia adsh04gmailcomAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Baraki AG Tessema GM Demeke EA High burden of depression among cancerpatients on chemotherapy in University of Gondarcomprehensive hospital and Felege Hiwot referralhospital Northwest Ethiopia e0237837 101371journalpone0237837Editor Nu¨lu¨fer Erbil Ordu University TURKEYReceived September Accepted August Published August Copyright Baraki This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data containspotentially identifying characteristics as well assensitive patient information eg HIV statusTherefore please send all data requests to theDirector of School of Medicine Dr MezgebuSilamsew at msilamsawgmailcom orPostgraduate committee Mr Getasew Amare atgetasewa23gmailcomFunding The authors received no specificfunding for this workIntroductionCancer the most stressful event a person may experience often triggers depressionDepression among these groups of people in turn affects chemotherapy adherence lengthof hospitalization quality of life and cancer treatment outcome Even though the problem isenormous studies that address it are limited Therefore this study was conducted to determine the prevalence of depression and associated factors among cancer patients on chemotherapy in FelegeHiwot referral hospital and University of Gondar referral hospitalNorthwest EthiopiaMethodsAn institutionbased crosssectional study was conducted from April to May A total of cancer patients on chemotherapy were included Depression was assessed using thepatient health questionnaire PHQ9 Binary logistic regression was used to select variablesand determine Crude Odds Ratio COR Variables with P value were entered into multivariable logistic regression Adjusted Odds Ratio AOR with confidence intervals forvariables with Pvalue was estimated to show factors affecting depression amongcancer patients The fitness of the model was checked by using the HosmerLemeshowgoodnessoffit testResultsThe prevalence of depression among cancer patients on chemotherapy was CI Educational status of college and above AOR CI Jobless AOR CI UnderweightAOR CI PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaCompeting interests The authors have declaredthat no competing interests existAbbreviations AOR Adjusted Odds Ratio CICrude Odds Ratio COR Crude Odds Ratio PHQPatient Health Questionnaire SD StandardDeviation UoGCSH University of GondarComprehensive Specialized Hospitalchemotherapy duration � months or more AOR CI were notablyassociated with depressionConclusionThe burden of depression among cancer patients in this study was high We recommendconcerned bodies working to curve the problem to intervene based on the identified risk factors Improving educational status reducing work stress and maintaining normal weightwould reduce depressionIntroductionThe global burden of cancer has risen to million new cases and million deaths in Worldwide the total number of people who are alive within years of a cancer diagnosis isestimated to be million []Depression is a common mental disorder characterized by persistent sadness and a loss ofinterest in activities that one normally enjoys accompanied by an inability to carry out dailyactivities for at least two weeks More than million people are now living with depressionan increase of more than between and [] The national prevalence of depression among the general population in Ethiopia was []Cancer the most stressful event that a person may experience often triggers depression [] The prevalence of depression among cancer exceeds that observed in the general population [] and it ranges from to [ ] Depression among cancer patientsaffects treatment since they have to take medications for both cancer and depression [] affectacceptance of adjuvant cancer treatment [] adherence [] extend hospitalization reducesthe quality of life [ ] and increases the risk of suicide [] Depression also predicts cancerprogression and mortality [ ]Several factors affect depression among cancer patients these include age sex marital status educational status occupation pain type of cancer phase of treatment [ ] andsocial support [ ]Even though routine screening of distress is recommended internationally for good cancercare [] less emphasis is given in the study area and most of the care focuses on cancer Studies on the magnitude and the contributing factors are also limited Therefore this study wasconducted to fill this information gap by determining the prevalence of depression among cancer patients and factors affecting itMethodsStudy design and periodAn institutionbased crosssectional study was conducted among cancer patients from Aprilto May Study areaThis study was conducted on cancer patients who are getting treatment and have followed upat the oncology unit of the University of Gondar comprehensive specialized hospitalUoGCSH and FelegeHiwot referral hospital FHRH The two hospitals are found in theAmhara region northwest Ethiopia km and km away from the capital Addis AbabaPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiarespectively The oncology unit of UoGCSH currently has beds for the management of cancer patients whereas the oncology unit of FHRH has currently beds for inpatient treatmentof cancer patientsParticipantsThe source populations were all adult cancer patients visiting the oncology unit and treatedwith chemotherapy in these hospitals All adults with any type of cancer patients under chemotherapy treatment and follow up during the study period were included in the studySample size and sampling procedureA final sample size of was found by using single population proportion formula with population correction for total cancer patients of in the two hospitals using the followingassumptions the prevalence of depression Za2 for confidence interval andmargin of error of The final sample size was proportionally allocated to the two hospitals for UoGCSHand 176FHRH A systematic random sampling method was employed to select every 3rdpatients who were coming to the oncology unit during the data collection period and full fillthe inclusion criteria We had a plan to randomly select and replace the nonresponders butno study participant refused to participateVariablesThe dependent variable of depression was measured using the widely used Patient HealthQuestionnaire PHQ9 The Amharic Local language version of the scale has been validatedin Ethiopia sensitivity and specificity [] We have used a cutoff point of toclassify patients as having depression or notIndependent variables like Age sex marital status average monthly income educationallevel smoking habit alcoholic habit physical activity were collected by intervieweradministered questionnaire whereas variables like Body Mass Index Type of cancer clinicalstagetype of chemotherapy Duration of chemotherapy and comorbidities like Hypertension DMHIV and Anemia were collected from patient charts The smoking and Alcohol use habitswere assessed by asking the patients if they ever smoke cigarette or drink alcohol and socialsupport was evaluated using the Oslo item social support scale with scores ranging from to poor moderate and strong []Data collection procedure and quality assuranceThe data was collected by interviewing the participants using a structured pretested questionnaire and chart review The data was collected by three nurses working in each oncology unitData collectors were trained for one day about the objective of the study and ethical considerations Data collectors were supervised by the principal investigator Data was reviewed andchecked for completeness accuracy and consistency after each day of data collectionData processing and analysisData were entered into Epiinfo version and STATA version was used for analysis Frequencies and percentages were computed for all variables Data were presented in tables andgraphs Binary logistic regression was used to select variables and to determine Crude OddsRatio COR Variables with P value were entered into multivariable logistic regressionAdjusted Odds Ratio AOR with confidence intervals for variables with Pvalue PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiawas estimated to show factors affecting depression among cancer patients The fitness of themodel was checked by using the HosmerLemeshow goodnessoffit testEthics statementEthical approval to conduct the study was received from the University of Gondar ethicalreview board School of Medicine Reference number SOM12372019 A permission letterwas received from the two hospitals To keep the privacy of participants name and other personal identifiers were not collected Consent to participate in the study was also orally takenfrom patients Patients with depression were also linked to the psychiatry clinicResultsSociodemographic characteristic of study participantsA total of patients participated in the study From the total respondents the majority ofthem were females were married and were housewivesRegarding to age distribution the mean and standard deviation of participants age was SD Table Behavioral and comorbidity characteristicsFour participants were declared that they were smoking cigarettes daily and were alcohol consumers Most of the participants were physically active Most ofthe participants were in normal weight category based on their BMI whereasTable Baseline characteristics of cancer patients in UoGCSH and FHRH FrequencyPercentageVariablesSexMarital statusMaleFemaleSingleMarriedDivorcedWidowedEducational levelOccupationNo educationPrimary educationSecondary educationCollege and aboveGovernment employeeMonthly Income ETBFarmerMerchantUnemployed 101371journalpone0237837t001PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Behavioral factors and comorbidities among study participant at UoGCSH and FHRH VariablesSmoking statusNoYesAlcohol drinkingNoYesPhysical activityNoYesBody mass index ComorbidityNoYesDiabetesNoYesHypertensionNoYesAnemiaNoYesHIVAIDSNoYesFrequencyPercentage101371journalpone0237837t002 and were underweight and overweight respectively Ninetytwo participants had additional comorbidity Table Type of cancer and treatmentrelated characteristicsBreast cancer was the commonest cancer Whereas cervical cancer colorectal cancer35 and lung cancer are ranked second to fourth Most of thepatients were diagnosed with the disease in the past six months prior to the studyRegarding the clinical stage of the disease the third stage accounts for patients Atotal of participants have taken chemotherapy for less than three months Table Prevalence of depression among cancer patientsIn this study patients had depression making the prevalence CI The prevalence of depression among male cancer patients was CI whereas it was CI among female patients From the totalpatients with depression and had moderate moderatelysevere and severe depression respectively The magnitude of depression has also shown thedifference among different types of cancer Fig PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Type of cancer and treatmentrelated characteristics of study participant at UoGCSH and FHRH VariablesFrequencyPercentageType of cancerBreast cancerLung cancerColorectal cancerGastric cancerCervical cancerHead and Neck cancerEsophageal cancerBlood cancerSkin cancerThyroid cancerBladder cancerLymphomaLiver cancerSarcomaTesticular cancerClinical stageStage Stage Stage Stage UnknownDuration since diagnosis months months monthsDuration since start of chemotherapy months months months101371journalpone0237837t003Factors associated with depression among cancer patientsIn bivariable logistic regression age sex marital status educational status occupation BMISocial support and duration of chemotherapy were found to have Pvalue 02subsequentlythese variables were subjected to multivariable analysis and educational level occupational status BMI status and duration of chemotherapy were statistically associated with depressionamong cancer patientsThe odds of depression among patients who attended college and above was significantlyreduced when compared to those with no education AOR CI Whencompared to government employees patients who are unemployed had less risk of depressionAOR CI Underweight patients had 239AOR CI times higher odds of depression as compared to those with normal body mass indexPatients who took chemotherapy for six months or more had AOR CI times higher odds of depression as compared to their counterparts Table PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaFig Prevalence of depression among cancer patients on chemotherapy in UoGCSH and FHRH northwest Ethiopia101371journalpone0237837g001DiscussionIn this study we have assessed the magnitude of depression among cancer patients and the factors affecting it We have found prevalence and occupation educational status bodymass index and duration of chemotherapy were found to be independent predictors ofdepressionThe magnitude of depression in this study was consistent with other studies conductedamong Chinese cancer patients [] whereas this figure was higher than a study conducted in Addis Ababa [] Iran [] and metaanalysis done by Krebber [] This discrepancy could be attributable to the difference in the study populations in terms of types ofcancer the tool used for screening or other sociodemographic variations and severity ofdepression consideredThe odds of depression was significantly reduced in patients who are unemployed whencompared to government employees This piece of evidence is supported by another multicenter study [] This could be related to workrelated stress which worsens feelings of inadequate control over ones work frustrated hopes and expectations leading to depression []The odds of depression among patient who attended college and above was reduced whencompared to those who have no education This finding is supported by a study from China[] Atlanta [] and Greece [] The possible reason could be these patients may have a betterunderstanding of the disease and have early screening which increases their recovery A higherproportion of educated people are in the first or second clinical stage of cancer ascompared to of patients without educationUnderweight cancer patients had more than double odds of depression as compared tothose who have a normal body mass index This finding is supported by several single studies[ ] and systematic review and metaanalysis [] showing underweight people at higherrisk of depression This shows malnutrition has a significant role in the mental health of peopleand maintaining a healthy weight is essential to improve health in general and mental healthin particularEven though the chemotherapy duration has shown no significant association with depression in few pieces of literature [ ] The odds of depression among cancer patients whoPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaTable Factors affecting depression among cancer patientsat UoGCSH and FHRH VariablesDepressionCOR CIAOR95 CIYesNoAge mean sd SexMarital statusMale Female Single Married Divorced Widowed Educational levelNo education Primary Secondary College OccupationGovernment Farmer Merchant Unemployed Body mass indexUnderweightNormalOver weight Social supportPoorModerateStrongDuration of chemotherapy� months months� Pvalue � � � �101371journalpone0237837t004took chemotherapy for more than six months was higher than their counterparts in this studyThis could be a side effect of chemotherapy [] or it could be also associated with the staggering cost of chemotherapy which makes these patients stress to buy it for an extended durationThis study assessed the frequently ignored aspect of cancer comorbidity depression Butthe study has some limitations as it was a crosssectional study The causeeffect relationshipsare not guaranteed in these studies therefore we recommend a prospective study Even thoughwe have used a validated tool some of the symptoms used in PHQ like weight loss andtiredness might be related to cancer itself and may overestimate depressionConclusionThe burden of depression among cancer patients in this study was high Occupation educational status body mass index and duration of chemotherapy were found to be independentlyassociated to depression We recommend concerned bodies working to curve the problem toPLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest Ethiopiaintervene based on the identified risk factors Improving educational status reducing workstress and maintaining normal weight would reduce depression Clinicians shall also provideintegrated care of mental health and cancer treatmentAcknowledgmentsWe would like to thank the University of Gondar Oncology unit staff of FelegeHiwot referralhospital and University of Gondar comprehensive specialized hospitals and all data collectorsAuthor ContributionsConceptualization Adhanom Gebreegziabher Baraki Getahun Mengistu Tessema EyayawAdisu DemekeData curation Eyayaw Adisu DemekeFormal analysis Adhanom Gebreegziabher Baraki Eyayaw Adisu DemekeInvestigation Adhanom Gebreegziabher BarakiMethodology Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaProject administration Eyayaw Adisu DemekeSoftware Adhanom Gebreegziabher BarakiValidation Getahun Mengistu TessemaVisualization Getahun Mengistu TessemaWriting original draft Adhanom Gebreegziabher BarakiWriting review editing Adhanom Gebreegziabher Baraki Getahun Mengistu TessemaEyayaw Adisu DemekeReferences Bray F Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA a cancer journal for clinicians p World Health anization Mental Health [cited August ] wwwwhointmental_healthmanagementdepressionen Hailemariam S The prevalence of depression and associated factors in Ethiopia findings fromthe National Health Survey International journal of mental health systems p 10118617524458623 PMID Nikbakhsh N Prevalence of depression and anxiety among cancer patients Caspian journal ofinternal medicine p PMID AlShakhli H Harcourt D and Kenealy J Psychological distress surrounding diagnosis of malignantand nonmalignant skin lesions at a pigmented lesion clinic Journal of Plastic Reconstructive Aesthetic Surgery p Sotelo JL Musselman D and Nemeroff C The biology of depression in cancer and the relationshipbetween depression and cancer progression Int Rev Psychiatry p PMID Krebber A Prevalence of depression in cancer patients a metaanalysis of diagnostic interviewsand selfreport instruments PsychoOncology p 101002pon PMID Hong JS and Tian J Prevalence of anxiety and depression and their risk factors in Chinese cancerpatients Supportive care in cancer p 101007s005200131997y PMID Mitchell AJ Prevalence of depression anxiety and adjustment disorder in oncological haematological and palliativecare settings a metaanalysis of interviewbased studies The lancet oncology p 101016S147020451170002X PMID PLOS ONE 101371journalpone0237837 August PLOS ONE 0cHigh burden of depression among cancer patients on chemotherapy in Northwest EthiopiaLinden W Anxiety and depression after cancer diagnosis prevalence rates by cancer type gender and age J Affect Disord p 101016jjad201203025PMID JimenezFonseca P Factors associated with anxiety and depression in cancer patients prior toinitiating adjuvant therapy Clin Transl Oncol p 101007s1209401818739 PMID Alemayehu M Deyessa N Medihin G Fekadu A A descriptive analysis of depression and pain complaints among patients with cancer in a low income country PloS one Colleoni M Depression and degree of acceptance of adjuvant cytotoxic drugs Lancet p 101016S014067360002821X PMID Pitman A Depression and anxiety in patients with cancer Bmj p k1415 101136bmjk1415 PMID Prieto JM Psychiatric morbidity and impact on hospital length of stay among hematologic cancerpatients receiving stemcell transplantation J Clin Oncol p 101200JCO200207101 PMID Tian J Chen ZC and Hang LF The effects of psychological status of the patients with digestive systemcancers on prognosis of the disease Europe PMC p Yousaf U Suicides among Danish cancer patients British journal of cancer p 101038sjbjc6602424 PMID Spiegel D and GieseDavis J Depression and cancer mechanisms and disease progression Biologicalpsychiatry p 101016s0006322303005663 PMID Ng CG Anxiety depression perceived social support and quality of life in Malaysian breast cancer patients a 1year prospective study Health and quality of life outcomes p Burgess C Depression and anxiety in women with early breast cancer five year observationalcohort study Bmj p 101136bmj38343670868D3 PMID Grassi L Screening for distress in cancer patients a multicenter nationwide study in Italy Cancer p 101002cncr27902 PMID Gelaye B Williams MA Lemma S Deyessa N Bahretibeb Y Shibre T Validity of the patienthealth questionnaire9 for depression screening and diagnosis in East Africa Psychiatry research Dec 101016jpsychres201307015 PMID Dalgard OS Dowrick C Lehtinen V VazquezBarquero JL Casey P Wilkinson G Negative lifeevents social support and gender difference in depression Social psychiatry and psychiatric epidemiology Jun 101007s0012700600515 PMID Iacovides A The relationship between job stress burnout and clinical depression Journal ofaffective disorders p 101016s0165032702001015 PMIDTorres MA Predictors of depression in breast cancer patients treated with radiation role of priorchemotherapy and nuclear factor kappa B Cancer p 101002cncr28003 PMID Polikandrioti M EVALUATION OF DEPRESSION IN PATIENTS UNDERGOING CHEMOTHERAPY Health Science Journal De Wit LM Depression and body mass index a ushaped association BMC public health p MartinRodriguez E Relationship between body mass index and depression in women a 7yearprospective cohort study The APNA study European Psychiatry p 101016jeurpsy201511003 PMID Jung SJ Association between body size weight change and depression systematic review andmetaanalysis The British Journal of Psychiatry p 101192bjpbp116186726 PMID Ciaramella A and Poli P Assessment of depression among cancer patients the role of pain cancertype and treatment PsychoOncology Journal of the Psychological Social and Behavioral Dimensionsof Cancer p Atag E Prevalence of depressive symptoms in elderly cancer patients receiving chemotherapyand influencing factors Psychogeriatrics p 101111psyg12329PMID Thornton LM Delayed emotional recovery after taxanebased chemotherapy Cancer Interdisciplinary International Journal of the American Cancer Society p PLOS ONE 101371journalpone0237837 August PLOS ONE 0c' | 2 |
ammatory bowel disease ibd is a chronic immunemediated disease mainly consisting of two diseases crohnsdisease cd and ulcerative colitis uc cd is active in anypart of the gastrointestinal tract from the mouth to the anuswhile uc is restricted to the colonic mucosa e etiologyand pathology of ibd remain largely unknown but a complicated interplay of genetic environmental immunologicaland lifestyle factors has been associated with this condition[ ] clinical symptoms of ibd involved but not limitedmultiple ans and systems throughout the body such asnodular erythema osteoporosis and ibdrelated arthropathye current medical treatment in ibd is available foribd patients drugbased therapies such as nonsteroidalantiammatory drugs sulfasalazine and masalazinebiological therapies such as antitnfα antibodies andimmunomodulatorssuch as methotrexate meanwhilesurgical management is used to control the progression ofsevere disease unfortunately these drugs also cause adverseeï¬ects in ibd patients and the high disease cost with a longtreatment process has brought economic burden for patients for example biological therapies such as antitnfαmay increase the risk of infection and the costs of thesedrugs vary from to us dollars erefore it isnot surprising that patients seek complementary and adjuvant therapies in ibdin this review we focus on rational dietary structureschinese traditional medicine suitable smoking alcohol andphysical activities rational dietary structure has beenspeculated to be a pivotal factor in the pathogenesis of ibdand may be important in managing disease symptoms ibdpatients choose various dietary strategies to minimize gastrointestinal distress and improve overall health dietaryproducts are the most common antigens in the intestinealtering the composition of the intestinal ï¬ora and changingthe permeability of gastrointestinal tract dietary interventions may not be appropriate alternatives to conventional medical therapy but are eï¬ective complementaryapproaches for ibd treatment in this review we focus ondiï¬erent dietary components which are reported to beneï¬tpatients symptoms 0ccanadian of gastroenterology and hepatologye popularity of chinese traditional medicine camhas been systematically increasing among various complementary and adjuvant medicine approaches for the treatment of gastrointestinal disorders mainly because it isnatural and eï¬ective cam products range from homeopathy herbal medicine acupuncture and moxibustion although the cam is often of yet unknown eï¬cacy andmechanism the induction and maintenance of disease remission in uc and cd have been investigatedover the last few years it is controversial whether ibdpatients should quit smoking and alcohol cigarette smokinghas been shown to worsen disease activity in cd while inuc smoking decreases the extent of disease anotherimportant factor is alcohol alcohol has been previouslyshown to be associated with worsening gi symptoms but acasecontrol study illustrated that moderate red wine consumption by patients is linked with a lower risk in ibd physical activity is one candidate complementary intervention that is of potential beneï¬t in various chronicdiseases previous studies showed that ibd patients perceivephysical activities as a helpful management in reducingsymptoms and complications of ibd ere are physiologic beneï¬ts to physical activity such as improved bonedensity decreased incident of colitis associated colorectalcancer and the prevention of obesity swimmingwalking and chinese martial arts such as qigong and tai chihave been shown to improve the qol balance internally forhealing and improve bone density we will summarizethe role of diï¬erent physical activities in the developmentand course of ibds dietarydiet is closely linked with ibd especially in western dietcurrently dietary interventions have been studied in ibd toalleviate active disease and maintain remission e mediterranean diet pattern has been shown to be protective inibd as the incidence of ibd in the south of europe is lowerthan in the northern europe some of the components in themediterranean diet pattern such as olive oil ï¬sh oil fruitsand vegetables have been shown to be eï¬cacious and patientfriendlyepidemiological studies illustrated that the intake of ahigher ratio of n polyunsaturated fatty acids n pufas and a lower ratio of n polyunsaturated fatty acidsn pufas was associated with an increased risk ofdeveloping ibd [ ] researches to date have demonstrated that n pufas may oï¬er a promising approach toimproving dysbacteriosis reducing the likelihood of relapseand lowering the mortality of colitis [ ] moreover theirprotective eï¬ect in ibd is hypothesized to be derived fromthe balance in the ratio of n 6n pufas or higher e hypothesized machanism underlying the antiammatory eï¬ect is to release proammatory mediatorsreduce freeradical generation and platelet activating factorformation all of which are increased in ibd [ ]currently ï¬sh peptide seems to have tissue reparativeproperties based on several studies in rodents and humansalmon ï¬llets contain n pufas and marine collagenpeptide has an antioxidative eï¬ect a previous studyshowed that a regular intake of salmon in patients with uc isbeneï¬cial based on the improved simple clinical colitisactivity index sccai and antiammatory fattyacidindex aifai e combination of ï¬sh peptides andï¬sh oil diet was more eï¬cient than pure ï¬sh oil in an animalmodels study compared with the pure ï¬sh oil addingthe ï¬sh peptides diet eï¬ectively reduces the production ofproammatory cytokines and increases the level of pge3in plasma additionally other dietary peptides also havedemonstrated an antiammatory eï¬ect in ibd animalmodels machbank that dish hydrolysate was suggested to have an intestinal protective eï¬ect in mice based on these observations the ï¬sh peptide diet may be aneï¬ective way to maintain remission ibddietary ï¬ber is more commonly used as a supplement forthe management of ibd rational intake of ï¬ber may reducecd risk especially that which originated from fruits fibers from fruit have antiammatory properties andpositive modulation of the intestinal microbiota fibersfermented by bacteria in the colon produce the shortchainfatty acids inhibiting the activation of transcription ofproammatory mediators according to a casecontrol study high ï¬ber intake respondents were percentless likely to develop cd than those with low ï¬ber intakemedian gday however ï¬ber intake is controversial scientists found that the risk of ï¬ber was15 gdaywhile ornton showed there was no diï¬erence betweenpatients and controls e future study should pay moreattention to the amount of ï¬ber and ibdvitamin d is a group of fatsoluble vitamins which playsa key role in ibd treatments recent studies have reported that vitamin d was linked to the protection againstinfection and the control of the gut commensal microbialcomposition [ ] clinical trials have suggested a positivecorrelation between vitamin d deï¬ciency and ibd thatnearly half of the patients had hypovitaminosis d []vitamin d deï¬ciency may reduce the expression of a tightjunction in the intestinal epithelium decrease the clearanceof colonic bacteria directly and aï¬ect the gut barrier andimmune system functions that impact the onset and progression of ibd [] to correct vitamin d deï¬ciencyresearchers found that after ingestion of iu vitamind3 daily for months and iu vitamin d2 weekly for weeks the vitamin d status was significantly improved and of the patients cd activity index cdai had a drop ofless than points compared with placebo among childrenand adolescents with ibd erefore in cd the effective dose of vitamin d3 was determined at iud traditional chinese medicinein recent years traditional chinese medicine tcm including herbal medicine acupuncture and hemopathy hasbeen commonly used among ibd patients from all agegroups it is estimated that the percentage of ibd patientsusing tcm in north america at might increase up toeven herbal medicine is the most common tcmmodality with lower cost and higher eï¬ciency tcm herbal 0ccanadian of gastroenterology and hepatologyenema has been proved to be the most eï¬cient method fortherapies because of the regulation of immune responses inthe colon mucosa e mechanism of tcm in ibdincludes improving antiammatory activities and reducing the level of proammatory cytokines yun nan bai yao ynby is a chinese herbal remedyused for treating wounds for its hemostatic properties in the recent years researchers showed that ynby caneï¬ectively reduce the severity of experimental colitis by theimmunosuppression and wound healing mechanisms it wasshown that ynby significantly suppresses the growth oft lymphocytes and b lymphocytes thus decreasing severalproammatory cytokines such as tnfα which wereclosely correlated with ibd a dosedependent hemostatic eï¬ect was also revealed by researchers through rabbitsmodels erefore for patients suï¬ering from gastrointestinal bleeding giving ynby enterally may serve as aneï¬ective adjunctive therapy ynby is also capable of reducing intraoperative blood loss e dosage of ynby oncd is still unclear and future studies should focus on thedosage and indications for ibd patientstripterygium wilfordii hook f twhf aloe vera andtormentil are tcm herbal remedies with antiammatory activities it has been shown that twhf achieves thesame level in preventing the postoperative recurrence ofcd in the previous research the relapsed patients inthe tw and mesalazinetreated groups were versus in months and versus in yearmoreover the cdai was decreased during the ï¬rst weeksand reached the minimum in week it was alsoreported that respondents symptoms are alleviatedthrough using aloe vera tormetil extracts weresuitable for chronic ibd patients which was proved to besafe up to mgday for weeks with minor side eï¬ects another herbal therapy such as food allergy herbalformula2 fahf2 which originated from wu mei wanthat has long been used in china to treat colitis may have thepossibility to be served as a novel treatment of cd composed of aconitum coptis ginger ginseng cinnamomum angelica and ganoderma lucidum it was found to beable to inhibit both adaptive and innate immune proammatory cytokine responses in amed cd mucosa andvalid in halting progression of colitis in a murine modelwhich indicated that fahf2 may be safe and eï¬ective forcd to maintain remission and avoid the need for pharmacologic escalation in therapy with medications that havepotentially severe side eï¬ects [ ]c longa l turmeric is a plant whose root segment iscommonly used as a seasoning and in traditional chinesemedicine for thousands of years curcumin is the chiefbiologically active derivative of turmeric in a series of invitro and in vivo studies curcumin has recently caught muchattention for its antiammatory characteristic curcuminis capable of correcting abnormal immune response in ibdthrough decreasing proammatory cytokines synthesisdownregulating the transcription of the proammatorygene by inhibiting nfκβ and upregulating antioxidantenzymes a placebocontrolled doubleblind study proved that aproper combination of curcumin and mesalazine can effectively induce remission in mesalazinetolerant uc patients whose disease course fail to improve under maximumdose of mesalazine for weeks after standard addoncurcumin therapy gday in capsules for month of patients in the curcumintreated group achievedclinical remission and presented ameliorationin endoscopical performance evaluated by the endoscopicmayo index subscore while none achieved remission in the placebo group curcumin is nontoxic even at relatively high doses withno known toxic side eï¬ects in humans up to doses of gday and even pediatric patients with ibd are able to tolerate a curcumin dose up to g twiceday and some of themdemonstrate improvement in the disease course [ ]currently popular antiammatory therapy can leadto tremendous cost while curcumin remedy is much moreaï¬ordable with a price less than per week for its effectiveness safety and aï¬ordability curcumin could be anideal agent for curing ibd however it is hard to maintaintherapeutic curcumin concentration pattern in human bodyfor its rapid metabolism and dissatisï¬ed biodistribution more eï¬ort should be made to determine a feasible methodof administration as well as improve its absorption andbiodistribution before curcumin can fully beneï¬t ibdpatients smokingsmoking aï¬ects these cd and uc diï¬erently many casestudies suggest that smoking is a risk factor for cd while ittends to confer a protective eï¬ect against uc [ ]compared to neversmokers the incidence and severityof uc are lower in smokers in many studies and smokingexerts protective eï¬ects on both the development and theprogression of uc [ ] e relapse rate hospitalizationrates and the need for oral steroids and the colectomy rateswere found to be lower in current smokers rather thannonsmokers it was observed that high cigarette dosesuch as cigarettes per day was correlated with less extensive colitis and lower treatment needs however theprotective role of smoking in uc sustains until yearsafter smoking cessation and then the risk goes up interestingly the risk paralleled past cumulative exposure with anincrease in the disease activity and the need for hospitaladmission and major medical therapy [ ]as for the uence of smoking on cd most studieshave suggested that current smokers have a higher risk ofdeveloping cd than those who have never smoked [ ]besides previous data have demonstrated that smoking wasassociated more frequently with complicated disease such aspenetrating intestinal complications and a higher relapserate it was shown that patients with a high life timetobacco exposure cigarette years and heavy smokers cigarettesday had small bowel disease more oftenthan the patients with both lower life time exposure cigarette years and smoking¤ cigarettesday actually the increased risk of disease relapse is significantly 0ccanadian of gastroenterology and hepatologyover a threshold cigarettes per day in additioncompared with nonsmokers the needs of steroids andimmune suppressants rise in smokers [] e impact ofcigarette smoking on cd is temporary smoking cessationimproves the course of the disease and it has been estimatedthat after years of smoking cessation former smokersunderwent a process similar to that of patients who havenever smoked and the ï¬areup rate was decreased [ ]notwithstanding it has been noted that the use of snuswas not associated to the development of either uc or cdwhich underlines diï¬erences in combusted and noncombusted tobacco in the genesis of ibd [ ] in addition the fact that snus users have higher levels of thenicotine metabolite cotinine also implies that nicotine byitself may not be involved in the pathogenesis alcoholalcohol is another potential trigger for ï¬aring ibd sincealcohol in diï¬erent amount of consumptions aï¬ects theimmune system and results in various imbalanced answith ammation [] furthermore alcohol consumption has an eï¬ect on gut permeability and plasma levelsof gutderived bacterial products such as lipopolysaccharides and peptidoglycans however compared withnever drinking light drinking has a protective eï¬ect on thedevelopment of uc it stands to reason that hazardousalcohol intake it is deï¬ned as more than g of alcohol perday for men and g for women is detrimental for ibdpatients while moderate red wine consumption lasted for aweek is linked with a lower risk in ibd e mechanism isthe decrease in stool calprotectin which is known as anantioxidant with antiammatory eï¬ects [ ] a research showed that fecal stool calprotectin was decreasedcompared with baseline after week of drinking in ibdpatients antioxidants with additional antiammatory actions may beneï¬t the treatment in ibd on account ofthe mechanism in ammation caused by oxidative stress[ ] oxidative stress shows a deï¬nition of the imbalancebetween oxidants reactive oxygen species and reactivehydrogen species and antioxidants which is linked tochronic intestinal ammation in the early stage of ibd meanwhile alcohol inhibits the immune system byreducing interleukin il12 and increasing interleukin il production which can aï¬ect the induction on or immune responses [ ] moreover autocrine il10production can prevent maturation of dendritic cells andinduce anergy in the tcells responder which is an antiammatory cytokine closely related to the immune system however alcohol consumption was controversialnowadays a report showed that although beer is beneï¬cialfor some people though only a few more than percentof the participants reported worsening symptoms fromdrinking moreover only about percent patients wastolerant to wine while more than percent of the subjectsshowed an increase in red wine symptoms ereforethe future study should focus more on the dosage and sideeï¬ect of alcohol consumption physical activityphysical activity may potentially play a role in alleviatingsymptoms related to extraintestinal manifestations of ibd previous study showed patients with at least metabolic equivalent task met hours per week of physicalactivity have a reduction in risk of developing crohnsdisease compared with those with3 met·hwk exercise can be beneï¬cial for intestinal and extraintestinalmanifestations of ibd that regular physical exercise couldimprove physiological health maintain their weight improve bone mineral density and alleviate the anorexiacaused by ibd [ ]lowtomoderate intensity physical activities have beenproved to be safe and suitable that it was well tolerated byibd patients especially those who were in remission anddid note provoke subjective symptoms moderateintensity physical activity has beneï¬cial eï¬ects on thegastrointestinal system patients with regular exerciseduring the previous years may have lower chance ofdeveloping cd especially if the exercise is performed dailyadditionally it was also demonstrated that patients whoperformed exercise were less likely to develop the diseaseactivity in uc several possible mechanisms mayexplain the antiammatory uence of physical activitiesfromskeletal muscleinhibiting the tnf production andstimulating the release of il10 [ ] however extremeexercise may contribute to intestinal ammation by themeansandcd8 lymphocytes natural killer cells and the level ofreactive oxygen species it seems that the eï¬ect ofphysical activity depends on the intensity and duration ofthe physical activityincluding releasing interleukin6 il6increasingof cd4some of the aspects were also observed at experimentalconditions because moderate voluntary treadmill exercisecould significantly accelerate the healing of colitis in ibd arecent study by cook showed that sessions of forcedtreadmill exercise training exacerbated ammation indextran sodium sulfateinduced colitis proved by excessivediarrhea episodes and increased animal mortality in aprevious study a longterm physical activity of 6weekrunning attenuated the colonic tnfα protein contentindicating the antiammatory eï¬ect of physical exercise[ ] according to the previous rodent studies exercisecould downregulate the expression of interleukin 1β il1βand tnfα in both colonic mucosa and plasma moreover itwas demonstrated that leptin levels were significantly decreased which diminished the severity of colonic damagemediated and exerted an antiammatory eï¬ect on anamed colon swimming and cycling are two eï¬ective aerobic exercisesthat can be beneï¬cial with fewer gastrointestinal symptomsby the means of ammatory modulation and apoptosis[ ] in addition walking min at of maximalheart rate days per week along with resistance training times per week is advocated in many studies and it may havethe potential to decrease the risk of active disease at sixmonths [ ]ofthenumber 0ccanadian of gastroenterology and hepatologyqigong and tai chi are traditional chinese physicalactivities which coordinate the body and mind ese approaches have been shown to be eï¬ective in reducingsymptoms such as fatigue depression and pain and improving qol in a way of moderate exercise qigongcould improve immune functions and reduce ammationproï¬les such as proammatory cytokines tnfα whichwere correlated to ibd lymphocytes thyroidstimulatinghormone and igg were found to be modulated in responseto practicing tai chi researchers demonstrated that a minute weekly session of qigong with a duration of weeksis recommended moderate tai chi and qigong may enhance physiological and psychological function and thefuture study may concentrate more on the suitable amountof exercise in tai chi and qigong discussionconsidering the past few years the adjuvant treatments foribd have become increasingly important to better the immunity and ammation in the intestine more and moreinvestigations illuminated the signiï¬cance of diet traditionalchinese medicines and proper exercise which are capableof taking the edge oï¬ withdrawal symptoms in ibd furthermore smoking and alcohol mainly act as two environmental factors in adjuvant treatments ough unable to replace the conventional ibd therapiestreatment still plays an instrumentaltotally adjuvantsupplement role in treating the disease some patientfriendly components of the mediterranean diet have beenproven to aï¬ect the intestinal barrier and immune systemfunction thus aï¬ecting the progress of ibd traditionalchinese medicine can eï¬ectively reduce ammatory cytokines and alleviate ibd moderate exercise such as qigongtai chi swimming and walking are eï¬ective treatments inreducing the risk of pain and complications with less expenditure moreover achieving smoking cessation is alsoan important goal of ibd treatment for the beneï¬cial eï¬ectsof smoking on disease are oï¬set by the harmful eï¬ects oftobacco on the respiratory and cardiovascular system[ ] whereas reasonable alcohol drinking beneï¬ts cdpatients by not aï¬ecting the immune system [ ]however as a novel treatment for cd several weaknesses have been limited by the application of adjuvanttreatments it is of ambiguity whether adjuvant treatment issuitable or curable for all types of ibd patients for examplesome changes associated with diet on the maintenance ofibd remission are ambiguous and the aï¬ecting dosage foribd of each beneï¬cial dietary component is unknown forherbal medicines there has been no speciï¬c research toconï¬rm a particular dose or prescription has significanteï¬ects on patients in addition although previous studieshave vitriï¬ed the beneï¬ts of exercise in ibd treatment it hasnot been deï¬ned as the most appropriate adjuvant for alltypes of ibd especially in qigong and tai chi for ucpatients smoking was found to be more conducive while ithas strong linkage with pernicious diseases all the time soconducting highquality clinicaltrials with appropriateblinding and large number of patients is necessary to obtainmore conclusive results on the curative eï¬ect of adjuvanttreatment in ibdis paper systematically expounds the signiï¬cance anddiï¬erent factors of ibd adjuvant treatments within the ï¬eldof comprehensive treatments in ibd adjuvant treatment ismost approbated among the public nowadays for its relatively low cost and minor side eï¬ects compared with traditional remedies moreover this review also aims to drawthe attention of the public to engage patients in a discussionof adjuvant treatment and underline their role as a complement to conventional ibd therapies physicians are urgedto explore the use of adjuvant treatment and provide appropriate information and guidance to patients in order todevelop highquality care for patients with ibddata availabilitye data used to support the ï¬ndings of this study areavailable from the corresponding author upon requestconflicts of intereste authors declare that they have no conï¬icts of interestauthors contributionsqiyue wang and shuyi mi contributed equally to this workacknowledgmentsis study was funded by grants from the zhejiang university student science and technology innovation projectno 2018r401194supplementary materialssupplementary materials the supplementary material ï¬le isa ï¬gure describing the mechanism and eï¬ect of physicalactivities that could alleviate gastrointestinal symptoms andimprove patients quality of life physical activities such asswimming walking tai chi and qigong may induce antiammatory modulation in releasing the interleukin6 il interleukin10 il10 lymphocyte and immunoglobulin g igg moreover the expression of interleukin 1βil1β and the tnfα protein content would be downregulated due to moderate physical activities in additionthe physiological health would be better and the hospitalization time could be shortened through physical activitiessupplementary materialsreferences v andersen a olsen f carbonnel a tjønneland andu vogel diet and risk of ammatory bowel diseasedigestive and liver disease vol no pp 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dietary beliefs of people with ulcerative colitisand their eï¬ect on relapse and nutrient intake clinicalnutrition vol no pp j g hashash and d g binion exercise and ammatorybowel disease gastroenterology clinics of north americavol no pp v ng w millard c lebrun and j howard exercise andcrohns disease speculations on potential beneï¬ts canadian of gastroenterology vol no pp r jahnke l larkey c rogers j etnier and f lin acomprehensive review of health beneï¬ts of qigong and taichi american of health promotion vol no pp e1e25 c a chapmankiddell p s w davies l gillen andg l radfordsmith role of diet in the development ofammatory bowel disease ammatory bowel diseasesvol no pp e scaioli e liverani and a belluzzi e imbalance between n 6n polyunsaturated fatty acids and ammatory bowel disease a comprehensive review and futuretherapeutic perspectives international of molecularsciences vol no p c calder polyunsaturated fatty acidsammatoryprocesses and ammatory bowel diseases molecular nutrition food research vol no pp r reifen a karlinsky a h stark z berkovich anda nyska αlinolenic acid ala is an antiammatoryagent in ammatory bowel disease e of nutritional biochemistry vol no pp a belluzzi c brignola m campieri a pera s boschi andm miglioli eï¬ect of an entericcoated ï¬shoil preparationon relapses in crohns disease new england ofmedicine vol no pp k hillier r jewell l dorrell and c l smith incorporation of fatty acids from ï¬sh oil and olive oil into colonicmucosal lipids and eï¬ects upon eicosanoid synthesis in ammatory bowel disease gut vol no pp d camuesco m comalada a concha intestinalantiammatory activity of combined quercitrin and dietary olive oil supplemented with ï¬sh oil rich in epa anddha n polyunsaturated fatty acids in rats with dssinduced colitis clinical nutrition vol no pp k azuma t osaki and t tsuka eï¬ects of ï¬sh scalecollagen peptide on an experimental ulcerative colitis mousemodel vol no pp t grimstad b bjørndal d cacabelos a salmonpeptide di leviates experimental colitis as compared withï¬sh oil of nutritional science vol p e2 t marchbank g elia and r j playford intestinal protectiveeï¬ect of a commercial ï¬sh protein hydrolysate preparationregulatory peptides vol no pp a n ananthakrishnan h khalili g g konijeti aprospective study of longterm intake of dietary ï¬ber and riskof crohns disease and ulcerative colitis gastroenterologyvol no pp a wedrychowicz a zajac and p tomasik advances innutritional therapy in ammatory bowel diseases reviewworld of gastroenterology vol no pp k m maslowski and c r mackay diet gut microbiota andimmune responses nature immunology vol no pp v andersen s chan r luben fibre intake and thedevelopment of ammatory bowel disease a europeanprospective multicentre cohort study epicibd ofcrohns and colitis vol no pp m t palmer and c t weaver linking vitamin d deï¬ciencyto ammatory bowel disease ammatory bowel diseasesvol no pp a barb´achano a fern´andezbarral g ferrermayaa costalescarrera m j larriba and a muñoz e endocrine vitamin d system in the gut molecular and cellularendocrinology vol pp d statovci m aguilera j macsharry and s melgar eimpact of western diet and nutrients on the microbiota andimmune response at mucosal interfaces frontiers in immunology vol p m ardesia g ferlazzo and w fries vitamin d and ammatory bowel disease biomed research internationalvol article id pages r del pinto d pietropaoli a k chandar c ferri andf cominelli association between ammatory boweldisease and vitamin d deï¬ciency ammatory boweldiseases vol no pp m sadeghian p saneei f siassi and a esmaillzadehvitamin d status in relation to crohns disease metaanalysisof observational studies nutrition vol no pp h m pappa p d mitchell h jiang treatment ofvitamin d insuï¬ciency in children and adolescents withammatory bowel disease a randomized clinicaltrialcomparing three regimens e of clinical endocrinology metabolism vol no pp m nakano k tominaga a hoshino t sugaya k kankeand h hiraishi erapeutic eï¬cacy of an elemental diet forpatients with crohns disease and its association with aminoacid metabolism saudi of gastroenterology vol no pp r teschke a wolï¬ c frenzel a eickhoï¬ and j schulzeherbal traditional chinese medicine and its evidence base ingastrointestinal disorders world of gastroenterologyvol no pp m salaga h zatorski m sobczak chinese herbalmedicines in the treatment of ibd and colorectal cancer areview current treatment options in oncology vol no pp 0ccanadian of gastroenterology and hepatology e j ladas j b karlik d rooney topical yunnanbaiyao administration as an adjunctive therapy for bleedingcomplications in adolescents with advanced cancer supportive care in cancer vol no pp a triantafyllidi herbal and plant therapy in patients withammatory bowel disease annals of gastroenterologyvol no pp f ke p k yadav and l z ju herbal medicine in thetreatment of ulcerative colitis saudi of gastroenterology vol no pp y song d dunkin s dahan antiammatoryeï¬ects of the chinese herbal formula fahf2 in experimentaland human ibd ammatory bowel diseases vol no pp g h n towers fahf1 purporting to block peanutinduced anaphylaxis of allergy and clinical immunology vol no p r mazieiro r r frizonandr d a goulart is curcumin a possibility to treat ammatory bowel diseases of medicinal food vol no pp s m barbalho a lang n salomon j c y wu curcumin incombination with mesalamine induces remission in patientswith mildtomoderate ulcerative colitis in a randomizedcont | 0 |
" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim | 0 |
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | 0 |
Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA STOP questions snoring tirednessobserved apnea and high blood pressure and Bang BMI kgm2 Age years Neckcircumference cm and Gender male [ ] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patients potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arms purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participants admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcares outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this studys intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patients EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participants electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemars test with correction for continuity was used to compare the participants CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [] This studys findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ ] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatients likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting original draft Kathleen WhittingtonWriting review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J The STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol 65B 105664JCSM1306 PMID Manne Mahesh B Obstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg 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Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain 10109701ajp000012975731856f7 PMID Cozowicz Crispiana Opioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief 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Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Coxsproportional hazard analysis Sleep Med 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c' | 2 |
" to improve the postoperative prognosis of patients with lung cancer predicting the recurrence highrisk patients is needed for the efficient application of adjuvant chemotherapy however predicting lung cancerrecurrence after a radical surgery is difficult even with conventional histopathological prognostic factors thereby anovel predictor should be identified as lipid metabolism alterations are known to contribute to cancer progressionwe hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors this studyaimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgerymethods frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radicalsurgery were retrospectively reviewed recurrent and nonrecurrent cases were assigned to recurrent n andnonrecurrent n groups respectively extracted lipids from frozen tissue samples were subjected to liquidchromatographytandem mass spectrometry analysis the average total lipid levels of the nonrecurrent andrecurrent groups were compared candidate predictors were screened by comparing the folding change and pvalue ofttest in each lipid species between the recurrent and nonrecurrent groupsresults the average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp a total of lipid species were increased folding change ¥ p and lipid species were decreasedfolding change p in the recurrent group among these candidates increased sphingomyelin smd351 inthe recurrent group was the most prominent candidate predictor showing high performance of recurrence predictionauc sensitivity specificity accuracy we propose smd351 as a novel candidate predictor for lung adenocarcinoma recurrence our finding cancontribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomastrial registration this retrospective study was registered at the umin clinical trial registry umin000039202 on 21stjanuary keywords lung adenocarcinoma prognostic factor recurrence prediction lipid mass spectrometry correspondence kahyohamamedacjp1department of cellular and molecular anatomy hamamatsu universityschool of medicine handayama higashi ward hamamatsu shizuoka japan5international mass imaging center hamamatsu university school ofmedicine handayama higashi ward hamamatsu shizuoka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctakanashi bmc cancer page of lung cancer is one of the leading causes of cancerrelatedmortality worldwide radical resection is the standardtreatment for stage iii nonsmall cell lung cancer nsclc however the postoperative survival rate remainsunsatisfactory despite complete resection among patientswith nsclc who received complete resection experience local or distant disease recurrence thereforeadjuvant chemotherapy should be administered to improve survival after a radical surgery adjuvant chemotherapy has been shown to reduce therisk of death due to lung cancer recurrence [] nonetheless not all patients who underwent radical surgerybenefit from adjuvant chemotherapy because some ofthem are already successfully healed without adjuvantchemotherapy therefore patients highly at risk for recurrence who are likely to benefit from adjuvant chemotherapy should be identified for the efficient applicationof adjuvant chemotherapyadenocarcinoma is the most common histologicaltype of nsclc accounting approximately of allnsclc cases in lung adenocarcinomas severalprognostic factors obtained by histopathological evaluations of surgical specimens have been reported to datesuch as lymph node metastasis pleural invasion lymphatic vessel invasion [ ] blood vessel invasion[ ] adenocarcinoma subtype of micropapillary pattern and spread through air space stas [ ]however predicting lung cancer recurrence after radicalsurgery is still difficult because data on the direct relationship between conventional prognostic factors and recurrence are limited furthermore subjective judgmentsof conventional prognostic factors are considered to hinder accurate recurrence prediction and its retrospectivevalidation accordingly novel recurrence predictors withhigh objectivity are strongly expectedexampleprevious studies demonstrated that lipid metabolismalterations in cancer contribute to cancer cell proliferation and invasion [ ] and some lipids have beensuggested as prognostic factors in several cancer typesforthe number of phosphatidylcholinepc321 in recurrent cases of primary triplenegativebreast cancer tnbc is higher than in that of nonrecurrent cases and thereby pc is suggested as acandidate predictor for tnbc recurrence oleicacid attenuation is correlated with shorter progressionfree period in clear cell renal carcinoma with regard to lung cancer although nsclc is reportedly characterized by drastic changes in phospholipid profiles ascompared to the normal lung tissue and contains different lipid profiles according to the histologic subtypes no lipidomic approach to investigate the prognosticfactor for nsclc has been used based on these previous studies we hypothesized that lung adenocarcinomaswith high recurrence risk have different lipidomes fromthat of lung adenocarcinomas with low recurrence riskand specific lipids that can be considered as candidatesas novel predictive factors for recurrencein this study lipid species that can be considered as potential predictors for lung adenocarcinoma recurrence aftera radical surgery were identified by comparing lipidomes ofprimary lung adenocarcinomas between recurrent andnonrecurrent cases using liquid chromatographytandemmass spectrometry lcmsmsmethodspatients and tissue samplesretrospective frozen tissue samples of primary lungadenocarcinoma obtained from patients who underwentradical surgery from january to december athamamatsu university hospital were examined radicalsurgery was defined as complete resection performedwith lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage i or ii and ascomplete resection achieved by segmentectomy orwedge resection with or without lymph node samplingat stage i tissue samples of primary tumors were collected immediately after the resection and stored at °c after a rapid freezing in liquid nitrogen histopathological diagnosis was performed by experienced pathologists according to the world health anizationcriteria pathological staging was identified based on the8th edition ofthe tnm classification for lung andpleuraltumors patients were followedup withcomputed tomography ct of the body trunk and biochemicalantigencea every months during the first years thenevery months until more than years after the surgerywhen cea was elevated ¥ ngml without any ctfindings of recurrence head magnetic resonance imaging and systemic positron emission tomography wereperformed for the detection of brain metastasis or bonemetastasiscarcinoembryonicexamination ofin patient selection clinical records of these tissuesamples were retrospectively reviewed patients withpathological stage i or ii indicated for radical surgeryand with major histological subtypes of invasive adenocarcinoma lepidic papillary acinar or solid predominant were analyzed patients who received inductionchemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excludedthencases withoutand with recurrence wereassigned to nonrecurrent and recurrent groups respectively recurrence was defined as radiological imagingbased findings of distant or locoregional recurrencewithin years whereas no recurrence was defined as nofindings of distant or locoregional recurrence in ¥ yearsafter the radical surgery in the nonrecurrent group 0ctakanashi bmc cancer page of cases with followup period of years were excludedin the recurrent group cases with recurrence in theform of pleural dissemination were excluded assumingthe possible attribution with insufficient surgical marginfinally cases for recurrent and for nonrecurrentgroups were subjected for analysishistological evaluationparaffinembedded tissue blocks were sectioned at μmthick sections stained by hematoxylineosin he wereexamined for adenocarcinoma subtypesizelymph node metastasis and stas d2 stain wasused to evaluate lymphatic vessel invasion and elasticavan gienson stain to evaluate blood vessel invasion allhistologicalsections were reviewed by experiencedpathologiststumorchemicalsmethanol chloroform glacial acetate and ultrapurewater were purchased from wako pure chemical industries osaka japan the 12dilauroylsnglycero3pcavanti polar lipids alabaster al pc 120_120 wasused to calibrate standard lipid levelslipid extraction from the cancer tissueeach weight of the frozen tissue samples was measuredusing sartorius analytical lab balance cpa224s sartorius ag göttingen germany additional file supplemental table after the weight measurement modifiedblighdyer methods were performed for lipid extractiontissue samples were transferred into glass tubes and ml of methanol ml of chloroform and mlof m glacial acetate were subsequently addedthen mmol of pc 120_120 per mg of sampletissue was added and subsequently followed by 10minextraction at room temperature after the extraction ml of chloroform was added and vortexed sequentially ml of m glacial acetate was added andvortexed extracted samples were subjected to centrifugation at rpm for min extracted anic layerswere transferred into new glass tubes and were evaporated until completely dried using mivac duo lv genevacextracted lipid wasdissolved with μl of methanol and μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so thatthe concentration of pc 120_120internal controlwill be as similar as possible among casesipswich england thelipid analysis by liquid chromatographytandem massspectrometry lcmsmsextracted lipids from collected frozen tissue sampleswere analyzed using q exactive¢ hybrid quadrupoleorbitrap¢ massanequipped withspectrometerelectrospray ionization source and connected to an ultimate system thermo scientific μl of the extracted lipid samples were injected and separated onacculaim c18 column mm à mm μmthermo scientific components of mobile phase awere as follows wateracetonitrilemethanol vvv mm ammonium formate and formic acidthe components of mobile phase b were as followsacetonitrileisopropanol vv mm ammonium formate and formic acid for elution the flow ratewas set at μlmin a set of linear gradient startingat solvent b was used and linearly increased to b in min maintained at b until minthen decreased linearly to b from min to min and finished with b for the last min theoverall run time was min ms instrument conditionswere as follows sheath gas flow rate auxiliary flowrate sweep gas flow rate capillary temperature °c slens rf level probe heater temperature °c and spray voltage of kv in positive mode and kv in negative mode fullms mode conditions forquantification were as follows ms scan range resolution agc target à and maximum injection time was ms for identification top datadependent ms2 method with a resolution of was used the agc target was à and themaximum injection time was ms stepped normalizedcollision energies of and for the positivemode and and for the negative mode wereapplied spectral data were acquired in the mz range of mz using an xcalibur v30 software thermoscientifictolerance ppmlipid identification and quantificationlipidsearch¢ software version mitsui knowledgeindustry tokyo japan was used to identify and quantify lipid species parameter settings for identificationwere followings database hcd retention time min search type product_qex precursor tolerance ppm and productidentificationquality filters of a b and c were used quantificationwas performed at mz tolerance of ± with retentiontime range from min to min alignment of theidentified lipid species among cases was performedwith retention time tolerance of molecules that areannotated as redundant lipid names with different calculated mz and retention times were regarded as independentisomersinadditional file duplicationannotatedasdata processingtrend analysis between the nonrecurrent and recurrentgroups was performed by comparing the average totallipid level between the two groups and principal 0ctakanashi bmc cancer page of component analysis pca intensities of lipids recordedin the xcalibur v30 software and monoisotopic peakarea values of lipid species identified by lipidsearch¢software were normalized by dividing with the areavalues of internal control pc 120_120 the total lipidlevel of each case was defined as an accumulation ofnormalized intensities of lipids normalized area valueswere subjected for pcafor respective lipid species pvalues were calculatedusing the student ttest to compare area values betweenthe two groups to screen candidate lipids for recurrence prediction lipidomes were compared between thenonrecurrent and recurrent groups by describing volcano plots with log10 pvalue for vertical axis andlog2 folding change for horizontal axis the foldingchange for a lipid was defined as an average area valueof the recurrent group divided by that of the nonrecurrent group significance was determined at pvaluesof folding change of ¥ or statistical analysisdemographic information and associations with clinicalcharacteristics were evaluated using the fisher exact testcategorical variables or the mannwhitney utest forcontinuous variables the student ttest was used tocompare the average totallipid amounts of the nonrecurrent and recurrent groups and to describe volcanoplots recurrentfree survival rfs was determined asthe time from operation until the first disease recurrenceor death survival curve was described using thekaplanmeier method the optimal cutoff values todiscriminate the two groups were determined using thereceiver operating characteristic roc curve analysisthe area under the roc curves aucs were calculatedto validate the discrimination abilities of candidate lipidsspearmans rank correlation analysis was used to validatethe correlation among candidate lipid predictors allstatistical analyses except for the ttest were performedusing r the r foundation for statistical computingvienna austria version the student ttest wasperformed with ttest of excel¢ microsoft redmond usa pvalues of were considered assignificantresultsclinicopathological characteristics of patient cohortclinicopathological characteristics of patients are shownin table in this study cohort tissue samples from nonrecurrent and recurrent cases were analyzedamong the characteristics of these two groups differences in pathological stage p lymph node metastasis p and blood vessel invasion p were statistically significant the and 2year rfs rateof the recurrent group was and with median rfstime of range monthsrespectivelyfile supplemental fig the medianadditionalfollowup time ofthe nonrecurrent and recurrentgroups was range and range months respectivelytrend analysis between the nonrecurrent and recurrentgroupsthe frozen tissue samples were subjected to lcmsms and the total lipid level of cases was calculated byaccumulating normalized intensities of lipids notablythe average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp fig a total of lipid species wereidentified and quantified by analyzing the mass spectraldata using a lipidsearch¢ software the full list of identified lipid species is presented as additional file which were also subjected to pca the pca plot didnot show clear separation between the recurrent andnonrecurrent groups however the recurrent group exhibited partial separations between the first three principal components additional file supplemental fig these results suggested differences of lipidome betweenthe recurrent and nonrecurrent groups which urged usto screen lipids to distinguish the two groupsscreening of candidate lipids for recurrence predictionto screen lipids with different levels between the twogroups volcano plots of the identified lipids were described first and lipidomes between the nonrecurrentand recurrent groups were compared fig the volcano plotidentified lipid species with relativeamounts significantly different between the two groupsfolding change ¥ or pvalues thenumber of lipids that increased and decreased in the recurrent group was and respectively these increased or decreased lipid species consisted of varioushead groups additional file increased lipid speciesshown in red decreased lipid species shown in greenthen based on prominent distributions of the volcanoplot we narrowed the candidate lipids increased inthe recurrent group to the following molecules fig biotinylbluephosphoethanolamineceramidecerd420 sphingomyelin smd351 cerd180_240pc monoether phosphatidylcholine mepc346echolesterol ester che241 mepc 408e and che20 as for the lipids that decreased in the recurrent groupthe following four molecules were annotated fig bluearrows pointing to green plots monohexosylceramidehex1cert421 otriglyceride tg150_140_140pc 182_182 and lysophosphatidylcholine lpc120biotinylpe303arrowsplotspointingtoredthe relative amounts of these lipid species were evaluated with their distributions by comparing the two 0ctakanashi bmc cancer page of table clinicopathological characteristics of the nonrecurrent and recurrent groupscharacteristicsmedian age rangenonrecurrent n gender malefemalesmoking history pathological stage iiimedian tumor size mm rangeadenocarcinoma subtypelepidicpapillaryacinarsolidlymph node metastasis pleural invasion lymphatic vessel invasion blood vessel invasion micropapillary component spread through air space driver gene mutationegfr alk surgical procedurelobectomywedge resectionadjuvant chemotherapyindication stage ia3iibreceivedrecurrent stylelocoregionaldistant recurrent n pvalueabbreviations alk anaplastic lymphoma kinase egfr epithelial growth factor receptroups fig 3a and b in all tested lipids distributionsbetween the two groups were well separated enough toestablish the cutoff values whereas only few markedoutliers were foundwe next calculated the cutoff values and auc of these lipids to evaluate their discrimination ability for diseaserecurrence and the following final candidates with topthree auc were selected smd351 cerd420 and tg 150_140_140 table respective lipidspecies can be found in additional file with the followingidentical numbers smd351 cerd420 andtg 150_140_140 these three final lipid candidates were annotated as the following ions [smd351 h] [cerd420 hcoo] and [tg 150_140_140 nh4] in the lipidsearch¢ software additional file msms for [smd351 h] [cerd420 hcoo]and [tg 150_140_140 nh4] demonstrated production peaks corresponding to phosphocholine severalfragments compatible with fragmentation of cerd42 with concomitant oxidation reaction two fragmentsproduced by neutralfatty acid fa140 orfa respectivelyadditional file supplemental fig consequentlythe annotations ofthe final candidates by lipidsearch¢ software were consistent with the results ofmsmsfrom tg 150_140_140loss ofamong these three candidate predictors smd351was found to be positively correlated with cerd420spearmans rank correlation coefficient[rs] p tg 150_140_140 was inversely correlated with smd351 rs p and tg150_140_140 was weakly inversely correlated withcerd420 rs p additional file supplemental fig 0ctakanashi bmc cancer page of conventionalpathologicalvalidation of recurrence prediction ability among thefinal lipid candidatestable shows the sensitivity specificity and accuracyof the final candidate lipid predictors compared withfactorstheprognosticlymph node metastasis and blood vesselinvasionwhich were identified as significant recurrent factorsin this cohort sensitivity of all three candidate lipidpredictors is superior to that of lymph node metastasis patients with lymph node metastasis all of themwere hilar or lobar lymph node metastasis corresponded to those in stage ii among the recurrentgroup in this study cohort half of the study population had stage i whereas the other half had stage iias lymph node metastasis can be detected amongstage ii cases the sensitivity of lymph node metastasiswas consequently lower than those of three candidatelipid predictors which detected both stage i and stageii hencethese three predictors were superior tolymph node metastasis for patient screening whencomparing the candidate lipid predictors and bloodvesselshowed predictionto those of blood vesselabilities higher or equalinvasion only smd351fig comparison of total lipid levels between the recurrent andnonrecurrent groups the average total lipid level of the recurrentgroup was times higher than that of the nonrecurrentgroup p fig volcano plots of identified lipid species each plot represents a lipid species to be identified the relative amount of lipid speciesred plots were increased fc ¥ right side of in the horizontal axis pvalue in vertical axis and that of lipid species greenplots were decreased fc left side of in the horizontal axis pvalue in vertical axis in the recurrent group nineincreased lipids showing prominent distributions and all decreased lipid species were annotated for candidate predictors blue arrowsabbreviations cer ceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepcmonoether phosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine sm sphingomyelin tg triglyceride 0ctakanashi bmc cancer page of fig comparisons of relative amount distributions between the nonrecurrent and recurrent groups are shown for increased a and decreasedb lipid species in the recurrent group boxplots show the upper percentile upper quartile median lower quartile and lower percentilemaximum and minimum values are shown in dots pvalues for significance and fcs are presented for each lipid species abbreviations cerceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepc monoetherphosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine tg triglycerideinvasion in allvalidation points therefore wepropose smd351 as the most hopeful candidate forrecurrence predictiondiscussionin this study candidate lipid predictors for lung adenocarcinoma recurrence after a radical surgery were retrospectively screened and smd351 was found as themost prominent predictor showing that the predictionability was superior to that of conventional pathologicalprognostic factors in this small cohortthe average total lipid level was significantly high inthe recurrent group in this study furthermore thenumber ofincreased lipid species was considerablyhigher than that of decreased lipid species in the recurrent group these results were consistent with that of 0ctakanashi bmc cancer page of table auc rank of candidate lipid predictors determined byroc curverankcutoff valuespeciessmd351cerd420tg150_140_140cerd180_240pc182_182che241pc412biotinylpe303lpc120hex1cert421 omepc408eche201mepc346eauc ci lipids with top three auc were selected as final candidate predictorsboldfaced notationsabbreviations auc area under the roc curve ci confidential interval rocreceiver operating characteristicprevious studies that showed an accelerated lipid synthesis in cancer cells contributing to tumor phenotypes such as cellular membrane building stimulationof signaling pathways for growth and proliferation orsurvival under hypoxic conditions by supporting glycolysis [ ] increased total lipid level in the recurrent group may be biologically plausible because theaggressiveness may be supported by accelerated lipidsynthesisthe number of smd351 and cerd420 two of finalcandidate predictors were increased in the recurrentgroup sm and cer are major bioactive components oflipid rafts on the cellular membrane sm is synthesized from cer by sm synthase sms which transfersthe phosphocholine head group from phosphatidylcholine to cer and results in concomitantly producingtable comparison of sensitivity specificity and accuracyamong the three final candidate predictors and conventionalhistopathological prognostic factorspredictors for recurrencecandidate lipid predictorsspecificitysensitivityaccuracysmd351cerd420tg150_140_140pathological prognostic factorslymph node metastasisblood vessel invasionsmd351 showed the most excellent prediction abilityabbreviations cer ceramide sm sphingomyelin tg triglyceridediacylglycerol dag sm reconversion to cer is catalyzed by sphingomyelinase smase increased smabundance and sms activity have been reported to playa critical role in cell proliferation and survival in severalcancer types [] with regard to lung cancer metabolic changes in sphingolipids are suggested to correlatewith chemoresistance phenotype and the total smlevel in cancer tissues is reportedly lower than that ofthe normallung tissue in patients with nsclc this is speculated in the report that decreased sm abundance in lung cancer tissues may be attributable to highconsumption of serine precursor by highly proliferatingcancer cells cer accumulation in the lungs has beensuggested to participate in both cell apoptosis andtumorigenesis under cigarette smokeinduced oxidativestress taking together these knowledge and significant positive correlation between smd351 h andcerd420 in this study increased synthesis flow of certoward sm in the recurrent group was suggested actually significant increase on the total sm p leveland increased tendency on total cer p anddag p levels in the recurrent group were observed in this study cohort additional file supplemental fig this result supports the suggestion ofstrong synthesis flow of cer toward sm the sm andcer levels were not compared between the tumor tissuesand normal lung tissues in this study because normallung tissue samples were lacking nonetheless increasedsmd351 and cerd420 in the recurrent group in thisstudy is consistent with previous studies [ ]based on the following explanation among lung adenocarcinomas with high sm and cer consumption casesthat can maintain increased sm and cer synthesis havehighly aggressive phenotypes resulting in recurrencedecreased tg 150_140_140 in the recurrent groupwas also included in the final candidate predictors although tg abundance in the lung cancer tissue has not yetbeen explored to date tg level in colon cancer is reportedto be lower as the disease progresses suggesting that energysupply for colon cancer with higher degree of malignancymay depend on tg hydrolysis inconsistent with theprevious study the total tg level in this study revealedno significant difference between the nonrecurrent and recurrent groups p possible explanation for decreased tg 150_140_140 in the recurrent group is thataggressive recurrent lung adenocarcinoma that may preferably consume specific tg species for energy supplythe difficulty of predicting lung cancer recurrenceusing histopathological prognostic factors may be partlyattributed to subjective judgement in addition althoughthe degree of histopathological prognostic factors widelyvaries their judgements have been performed qualitatively [ ] thereby these methods may hinder accurateretrospectiverecurrence prediction and its 0ctakanashi bmc cancer page of between representativerecurrentimages of papillarytype adenocarcinomavalidation conversely excellent prediction ability ofsmd351 that is superior to histopathological factorswas considered for its high objectivity and quantitativevalues actuallyit was difficult to predict recurrentprognosis objectively from the conventional histopathologicalthemost popular tissue subtype with no significant differenceand nonrecurrent cases whereas the mass spectrum intensitiesof [smd351 h] were markedly higher in the recurrent case to help recurrence prediction additional file supplemental fig furthermore as high smd351level was detected in all recurrent cases including stagei and stage ii cases with high specificity and accuracysmd351 was considered to be widely applicable for recurrence prediction in postoperative patients whounderwent radical surgeryseveral limitations in this study should be acknowledged first this retrospective study is performed on asmall sample size due to difficulty of obtaining frozensurgical specimens with clinical information that meetour inclusion criteria thereby verifying the reproducibility of using other validation cohorts was difficult thusidentified lipid predictors did not exceed above the candidate levels and further large cohort studies should beconducted to validate candidate predictors identified inthis study as rigid predictors for lung adenocarcinomarecurrencebecause a large number of candidate lipid species species relative to the small number of samplesize cases were screened for candidate predictorsone candidate that shows nearperfect discriminationability can be bound to be identified third adjacentnormal lung tissue samples were lacking hence the difference between the abundance of the identified candidate lipid predictors in the normal lung tissue of therecurrent group and that of the nonrecurrent groupwas not able to be compared fourth because the nonrecurrent group in this study included five cases that received adjuvant chemotherapy the nonrecurrent groupmay possibly include the recurrence highrisk casesamong them recurrence might be prevented by adjuvantchemotherapy moreover the nonrecurrent group inthis study included two cases with recurrence predictionpositive for smd351 additional file supplementalfig among the two cases one patient received adjuvant chemotherapy and the other did not the formercase may be considered as highly at risk for recurrencewhich was prevented by adjuvant chemotherapy thelatter may be an exceptional case that cannot be ruledout by smd351 fifth because lcmsms is not auniversal examination in the clinical field examining alarge number of surgical specimens for recurrence prediction using lcmsms is difficult to utilize thefindings of this study in a clinical field lipid predictorsshould be replaced with other molecules that can be examined by universal methods such as immunohistochemistry of sms or smase involved in the smmetabolism additionallyin thisstudy included histopathological type of adenocarcinomaonly as a topic for future study squamous cell carcinoma a major histological subtype behind adenocarcinomarecurrent predictorsshould be explored forthrough the lipidomic approachthe sample cohortswe propose that smd351 is a hopeful candidate predictor for lung adenocarcinoma recurrence after a radical surgery our findings provide novel insights on themechanisms oflung adenocarcinoma recurrence andcan contribute to the development of precise recurrenceprediction and qualified postoperative therapeutic strategy for lung adenocarcinomasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020073061additional file supplemental table weights of the frozen tissuesamples each weight of the frozen t | 0 |
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir15a5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a wellknown oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir15a5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at Ëc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at Ëc with co2reverse transcriptionquantitative pcr rtqpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at Ëc for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at Ëc for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions Ëc for min followed by cycles at Ëc for sec and Ëc for sec and a final extension at Ëc for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir15a5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at Ëc for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 siyap1 and the negative control targeting a nonspecific sequence siscramble were provided by thermo fisher scientific inc siha and c33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of siyap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck solution was added to each well and cultured for h at Ëc the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin vfitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdualluciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h posttransfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at Ëc overnight each membrane was probed with primary antibodies against yap1 cat no and βactin cat no at Ëc overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir15a5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir15a5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir15a5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir15a5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir15a5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir15a5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir15a5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir15a5p inhibits the invasion and migration of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c33a cells were significantly inhibited by mir15a5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir15a5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir15a5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcriptionquantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase activity was detected by a commercial caspase activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir15a5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdnayap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir15a5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells cotransfected with firefly luciferase constructs containing the yap1 wt or mut 'utrs and mir15a5p mimics mimics nc mir15a5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir15a5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir15a5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir15a5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s | 0 |
" to improve the postoperative prognosis of patients with lung cancer predicting the recurrence highrisk patients is needed for the efficient application of adjuvant chemotherapy however predicting lung cancerrecurrence after a radical surgery is difficult even with conventional histopathological prognostic factors thereby anovel predictor should be identified as lipid metabolism alterations are known to contribute to cancer progressionwe hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors this studyaimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgerymethods frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radicalsurgery were retrospectively reviewed recurrent and nonrecurrent cases were assigned to recurrent n andnonrecurrent n groups respectively extracted lipids from frozen tissue samples were subjected to liquidchromatographytandem mass spectrometry analysis the average total lipid levels of the nonrecurrent andrecurrent groups were compared candidate predictors were screened by comparing the folding change and pvalue ofttest in each lipid species between the recurrent and nonrecurrent groupsresults the average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp a total of lipid species were increased folding change ¥ p and lipid species were decreasedfolding change p in the recurrent group among these candidates increased sphingomyelin smd351 inthe recurrent group was the most prominent candidate predictor showing high performance of recurrence predictionauc sensitivity specificity accuracy we propose smd351 as a novel candidate predictor for lung adenocarcinoma recurrence our finding cancontribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomastrial registration this retrospective study was registered at the umin clinical trial registry umin000039202 on 21stjanuary keywords lung adenocarcinoma prognostic factor recurrence prediction lipid mass spectrometry correspondence kahyohamamedacjp1department of cellular and molecular anatomy hamamatsu universityschool of medicine handayama higashi ward hamamatsu shizuoka japan5international mass imaging center hamamatsu university school ofmedicine handayama higashi ward hamamatsu shizuoka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctakanashi bmc cancer page of lung cancer is one of the leading causes of cancerrelatedmortality worldwide radical resection is the standardtreatment for stage iii nonsmall cell lung cancer nsclc however the postoperative survival rate remainsunsatisfactory despite complete resection among patientswith nsclc who received complete resection experience local or distant disease recurrence thereforeadjuvant chemotherapy should be administered to improve survival after a radical surgery adjuvant chemotherapy has been shown to reduce therisk of death due to lung cancer recurrence [] nonetheless not all patients who underwent radical surgerybenefit from adjuvant chemotherapy because some ofthem are already successfully healed without adjuvantchemotherapy therefore patients highly at risk for recurrence who are likely to benefit from adjuvant chemotherapy should be identified for the efficient applicationof adjuvant chemotherapyadenocarcinoma is the most common histologicaltype of nsclc accounting approximately of allnsclc cases in lung adenocarcinomas severalprognostic factors obtained by histopathological evaluations of surgical specimens have been reported to datesuch as lymph node metastasis pleural invasion lymphatic vessel invasion [ ] blood vessel invasion[ ] adenocarcinoma subtype of micropapillary pattern and spread through air space stas [ ]however predicting lung cancer recurrence after radicalsurgery is still difficult because data on the direct relationship between conventional prognostic factors and recurrence are limited furthermore subjective judgmentsof conventional prognostic factors are considered to hinder accurate recurrence prediction and its retrospectivevalidation accordingly novel recurrence predictors withhigh objectivity are strongly expectedexampleprevious studies demonstrated that lipid metabolismalterations in cancer contribute to cancer cell proliferation and invasion [ ] and some lipids have beensuggested as prognostic factors in several cancer typesforthe number of phosphatidylcholinepc321 in recurrent cases of primary triplenegativebreast cancer tnbc is higher than in that of nonrecurrent cases and thereby pc is suggested as acandidate predictor for tnbc recurrence oleicacid attenuation is correlated with shorter progressionfree period in clear cell renal carcinoma with regard to lung cancer although nsclc is reportedly characterized by drastic changes in phospholipid profiles ascompared to the normal lung tissue and contains different lipid profiles according to the histologic subtypes no lipidomic approach to investigate the prognosticfactor for nsclc has been used based on these previous studies we hypothesized that lung adenocarcinomaswith high recurrence risk have different lipidomes fromthat of lung adenocarcinomas with low recurrence riskand specific lipids that can be considered as candidatesas novel predictive factors for recurrencein this study lipid species that can be considered as potential predictors for lung adenocarcinoma recurrence aftera radical surgery were identified by comparing lipidomes ofprimary lung adenocarcinomas between recurrent andnonrecurrent cases using liquid chromatographytandemmass spectrometry lcmsmsmethodspatients and tissue samplesretrospective frozen tissue samples of primary lungadenocarcinoma obtained from patients who underwentradical surgery from january to december athamamatsu university hospital were examined radicalsurgery was defined as complete resection performedwith lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage i or ii and ascomplete resection achieved by segmentectomy orwedge resection with or without lymph node samplingat stage i tissue samples of primary tumors were collected immediately after the resection and stored at °c after a rapid freezing in liquid nitrogen histopathological diagnosis was performed by experienced pathologists according to the world health anizationcriteria pathological staging was identified based on the8th edition ofthe tnm classification for lung andpleuraltumors patients were followedup withcomputed tomography ct of the body trunk and biochemicalantigencea every months during the first years thenevery months until more than years after the surgerywhen cea was elevated ¥ ngml without any ctfindings of recurrence head magnetic resonance imaging and systemic positron emission tomography wereperformed for the detection of brain metastasis or bonemetastasiscarcinoembryonicexamination ofin patient selection clinical records of these tissuesamples were retrospectively reviewed patients withpathological stage i or ii indicated for radical surgeryand with major histological subtypes of invasive adenocarcinoma lepidic papillary acinar or solid predominant were analyzed patients who received inductionchemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excludedthencases withoutand with recurrence wereassigned to nonrecurrent and recurrent groups respectively recurrence was defined as radiological imagingbased findings of distant or locoregional recurrencewithin years whereas no recurrence was defined as nofindings of distant or locoregional recurrence in ¥ yearsafter the radical surgery in the nonrecurrent group 0ctakanashi bmc cancer page of cases with followup period of years were excludedin the recurrent group cases with recurrence in theform of pleural dissemination were excluded assumingthe possible attribution with insufficient surgical marginfinally cases for recurrent and for nonrecurrentgroups were subjected for analysishistological evaluationparaffinembedded tissue blocks were sectioned at μmthick sections stained by hematoxylineosin he wereexamined for adenocarcinoma subtypesizelymph node metastasis and stas d2 stain wasused to evaluate lymphatic vessel invasion and elasticavan gienson stain to evaluate blood vessel invasion allhistologicalsections were reviewed by experiencedpathologiststumorchemicalsmethanol chloroform glacial acetate and ultrapurewater were purchased from wako pure chemical industries osaka japan the 12dilauroylsnglycero3pcavanti polar lipids alabaster al pc 120_120 wasused to calibrate standard lipid levelslipid extraction from the cancer tissueeach weight of the frozen tissue samples was measuredusing sartorius analytical lab balance cpa224s sartorius ag göttingen germany additional file supplemental table after the weight measurement modifiedblighdyer methods were performed for lipid extractiontissue samples were transferred into glass tubes and ml of methanol ml of chloroform and mlof m glacial acetate were subsequently addedthen mmol of pc 120_120 per mg of sampletissue was added and subsequently followed by 10minextraction at room temperature after the extraction ml of chloroform was added and vortexed sequentially ml of m glacial acetate was added andvortexed extracted samples were subjected to centrifugation at rpm for min extracted anic layerswere transferred into new glass tubes and were evaporated until completely dried using mivac duo lv genevacextracted lipid wasdissolved with μl of methanol and μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so thatthe concentration of pc 120_120internal controlwill be as similar as possible among casesipswich england thelipid analysis by liquid chromatographytandem massspectrometry lcmsmsextracted lipids from collected frozen tissue sampleswere analyzed using q exactive¢ hybrid quadrupoleorbitrap¢ massanequipped withspectrometerelectrospray ionization source and connected to an ultimate system thermo scientific μl of the extracted lipid samples were injected and separated onacculaim c18 column mm à mm μmthermo scientific components of mobile phase awere as follows wateracetonitrilemethanol vvv mm ammonium formate and formic acidthe components of mobile phase b were as followsacetonitrileisopropanol vv mm ammonium formate and formic acid for elution the flow ratewas set at μlmin a set of linear gradient startingat solvent b was used and linearly increased to b in min maintained at b until minthen decreased linearly to b from min to min and finished with b for the last min theoverall run time was min ms instrument conditionswere as follows sheath gas flow rate auxiliary flowrate sweep gas flow rate capillary temperature °c slens rf level probe heater temperature °c and spray voltage of kv in positive mode and kv in negative mode fullms mode conditions forquantification were as follows ms scan range resolution agc target à and maximum injection time was ms for identification top datadependent ms2 method with a resolution of was used the agc target was à and themaximum injection time was ms stepped normalizedcollision energies of and for the positivemode and and for the negative mode wereapplied spectral data were acquired in the mz range of mz using an xcalibur v30 software thermoscientifictolerance ppmlipid identification and quantificationlipidsearch¢ software version mitsui knowledgeindustry tokyo japan was used to identify and quantify lipid species parameter settings for identificationwere followings database hcd retention time min search type product_qex precursor tolerance ppm and productidentificationquality filters of a b and c were used quantificationwas performed at mz tolerance of ± with retentiontime range from min to min alignment of theidentified lipid species among cases was performedwith retention time tolerance of molecules that areannotated as redundant lipid names with different calculated mz and retention times were regarded as independentisomersinadditional file duplicationannotatedasdata processingtrend analysis between the nonrecurrent and recurrentgroups was performed by comparing the average totallipid level between the two groups and principal 0ctakanashi bmc cancer page of component analysis pca intensities of lipids recordedin the xcalibur v30 software and monoisotopic peakarea values of lipid species identified by lipidsearch¢software were normalized by dividing with the areavalues of internal control pc 120_120 the total lipidlevel of each case was defined as an accumulation ofnormalized intensities of lipids normalized area valueswere subjected for pcafor respective lipid species pvalues were calculatedusing the student ttest to compare area values betweenthe two groups to screen candidate lipids for recurrence prediction lipidomes were compared between thenonrecurrent and recurrent groups by describing volcano plots with log10 pvalue for vertical axis andlog2 folding change for horizontal axis the foldingchange for a lipid was defined as an average area valueof the recurrent group divided by that of the nonrecurrent group significance was determined at pvaluesof folding change of ¥ or statistical analysisdemographic information and associations with clinicalcharacteristics were evaluated using the fisher exact testcategorical variables or the mannwhitney utest forcontinuous variables the student ttest was used tocompare the average totallipid amounts of the nonrecurrent and recurrent groups and to describe volcanoplots recurrentfree survival rfs was determined asthe time from operation until the first disease recurrenceor death survival curve was described using thekaplanmeier method the optimal cutoff values todiscriminate the two groups were determined using thereceiver operating characteristic roc curve analysisthe area under the roc curves aucs were calculatedto validate the discrimination abilities of candidate lipidsspearmans rank correlation analysis was used to validatethe correlation among candidate lipid predictors allstatistical analyses except for the ttest were performedusing r the r foundation for statistical computingvienna austria version the student ttest wasperformed with ttest of excel¢ microsoft redmond usa pvalues of were considered assignificantresultsclinicopathological characteristics of patient cohortclinicopathological characteristics of patients are shownin table in this study cohort tissue samples from nonrecurrent and recurrent cases were analyzedamong the characteristics of these two groups differences in pathological stage p lymph node metastasis p and blood vessel invasion p were statistically significant the and 2year rfs rateof the recurrent group was and with median rfstime of range monthsrespectivelyfile supplemental fig the medianadditionalfollowup time ofthe nonrecurrent and recurrentgroups was range and range months respectivelytrend analysis between the nonrecurrent and recurrentgroupsthe frozen tissue samples were subjected to lcmsms and the total lipid level of cases was calculated byaccumulating normalized intensities of lipids notablythe average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp fig a total of lipid species wereidentified and quantified by analyzing the mass spectraldata using a lipidsearch¢ software the full list of identified lipid species is presented as additional file which were also subjected to pca the pca plot didnot show clear separation between the recurrent andnonrecurrent groups however the recurrent group exhibited partial separations between the first three principal components additional file supplemental fig these results suggested differences of lipidome betweenthe recurrent and nonrecurrent groups which urged usto screen lipids to distinguish the two groupsscreening of candidate lipids for recurrence predictionto screen lipids with different levels between the twogroups volcano plots of the identified lipids were described first and lipidomes between the nonrecurrentand recurrent groups were compared fig the volcano plotidentified lipid species with relativeamounts significantly different between the two groupsfolding change ¥ or pvalues thenumber of lipids that increased and decreased in the recurrent group was and respectively these increased or decreased lipid species consisted of varioushead groups additional file increased lipid speciesshown in red decreased lipid species shown in greenthen based on prominent distributions of the volcanoplot we narrowed the candidate lipids increased inthe recurrent group to the following molecules fig biotinylbluephosphoethanolamineceramidecerd420 sphingomyelin smd351 cerd180_240pc monoether phosphatidylcholine mepc346echolesterol ester che241 mepc 408e and che20 as for the lipids that decreased in the recurrent groupthe following four molecules were annotated fig bluearrows pointing to green plots monohexosylceramidehex1cert421 otriglyceride tg150_140_140pc 182_182 and lysophosphatidylcholine lpc120biotinylpe303arrowsplotspointingtoredthe relative amounts of these lipid species were evaluated with their distributions by comparing the two 0ctakanashi bmc cancer page of table clinicopathological characteristics of the nonrecurrent and recurrent groupscharacteristicsmedian age rangenonrecurrent n gender malefemalesmoking history pathological stage iiimedian tumor size mm rangeadenocarcinoma subtypelepidicpapillaryacinarsolidlymph node metastasis pleural invasion lymphatic vessel invasion blood vessel invasion micropapillary component spread through air space driver gene mutationegfr alk surgical procedurelobectomywedge resectionadjuvant chemotherapyindication stage ia3iibreceivedrecurrent stylelocoregionaldistant recurrent n pvalueabbreviations alk anaplastic lymphoma kinase egfr epithelial growth factor receptroups fig 3a and b in all tested lipids distributionsbetween the two groups were well separated enough toestablish the cutoff values whereas only few markedoutliers were foundwe next calculated the cutoff values and auc of these lipids to evaluate their discrimination ability for diseaserecurrence and the following final candidates with topthree auc were selected smd351 cerd420 and tg 150_140_140 table respective lipidspecies can be found in additional file with the followingidentical numbers smd351 cerd420 andtg 150_140_140 these three final lipid candidates were annotated as the following ions [smd351 h] [cerd420 hcoo] and [tg 150_140_140 nh4] in the lipidsearch¢ software additional file msms for [smd351 h] [cerd420 hcoo]and [tg 150_140_140 nh4] demonstrated production peaks corresponding to phosphocholine severalfragments compatible with fragmentation of cerd42 with concomitant oxidation reaction two fragmentsproduced by neutralfatty acid fa140 orfa respectivelyadditional file supplemental fig consequentlythe annotations ofthe final candidates by lipidsearch¢ software were consistent with the results ofmsmsfrom tg 150_140_140loss ofamong these three candidate predictors smd351was found to be positively correlated with cerd420spearmans rank correlation coefficient[rs] p tg 150_140_140 was inversely correlated with smd351 rs p and tg150_140_140 was weakly inversely correlated withcerd420 rs p additional file supplemental fig 0ctakanashi bmc cancer page of conventionalpathologicalvalidation of recurrence prediction ability among thefinal lipid candidatestable shows the sensitivity specificity and accuracyof the final candidate lipid predictors compared withfactorstheprognosticlymph node metastasis and blood vesselinvasionwhich were identified as significant recurrent factorsin this cohort sensitivity of all three candidate lipidpredictors is superior to that of lymph node metastasis patients with lymph node metastasis all of themwere hilar or lobar lymph node metastasis corresponded to those in stage ii among the recurrentgroup in this study cohort half of the study population had stage i whereas the other half had stage iias lymph node metastasis can be detected amongstage ii cases the sensitivity of lymph node metastasiswas consequently lower than those of three candidatelipid predictors which detected both stage i and stageii hencethese three predictors were superior tolymph node metastasis for patient screening whencomparing the candidate lipid predictors and bloodvesselshowed predictionto those of blood vesselabilities higher or equalinvasion only smd351fig comparison of total lipid levels between the recurrent andnonrecurrent groups the average total lipid level of the recurrentgroup was times higher than that of the nonrecurrentgroup p fig volcano plots of identified lipid species each plot represents a lipid species to be identified the relative amount of lipid speciesred plots were increased fc ¥ right side of in the horizontal axis pvalue in vertical axis and that of lipid species greenplots were decreased fc left side of in the horizontal axis pvalue in vertical axis in the recurrent group nineincreased lipids showing prominent distributions and all decreased lipid species were annotated for candidate predictors blue arrowsabbreviations cer ceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepcmonoether phosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine sm sphingomyelin tg triglyceride 0ctakanashi bmc cancer page of fig comparisons of relative amount distributions between the nonrecurrent and recurrent groups are shown for increased a and decreasedb lipid species in the recurrent group boxplots show the upper percentile upper quartile median lower quartile and lower percentilemaximum and minimum values are shown in dots pvalues for significance and fcs are presented for each lipid species abbreviations cerceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepc monoetherphosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine tg triglycerideinvasion in allvalidation points therefore wepropose smd351 as the most hopeful candidate forrecurrence predictiondiscussionin this study candidate lipid predictors for lung adenocarcinoma recurrence after a radical surgery were retrospectively screened and smd351 was found as themost prominent predictor showing that the predictionability was superior to that of conventional pathologicalprognostic factors in this small cohortthe average total lipid level was significantly high inthe recurrent group in this study furthermore thenumber ofincreased lipid species was considerablyhigher than that of decreased lipid species in the recurrent group these results were consistent with that of 0ctakanashi bmc cancer page of table auc rank of candidate lipid predictors determined byroc curverankcutoff valuespeciessmd351cerd420tg150_140_140cerd180_240pc182_182che241pc412biotinylpe303lpc120hex1cert421 omepc408eche201mepc346eauc ci lipids with top three auc were selected as final candidate predictorsboldfaced notationsabbreviations auc area under the roc curve ci confidential interval rocreceiver operating characteristicprevious studies that showed an accelerated lipid synthesis in cancer cells contributing to tumor phenotypes such as cellular membrane building stimulationof signaling pathways for growth and proliferation orsurvival under hypoxic conditions by supporting glycolysis [ ] increased total lipid level in the recurrent group may be biologically plausible because theaggressiveness may be supported by accelerated lipidsynthesisthe number of smd351 and cerd420 two of finalcandidate predictors were increased in the recurrentgroup sm and cer are major bioactive components oflipid rafts on the cellular membrane sm is synthesized from cer by sm synthase sms which transfersthe phosphocholine head group from phosphatidylcholine to cer and results in concomitantly producingtable comparison of sensitivity specificity and accuracyamong the three final candidate predictors and conventionalhistopathological prognostic factorspredictors for recurrencecandidate lipid predictorsspecificitysensitivityaccuracysmd351cerd420tg150_140_140pathological prognostic factorslymph node metastasisblood vessel invasionsmd351 showed the most excellent prediction abilityabbreviations cer ceramide sm sphingomyelin tg triglyceridediacylglycerol dag sm reconversion to cer is catalyzed by sphingomyelinase smase increased smabundance and sms activity have been reported to playa critical role in cell proliferation and survival in severalcancer types [] with regard to lung cancer metabolic changes in sphingolipids are suggested to correlatewith chemoresistance phenotype and the total smlevel in cancer tissues is reportedly lower than that ofthe normallung tissue in patients with nsclc this is speculated in the report that decreased sm abundance in lung cancer tissues may be attributable to highconsumption of serine precursor by highly proliferatingcancer cells cer accumulation in the lungs has beensuggested to participate in both cell apoptosis andtumorigenesis under cigarette smokeinduced oxidativestress taking together these knowledge and significant positive correlation between smd351 h andcerd420 in this study increased synthesis flow of certoward sm in the recurrent group was suggested actually significant increase on the total sm p leveland increased tendency on total cer p anddag p levels in the recurrent group were observed in this study cohort additional file supplemental fig this result supports the suggestion ofstrong synthesis flow of cer toward sm the sm andcer levels were not compared between the tumor tissuesand normal lung tissues in this study because normallung tissue samples were lacking nonetheless increasedsmd351 and cerd420 in the recurrent group in thisstudy is consistent with previous studies [ ]based on the following explanation among lung adenocarcinomas with high sm and cer consumption casesthat can maintain increased sm and cer synthesis havehighly aggressive phenotypes resulting in recurrencedecreased tg 150_140_140 in the recurrent groupwas also included in the final candidate predictors although tg abundance in the lung cancer tissue has not yetbeen explored to date tg level in colon cancer is reportedto be lower as the disease progresses suggesting that energysupply for colon cancer with higher degree of malignancymay depend on tg hydrolysis inconsistent with theprevious study the total tg level in this study revealedno significant difference between the nonrecurrent and recurrent groups p possible explanation for decreased tg 150_140_140 in the recurrent group is thataggressive recurrent lung adenocarcinoma that may preferably consume specific tg species for energy supplythe difficulty of predicting lung cancer recurrenceusing histopathological prognostic factors may be partlyattributed to subjective judgement in addition althoughthe degree of histopathological prognostic factors widelyvaries their judgements have been performed qualitatively [ ] thereby these methods may hinder accurateretrospectiverecurrence prediction and its 0ctakanashi bmc cancer page of between representativerecurrentimages of papillarytype adenocarcinomavalidation conversely excellent prediction ability ofsmd351 that is superior to histopathological factorswas considered for its high objectivity and quantitativevalues actuallyit was difficult to predict recurrentprognosis objectively from the conventional histopathologicalthemost popular tissue subtype with no significant differenceand nonrecurrent cases whereas the mass spectrum intensitiesof [smd351 h] were markedly higher in the recurrent case to help recurrence prediction additional file supplemental fig furthermore as high smd351level was detected in all recurrent cases including stagei and stage ii cases with high specificity and accuracysmd351 was considered to be widely applicable for recurrence prediction in postoperative patients whounderwent radical surgeryseveral limitations in this study should be acknowledged first this retrospective study is performed on asmall sample size due to difficulty of obtaining frozensurgical specimens with clinical information that meetour inclusion criteria thereby verifying the reproducibility of using other validation cohorts was difficult thusidentified lipid predictors did not exceed above the candidate levels and further large cohort studies should beconducted to validate candidate predictors identified inthis study as rigid predictors for lung adenocarcinomarecurrencebecause a large number of candidate lipid species species relative to the small number of samplesize cases were screened for candidate predictorsone candidate that shows nearperfect discriminationability can be bound to be identified third adjacentnormal lung tissue samples were lacking hence the difference between the abundance of the identified candidate lipid predictors in the normal lung tissue of therecurrent group and that of the nonrecurrent groupwas not able to be compared fourth because the nonrecurrent group in this study included five cases that received adjuvant chemotherapy the nonrecurrent groupmay possibly include the recurrence highrisk casesamong them recurrence might be prevented by adjuvantchemotherapy moreover the nonrecurrent group inthis study included two cases with recurrence predictionpositive for smd351 additional file supplementalfig among the two cases one patient received adjuvant chemotherapy and the other did not the formercase may be considered as highly at risk for recurrencewhich was prevented by adjuvant chemotherapy thelatter may be an exceptional case that cannot be ruledout by smd351 fifth because lcmsms is not auniversal examination in the clinical field examining alarge number of surgical specimens for recurrence prediction using lcmsms is difficult to utilize thefindings of this study in a clinical field lipid predictorsshould be replaced with other molecules that can be examined by universal methods such as immunohistochemistry of sms or smase involved in the smmetabolism additionallyin thisstudy included histopathological type of adenocarcinomaonly as a topic for future study squamous cell carcinoma a major histological subtype behind adenocarcinomarecurrent predictorsshould be explored forthrough the lipidomic approachthe sample cohortswe propose that smd351 is a hopeful candidate predictor for lung adenocarcinoma recurrence after a radical surgery our findings provide novel insights on themechanisms oflung adenocarcinoma recurrence andcan contribute to the development of precise recurrenceprediction and qualified postoperative therapeutic strategy for lung adenocarcinomasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020073061additional file supplemental table weights of the frozen tissuesamples each weight of the frozen t | 0 |
the ongoing pandemic coronavirus disease outbreak covid2019 stemming from the beta coronavirus class sarscov2 causing severe acute respiratory syndrome sars has created a global health emergency in countries around the globe huang nalla sarscov2 a novel coronavirus strain belonging to the sarbecovirus subgenus genus betacoronavirus family coronaviridae had first appeared in late in wuhan china infecting a large number of hosts million people with a mortality rate of ¥ neogi carter such putative etiopathogenic agents associated with the zoonotic viral transmission pathways are responsible for respiratory viral pneumonia and gastrointestinal infections leading to infected people or patients having multiple an failure in a b s t r a c t severe acute respiratory syndrome coronavirus sarscov2 a neoteric virus belonging to the beta coronavirus class has created a global health concern responsible for an outbreak of severe acute respiratory illness the covid19 pandemic infected hosts exhibit diverse clinical features ranging from asymptomatic to severe symptoms in their genital ans respiratory digestive and circulatory systems considering the high transmissibility r0 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for the clinical development of suitable antiviral nanotherapeutics ntps is incessant we are presenting a systematic review of the literature published between and to validate the hypothesis that the pharmacokinetics collateral acutechronic side effects of nano drugs and spike proteins arrangement of coronaviruses can revolutionize the therapeutic approach to cure covid19 our aim is also to critically assess the slow release kinetics and specific target site chemical synthesis influenced competence of ntps and nanotoxicity based antiviral actions which are commonly exploited in the synthesis of modulated nanomedicines the pathogenesis of novel virulent pathogens at the cellular and molecular levels are also considered which is of utmost importance to characterize the emerging nanodrug agents as diagnostics or therapeutics or viral entry inhibitors such types of approaches trigger the scientists and policymakers in the development of a conceptual framework of nanobiotechnology by linking nanoscience and virology to present a smart molecular diagnosis treatment for pandemic viral infections comorbidities till date no therapeutic drug has been discovered for the treatment of sarscov2 infection though the importance of monoclonal antibodies protease and helicase inhibitors and interferons ifnsα treatments have been highlighted in a handful of literature farzin torchilin palmieri and papi development of drugresistant strains host cell toxicitytarget specific actions costs associated with the serological diagnosis and therapeutics limit the widespread application of synthetic drugs nucleoside analogs palmieri and papi torchilin harnessing the potential of nanobiotechnology in the biomedical science development of engineered nanostructured materialsnanomedicines may lead to better drug delivery advanced therapeutics and medical diagnostics at nanoscale in recent years with the advancement of clinical practices the use of metal gold silver zinc and copper corresponding author discipline of earth sciences room no 336a block indian institute of technology gandhinagar gujarat india email addresses manishenvgmailcom manishkumariitgnacin m kumar 101016jenvres2020110119 received may received in revised form july accepted august environmentalresearch1912020110119availableonline23august2020001393512020elsevierincallrightsreserved 0cs mukherjee nanops in magnetic immunoassay viral diagnostics and microfluidic technology has driven the researchers to explore the potential of nanotherapeutics makvandi farzin letko and ahlawat and narayan have investigated the multistrain inhibition of sars coronavirus sarscov herpes simplex virus hsv1 and human immunodeficiency virus hiv of tcells by sulfonated nanops binding they have reported that small p size tunable surface charges biomimetic properties faster encapsulation have made the engineered nanops smart and stable colloidal carriers for the delivery of genes and drugs the mode of action of functionalized nanops can be explained by their covalent linkages with biological substrates such as peptides proteins antibodies and nucleic acids some researchers have reported virusnanops electrostatic nonspecific interactions elsewhere ie influenza a h5n1 strain inhibition alizadeh and khodavandi rothan and byrareddy nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha bachmaier balakrishna such surface proteins often act as a prime inducer of neutralizing antibodies as observed by boulware and chaturvedi and shrivastava where antigenic determinants enable nucleocapsid to take entry into the host cells as reported for in vitro sarscov inhibition of virus replication and the fusion between the viral and host cell endosomal membrane are often facilitated by some emerging nanobased technologies ie silver nanorod array surface enhanced raman spectroscopy sers substrate interferometric biosensor immunoassay chauhan chhikara choudhary the development of such biomolecular detectors enable the molecular binding of virus ps to target specific antigen phase antibodies coated waveguide which help in the fast and reliable detection of viral infections kirchdoerfer and ward cojocaru das considering the high transmissibility r0 and low to moderate pathogenicity of sarscov2 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for clinical development of suitable antiviral nanotherapeutics is need of table a summary of biocompatible nanomaterials and antiviral nanopharmaceuticals commonly used for biomedical drug delivery action as virucidal agents souce of data weiss udugama letko jamshidi gao dong nanocarriers aunps immobilized aunps tio2 nps modified tio2nps agnps engineered agnps sinps mesoporous si fullerenes modified fullerenes fenps engineered fenps acidbasic functionalized nanotube metal functionalized nanotube carbon dots graphene oxide go polystyrene nps virucidal action immunizationviral detection viral detection inactivation of virus by photolysis virus inhibitioninactivation viral entry inhibition viral replication deformation virus detection viral entry inhibition virus destabilization inhibition of virus entry host pathogen interaction viral detectionremoval virus inactivation immunization viral inhibition viral entry hindrance mucosal vaccine development target specific cell bhk21 helacdltr vero cells c636 balbc mice na mdck vero cells human rhabdomyosarcoma vero cells hek293t na supt1 na na ncih292 grass carp pk15 marc145 vero cells na virus types hin1 h3n2 hiv1 h3n2 dengue virus h5n1 h3n2 h5n1 h1n1 h7n3 feline calicivirus influenza papilloma virus hsv1 bacteriophage λ hiv1 wild and resistant type zika virus bacteriophage ms2 h5n2 h3n2 reovirus rsv pseudorabies virus porcine epidemic diarrhea virus hiv1 mode of antiviral activity binding with peroxidasemimic enzymes and viral gp120 antibody mediated inhibition viral capsid protein interaction viral surface protein interaction inhibit cd4based binding viral envelop rupture cell mediated immunenucleic acid inhibition hinder viral attachment viral capsidenvelop attachment and interaction impairing viral polyproteinhinder gag processing viral envelopprotein binding phosphatidylserine inhibit viral tropism photoactivated mediated viral inhibition destabilization vp7dna mediated inhibition type i interferon production inhibited negative single layered sharp edged p interaction with virus viral gp120 antigen binding and mannoselectin specific inhibition action immune system inhibition cd8 t cells inhibition antibodydependent cellmediated phagocytosiscytotoxicity viral transcriptase inhibition gene silencing action viral cell entry inhibition peroxidase inhibition viral entry inhibition coagulation results from virus surface protein interaction magnb mediated enzymeatic signaling inhibition cell cycle inhibition at g2m phase glycosaminoglycan binding affinity and in vitro replication inhibition chitosan coated nps peptide coated nps protein coated nps amide coated nps nanoliposomes nanomicelle aunpscarbon nanotubes polymeric micelle aunpsagnps aufenpscarbon nanotubes nanolipid carriers dendrimer na balbc strain balbc mice neuro 2a cell lines na apc49 huh75 na male wistar rats cell mdck na vk2e6e7 vero cells helfs rabies virus influenza a virus influenza a virus hiv na hepatitis c virus h3n2 hiv h1n1 h3n2 norovirus h1n1 papilloma virus hsv12 immunization nanoparticulate vaccine influenza vaccine antiviral therapy drug delivery immunomodulator antiviral activity and bioavailable vaccines viral detection by colorimetric assay oral bioavailable drugs viral inhibition and drug delivery action viral dna detection nontoxic viral inhibition mrna vaccine na not applicable aunpsgold nanops agnpssilver nanops fenpsiron nanops sinpssilica nanops tio2 titanium nanops gographene oxides environmentalresearch19120201101192 0ccome under anic nanocarrier systems based on the their function as therapeutic agents encapsulated chemical attachment adsorbed or dissolved grein neogi gacem fig demonstrates the comparative size range of nanops whereas for hybrid nanobased systems the molecular composition and target specific action of individual engineered nanoparticulate systems play a vital role in drug delivery actions table the mode of action of such nanobased therapeutic agents can be categorised based on the permeability of vasculature passive targeting for malignancy or attachment of bioactive ligands to the selective nanotherapeutics kirchdoerfer and ward gao gadade and pekamwar role of metallic nanops as in vivo and in vitro drug discharging agents s mukherjee the hour rosenberg nalla dung though it is noteworthy to mention that r0 is not data specific to the covid19 disease rather it depends on the place and the behavior of the population it is associated with social isolation hygienic habits among others and the variation must occur in different ways viceconte and petrosillo li the drug development for antiviral treatment of sarscov2 depends on several factors such as pharmacokinetics drug properties collateral acutechronic side effects and spike proteins s arrangement of coronaviruses virus properties these can act as therapeutic targets to prevent the fusion of the virus to host cell via binding with host cell receptors trimeric scaffolds such as nsp10 a viral transcription factor used along with secondary antigens in vaccines for lung and lower respiratory tract infections these are essential to nucleate and stabilize pseudosubdomains of protein and peptide antigens which are also responsible for cell infection and polycistronic expression that may help in the nanobased therapeutic vaccine development dong grein etman peginterferon α2a2b pegasys ifnα2b show excellent drug delivery actions against hepatitis virus c whereas cationiccrosslinked nanops biodegradable polymers such as mpolyethylene glycol mpegpla clpm are found suitable for hepatitis b and c virus infections synthetic and natural polymeric nanops pnps are widely used since the last decade for the effective control of pathogenic viruses based on their individual biochemical properties immuno biocompatibility biodistribution factors table low to zero toxicity profile high stability against proteinase degradation improved safety efficacy profiles and good internalization properties make such nanozymes effective against chronic infectious diseases and also help in exerting their cytoprotective actions against cytopathic effect polymeric nanops in hybridized forms have found their way in biomedical sectors as therapeutics and diagnostics of human adenovirus influenza virus and hiv therefore the widespread application of hybridized nanoformulation systems have been documented by kerry and kim where functionalized dnazyme along with cellular peptides encoding viral envelopes help in the knockdown of hsv2 hvc and influenza a viruses for the first time the purpose of the present systematic review lays out the framework for a developing a critical understanding of selfassembling metal nanops targeting a variety of fusion proteins for vaccine development b the spatial geometry three fold symmetry axis and radial distributions that drive the rapid antigen processing and render virucidal activity c building up a deep insight for biomarkers research in both prophylactic and therapeutic approaches d the critical assessment of nanops as therapeutics pathways biodegradation this review also provides a guide map for the regulation of metabolic enzymes on selected nanopharmaceuticals through which the multigenerational effects can be evaluated this publication is designed to provide vital information on biocompatible nanocarriers active vs passive targeted drug delivery action of nanomedicines and critically analyze the possible hybrid nanobased therapy for sarscov2 inhibition our current review also highlights the stateofart molecular fingerprinting techniques of virus identification through advanced biosensing methods which critically explore integral surveillance and monitoring of novel viral genotypes mode of action and biomedical applications of biocompatible nanocarriers the presence of multiple surface binding sites in vivo clinical interaction with the targeted sites composition based multiple interactions shape luminescence and large surfacevolume ratio of inanic nanops render their multifarious biomedical applications ignatov dong the virucidal activity of silver nanops agnps allows its wider application in the annihilation or amelioration of several viral infections such as poliovirus type1 coxsackievirus b3 influenza a virus etc the mode of action of such nanops can be described either inhibition of cd4dependent cellular bindingpathogenesis or by covalent linking with sulfhydryl group virion surface hill neogi elsheekh the viral internalization can also be prevented by merging nanops with viral genomecore protein that hinders viral replicationattachment release of viral core into the cytoplasm and governing conformational changes in viruscoreceptor association letko hu this type of viral replication inhibitions are quite effective in curing of dsrna viruses infectious hsv2 and bursal disease viruses iravani jamshidi on the other hand gold nanops aunps may also exert their antiviral efficacy through hindering of gp120 fusion to cd4 that are surrounded by capping agents encapsulated aunps table hybridized and charged aunps render virucidal mechanism after associating with nanobased formulation and mimicking peroxidase enzymatic reaction where inhibited viral entry is facilitated by blocking of transcription within the host cell isida kumar 2020a 2020b 2020c kang on the other hand stabilization of magnetic nanops is often carried out by biocompatible polymers which helps in the translation of magneto responsive nanoparticulate systems in clinical diagnostics bio imaging bioresonanceimaging and cell separation the use of superparamagnetic iron nanops γfe2o3fe2o3fe3o4 is not directly involved in the therapeutics but their indirect application may inhibit viral multiplication even at postentry cellular level as seen for zika virus h5n2 and hcv zhou 2020ab dong photothermal nanotherapy high refractive index photocatalytic activities and high solubility properties of titanium nanops tinps enable them to find their wide antiviral application for bacteriophages and h3n2 viruses grein kar the clinical application of functionalized silica nanops sinps drives the diverse antiviral therapeutics or diagnostics application of nanocarriers in inhibition of hiv hepatitis b virus and other recombinant viruses kim nucleic acid hybridization and fluorescent based virus viral protein detection methods are getting popular based on the antiviral efficacy of sinps though their meso to nanoporous biocompatible surfaces allow hostpathogen interaction the emerging demand for novel antiviral nanotherapeutics triggers the researchers to ponder about immunomodulation and immunization of host cells these are essential to regulate premature drug release or inhibition of viral entry through in vivo biodistribution kalantarzadeh kerry the different forms of nanomaterials that are mostly used as antiviral agents depend largely on the pathway of drug delivery system which provide versatile forms of nanobased carriers starting from complex anic hybrid nanosystems to simple inanic metallic composites nanocapsules nanocages and nanospheres are categorised under inanic nanop based systems though nanocapsules can sometimes through and associated drug metabolism environmentalresearch19120201101193 0cs mukherjee fig schematic of the size range of nanops commonly applied in clinical practice as drug delivery agents for gene and drug delivery system quantum dots qd a type of nanosized crystals have excellent nanobased sensing which allow them to be used as antiviral therapeutics or in vitro diagnosis of virulent pathogens where strong chemical interactionsbonding render their biochemical conjugation with therapeutic molecules kostarelos kumar et al 2020a 2020b 2020c and zhou 2020ab have shown that different metallic composition pb cu ga zn hg based qd showed target specific actions against hiv1 human tlymphotropic virus1 after their binding with nh2 receptorbiotin acceptor some researchers have shown that nanoformulations based qds crossed the bbb in vitro model along with target dna and saquinavir antiviral agent which have been widely utilized as highly active antiretroviral therapy kumar 2020a 2020b 2020c lembo evaluation of anic and hybrid nanops as therapeutics and in drug delivery action anic nanopsnanocarriers onp play an important role in the drug delivery action if the therapeutic compounds are of large sized molecules nm encapsulation of such nanoagents through environmentalresearch19120201101194 0cs mukherjee specific designstructure render offtarget toxicity which is required for target specific action lopez gacem slow release kinetics and specific target site chemical synthesis have direct influence on onps therapeutic competence kumar 2020a 2020b 2020c li polymeric nanops pnps have the clinical potential to carry the target drugs to its core or can coordinate with target molecules on its planar surface mainardes and diedrich nasrollahzadeh biocorona formation ease of biodegradation strong mechanicalthermal properties of carbonbased nanocarriers nanorods nanodots etc render their antiviral activities against respiratory syncytial virus rsv hiv1 ebola virus etc table in spite of having broad spectrum activities against antiviral infections the ratelimiting phase of these carbon materials limits their further biomedical applications nikaeen and nguyen have suggested that drug conjugated nanops having excellent antiviral activities can help in the development of an influenza vaccine with matrix protein m2e and hemagglutinin haamine functionalized gelatin surface coating though more research is required to validate the immunity and protection of cellular genes for such impeccable protein vaccines against infectious virulent pathogens like sarscov2 with the advancement of medical sciences the emergence of multifunctional nanobased lipid nanocarriers opens a new door in the field of novel antiviral nanotherapeutics table read and risitano have demonstrated that podophyllotoxin stearic acidglycol loaded lipid nanocarriers are useful in maintaining in vitro slow drug release nontoxic viral inhibition and hemocompatibility of vk2e6e7 hela cells such solidlipid nanops and other nanolipid carriers can fig a and b schematic of the internalization of nanodrugs through the plasma membrane and targeted drug release a and transcytosis of nanodrugs through cell barriers b nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes bloodbrain barrier and bloodtestis barrier and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha environmentalresearch19120201101195 0cs mukherjee become the exceptional antiviral drug discharging agents for infectious viral pathogens ie hpv hiv and hepatitis c virus núËnezdelgado prather hyperbranched monodispersed easily biodegradable anic dendrimers have gained importance for more than a decade in the field of nanomedicine because they act as targeted carriers for biological systems the antiviral activity of dendrimers is still under scientific investigations which may be facilitated through conjugated drug delivery mechanisms patil palestino the efficacy of such nanoparticulated dendrimer system can be observed for the inhibition of hsv1influenza virus where antibody mediated responsecd8 t cell activation and regulation of gene expressions are achieved through small rnas inactivation shang sportelli niosomes the nonionic surfactant based liposome alike anic nanovesicle are getting popular in the advanced biomedical sciences considering their nonimmunogenicity and stable optical properties which make them a suitable carrier of both hydrolipophilic drug molecules excellent bioavailability and controlled release of specific drugs at the targeted sites make such nanocarriers ideal antiviral agents hsv virus where nanoniosome was loaded with suitable antiviral drug acyclovir s´anchezl´opez et al improved drug delivery mechanism and suitable drug release kinetics may allow such nanocarriers to be used in infectious virus diseases nanomicelles fig supramolecular globular micelles exhibit colloidal stability and super encapsulation potential which help such polymeric micelles to show antiviral activity in vitro as observed from curcumin loaded bioavailable nanoformulations hepatitis c virus sivasankarapillai some researchers used graphene oxides conjugated adnps against infectious sars and bursal viruses where the drug resistance event after such antiviral efficacy was mediated by selenium nanops senps andor amantadine am arrangement te velthuis kumar 2020a 2020b 2020c diagnosis or detection of such virulent pathogens have been documented by viceconte and petrosillo and vazquezmunoz and lopez through thiolstabilized gold cluster or enteroviruses labeling with cysteine molecule a brief overview about antiviral nanomedicines rsv virus tremiliosi udugama the size and zeta potential of silver nanops can exert inhibition effects on different human parainfluenza three virus strains or on their replication event the nanocolloidal system of vivagel® is immensely used for the control of zika virus infection tkm130803 is widely used in the treatment of ebola virus utilizing the concept of rnaibased therapy for the lipidbased nanosystem it is well doccumented that for human norovirus treatment the employment of goldcopper sulfide coreshell capsid protein binding results into excellent virucidal activity ziaie on the other hand nanotrap ps are quite often used in the inhibition of infection of target cells by capturing viral rnaviral proteins ie influenza virus treatment zhou 2020ab zhang intrinsic in vivo instability poor immunogenicity and toxicity multiple therapeutic and prophylactic approaches can be overcome by nanovaccinology where cellular and humoral immune response drive the faster uptake of mucosagut associated lymphoid tissue slow controlled release of antigens is facilitated by surface modifications of nanovaccines with antibodiescarbohydrates which results in the target specific immune response by different immune cells additionally their small size and prolonged shelf life help in the faster recognition of the hostreceptor immune system ie hepatitis a virus hav and influenza virus where epaxalexapal is used with immuno targeting agents yu yang and wang nonresponsive immune systems high dose administration coldchain transport of parenteral vaccines limit their widespread application in drug therapy particularly for mucosally administered vaccines the chitosannanop embedded system might be useful for therapeutic proteins or antigens having negative charges which makes its wider application in vaccination against hbv virus through gene delivery systems table the utilization of mouse model employing humoral and mucosal immune responses helped in the liposomebased vaccine development in case of hepaxen used for hepatitis a c and e which further utilized the recombinant surface antigen as a prophylactic vaccine waris wang 2020a 2020b the usages of inflexal v and influvac as standard virosomal vaccine against influenza virus are getting quite popular considering its active biocompatible and immunogenicity wu weiss these types of licensed subunit nanovaccines are found quite useful for older infants and middleaged group people in terms of nanosafety issue as they mimic natural infections as seen in table a cysteineguanine rich oligonucleotide combination with extracellular m2egold conjugates renders molecular protection for pr8h1n1 influenza which was further activated by thiolgold interactions zhang zhou 2020ab from the discussion provided here it is clear that most of the research has been done for influenza virus vaccination but some scanty literature also report several nanomedicines including nanovaccines are under clinical trial or at least in the stage of commercialization for the cure of infectious viral diseases table in general the use of drugs for antiviral therapy is usually employed to target different life cycles of virulent pathogens ie hiv ebola virus or hsv1 valdiglesias and laffon alnrsv01 is a commonly used lipid nanop drug for lower tract respiratory disease which targets the nucleocapsid n gene of table commercial nanomedicines or under clinical trial for the antiviral therapytreatment souce of data neogi letko dong kalantarzadeh kang nanomedicines influvac plus tkmhbv cervisil doravirine dermavir inflexal v epaxal pegasys geovax novavax fluquit curevac peglntron vivagel a pepreclinical evaluation uceunder clinical evaluation hav hepatitis a virus hiv human immunodeficiency virus hbv hepatitis b virus hpv human papillomavirus hcv hepatitis c virus hsv herpes simplex virus mode of action presence of neuraminidase and hemagglutinin rnai therapeutics gene silencing reverse trancriptase inhibitor nonnucleoside dna immunogen with hiv specific t cell precursor antigens specific on speherical carriers surface natural process mimics peroxidases pegylation control stability of protein ankaravirus alike drug therapy clinical stage antiviral nanobiotechnology gene silencing mrna technique pegylation control stability of protein dendrimer with sulphonic acid group interaction disease indication influenza hbv hpv hiv hiv influenza hav hbv hcv sarscov2 sarscov2 influenza sarscov2 hcv hsv hiv biomedical application virosome vaccine solidlipid nanop sirna therapeutic nanoparticulate formulation therapeutic vaccine liposome vaccine liposome vaccine pegylated interferon antiviral therapy nanoparticulate therapeutics sirna therapeutic infections virus vaccine pegylated interferon dendrimer yearstage of development uce preclinical evaluation ucea uce pe pe pea pe uce environmentalresearch19120201101196 0cs mukherjee vaccine research for rotanoroebola virus hpv rsv and others dung das metabolic pathway of nanotherapeutics and their limitations in clinical practice pulmonary and hepatotoxicity studies are required to build a safety profile of such nanodelivery therapeutic or diagnostic agents nanobased approach for sarscov2 infection inhibition nanops uptake cellular process in nanotherapeutics are governed by their physicochemical properties along with cellular membrane characteristics which may have direct influence on the rate of administered drug dosages and structure of engineered nanops it is hypothesized that nanops with optimum diameter of nm and high surface charge density are quite effective in crossing the cellular membranes for hivderived tat cell penetrating peptides weiss waris immunoliposomes and other carbon based nanotubesnanocarriers play an important role in the activation of the complement pathway of host immune systems to deregulate in vitro utilization of nps neogi kumar 2020a 2020b 2020c antibodies that are specifically targeted at polyethylene glycol pegmacrogol polymers and peglike nanostructures can show independent therapeutic efficacy based on their individual immunotoxicology and risk assessment strategies letko read experimental findings wang 2020a 2020b hill with nanobased therapeutic agents reveal the urgent requirement of more rigorous scientific investigations to prove their clinical efficiency in reversing the drug resistance event ie h1n1 virus through seam biodegradation process of nanotherapeutics has gained special attention considering uniform biodistribution kinetics and sustained drug release which are essential for improved drug design process distribution metabolism absorption excretion are important pharmacokinetic features which rate of biochemical features are directly governed by hydrophobichydrophilic profile and tacticity of the nano based formulations at in vitro level patil exocytosis process plays a very important role in the clearance of the foreign nanops out of the cell depending on administered nanocarriers it was hypothesized that ps with nm diameter can | 0 |
neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing antiammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these ï¬nd ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the beneï¬t of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as insilico drug re purposing in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to ï¬nd out a new se ries of nep inhibitors to the best of our knowledge this is the ï¬rst study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of Ëa prior to docking and simulation studies the biological unit of protein was prepared using protein preparation wizard in schrodinger suite during the process of protein preparation the protein was subjected to import and reï¬ne review and modify and minimize processes in protein preparation wizard missing side chains and residues were ï¬lled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond Ëa were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force ï¬eld and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workï¬ow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force ï¬eld in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding aï¬nities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer ï¬le of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force ï¬eld to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme proï¬le the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coeï¬cient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of ï¬ve were calculated induced ï¬t docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedï¬t docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifdsp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which reï¬nement of all residues within Ëa of the ligands pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedï¬t protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula prime_energy glide score ifd score glide_ecoul molecular dynamics md simulation the ï¬exibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede ï¬ned spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol Ëa balanced at k tem perature and bar pressure via npt ensemble in the ï¬nal step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme proï¬le further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workï¬ow was used and the cubic box of speciï¬c dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the ï¬nal torsional reï¬nement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were ï¬ltered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identiï¬ed for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this ï¬nding indicates that the selected drugs may act as nep inhibitors induced ï¬t docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced ï¬t docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the ï¬exible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications ï¬rst it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd conï¬rmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme proï¬le was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of ï¬ve given in table all the selected drugs obey the lip inski rule of ï¬ve molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited ï¬exibility which is insuï¬cient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and lig ï¬t prot for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be Ëa and Ëa respectively the rmsd values were found to be in the acceptable range Ëa but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are Ëa and Ëa for protein and ligand respectively for complex the rmsd values were found to be Ëa for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of Ëa for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signiï¬ 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of ï¬ve sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is antiammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding aï¬nity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are ï¬rst it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drugs activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identiï¬ed based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0 | 0 |
"incidence of thyroid carcinoma is increasing all over the world Some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma However other studies have come to theopposite Therefore we studied the relationship between adipokines and thyroid carcinomaMethods DatabasesPubMed Cochrane Library SinoMed CNKI Wanfang and clinical trial registries weresearched A metaanalysis was then performed through a fixed or randomeffects model to calculate I values forheterogeneity analysisResults Twentynine s were finally included for analysis The level of serum tumor necrosis factoralpha TNFα [standardized mean difference SMD confidence interval CI to I2 P ]and the ratio of TNFα immunoreactivity in tissues [odds ratios OR CI to I2 P ]in thyroid carcinoma are significantly higher than those in control The serum interleukin6 IL6 in patients withthyroid carcinoma is higher than that in control SMD CI to I2 P There is nosignificant difference of the ratio of IL6 immunoreactivity in tissues between carcinoma and control OR CI to I2 P The ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma OR CI to I2 P However after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control P Conclusions Adipokines TNFα IL6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma However the association between adiponectin and thyroid carcinomaneeds further researchKeywords Thyroid carcinoma Adipokines TNFα IL6 Leptin Metaanalysis Correspondence liaolinsdueducn cwc_llsdueducn Junyu Zhao and Jing Wen contributed equally to this work1Department of Endocrinology and Metabology The First Affiliated Hospitalof Shandong First Medical University Shandong Provincial QianfoshanHospital Jinan China5Department of Endocrinology and Metabology Qilu Hospital of ShandongUniversity Cheeloo College of Medicine Shandong University Jinan ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhao BMC Cancer Page of BackgroundThyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis During the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] Someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason [] But this may be only partial andcan not totally explain the increased incidence of microcarcinoma Changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore Recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [] Various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic low grade inflammation altered immune response and DNA damage caused byoxidative stress [] Furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [] Adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphaTNFα interleukin6 IL6 and complement factor Dalso known as adipsin Adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin The concentration of adipokines such as TNFαIL6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index []It is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development At present many kindsof adipokines have been reported to be associated withthyroid carcinoma Rehem RA [] suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery Another envidence showed that adiponectin related with tumor size [] However the opposite resultswere also found in other studies [] Some researchesreported the expression of adipokines is lower in tumortissue than normal control [] It is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomaThese confunding factors above may be the cause of inconsistency results from different researches Additionaly the association between adipokines and thyroidcarcinoma are still not well documented Therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomaMethodsSearching progressWe conducted a search of all studies published until27th July regarding the association between adipokine and thyroid carcinoma Eligible casecontrol studieswere found by searching the database of PubMedCochrane library Sinomed CNKI and Wanfang and restricted to published results Clinical trial register centers httpwwwclinicaltrialsgov were also searchedThe following search terms Adipokine or Leptin oradiponectin or resistin or tumor necrosis factoralpha or Interleukin6 or Complement factor D orAdipocytokines or tumor necrosis factorα or TNFα or IL6 or adipsin and thyroid cancer or thyroid neoplasm or thyroid tumor or thyroid carcinoma or differentiated thyroid carcinoma or DTC orPapillary thyroid carcinoma or Thyroid carcinomapapillary or PTC or Thyroid cancer follicular orFTC or Thyroid Carcinoma Anaplastic or ATC orThyroid cancer medullary or MTC Hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews Eligible studies met the following criteria published in English or Chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue Studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate OR and95CI the type of study was not a casecontrol designhave not fulltext animal trialsStudy selection and data extractionTwo reviewers screened the studies and extracted dataindependently Any disagreement was resolved by discussion or consensus with a third senior reviewer Dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0cZhao BMC Cancer Page of scores were considered to be of high quality Disagreements were resolved by reevaluating and discussing between two reviewersinSearchingthis metaanalysisResultsSearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites Afterscreening the title and abstracts s were selected for fulltext review Finally studies were eligibleprogressincluded and excluded details are all shown in Fig Eighteen of these studies are published in Chinese[ ] and the rest are published in English[] Nineteen studies were conducted in Chinatwo in India and two in Turkey Brazil Greece IranItaly Denmark and Serbia each had one study Totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group The sample size ranges from to in the case group while to in the controlgroup All the thyroid carcinoma patients were confirmed by pathologically Among these studiesfourteen studies reported papillary thyroid carcinomaPTC eight studies reported differentiated thyroidcarcinoma DTCreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma MTC and the restfour studies did not show the pathological detailsThe detailed characteristics ofincluded studies aresummarized in Table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups Thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesStatistical analysisFor metaanalysis dichotomous outcomes were analyzedby using the odds ratios OR computed using the MantelHaenszel method fixed or random models Continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences WMD Otherwise standardized mean difference SMD were used for different scaleAll results were reported with confidence interval CI I2 was used to assess heterogeneity between studies and I2 values of and representing no lowmoderate and high heterogeneity respectively Visual inspection of the funnel plot was done to assess publicationbias The analyses were performed by Review Manager Cochrane Collaboration United Kingdom httpwwwcochraneQuality assessment and risk of biasThe methodological quality of casecontrol study wasassessed by the NewcastleOttawa Scale NOS Supplement Table which consists of the three parameterseight questions with nine possible scores Selection Exposure and Comparability A study can be awarded amaximum of one score for each numbered item withinthe Selection an Exposure categories A maximum oftwo scores can be got for Comparability A higher scoremeans better quality in methodology and five or moreFig Flow chart of the systematic search process 0cZhao BMC Cancer Page of Zhao Jianqiang []ChinaPTC FTC ATCand MTCthyroid adenoma andnormal healthUnknownUnknownTable Characteristic of included studiesFirst authorYearCountryPathologicaltype of thyroidcancerSource of controlsL Kayser []Denmark PTC and FTCCao Guangyao []ChinaUnknownMTrovato []ItalyDTC andundifferentiatedcarcinomamultinodular goitersadenomas Hashimotosthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesMelih Akinci []Wang Jingxia []ZhuangXiaoming []Yu Xiao []Hou Sen []SnezanaZivancevicSimonovic []Xu Xiaocheng []XeniProvatopoulou []TurkeyPTChealthy volunteersChinaPTC and FTCnormal thyroid tissuesChinaPTC FTC andMTCthyroid adenoma andnormal healthChinaPTCthyroid adenoma andnormal thyroid tissue nearcarcinomaChinaPTCthyroid adenomaSerbiaWDTChealthy subjectsChinathyroidcarcinomaGreecePTCthyroid adenomabenign thyroid disease andhealthy controlsSun Qinnuan []ChinaPTCnormal thyroid tissue nearcarcinoma and healthycontrolsChinaPTCthyroid adenomaChinaPTCthyroid adenomaMean age yearFemale Outcome indexNumber ofparticipants ncases control casesUnknowncontrolcontrolcasesUnknownUnknownUnknownUnknownUnknownTNFα tissueTNFα tissueIL6 tissueIL6ãTNFαblood ± ±Unknown leptinblood Unknown TNFα tissue Unknown IL6ãTNFαUnknownUnknownbloodleptintissueUnknown Unknown leptin ± ± tissueTNFαblood ± ± ± ± ± ± ± IL6blood IL6blood TNFαbloodandtissue ±Unknown Unknown leptinUnknownUnknowntissueadiponectintissueUnknown Unknown adiponectintissue IL6ãTNFαblood ± ± Zhang Zijie []Zhong Xiuxiu []Zhang Bo []Hu Jinhua []SnezanaZivancevicSimonovic []YanLan Fan []ChinaDTCChinaDTCnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±SerbiaPTCcontrol subjectsUnknownUnknownIL6bloodChinathyroidcarcinomanodular goitre Hashimotosthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesUnknownUnknownleptintissue 0cZhao BMC Cancer Page of Table Characteristic of included studies ContinuedFirst authorSource of controlsYearCountryPathologicaltype of thyroidcancerChinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseaseChinaPTCthyroid adenomaTurkeyPTChealthy volunteersIndiaPTCIndiaPTCbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsNumber ofparticipants ncases control cases ±Mean age yearFemale Outcome indexcontrol ±casescontrol TNFαtissue ± ±TNFα tissue IL6bloodUnknown Unknown TNFαbloodUnknown Unknown IL6bloodWangXinzheng []Song Runbo []Kemal Beksac []Toral PKobawala []Toral PKobawala []RaziyehAbooshahab []Zhang Bo []ZhouXiaodong []Ma Xiaokai []MarianaBonjiornoMartins []IranMTChealthy subjects ± ± leptinãadiponectinbloodChinaDTCnormal thyroid tissue nearcarcinomaUnknown Unknown leptintissueChinaDTChealthy subjects ± ±IL6ãTNFαbloodChinaPTCthyroid adenomaUnknown Unknown leptinBrazilDTCbenign thyroid nodules andhealthy controls ±tissue IL6blood ± ±ChinaIL6 Sun Zhenhua []tissueTNFα tumor necrosis factora DTC differentiated thyroid carcinoma IL6 interleukin6 PTC papillary thyroid carcinoma FTC follicular thyroid carcinoma ATCanaplastic thyroid carcinoma MTC medullary thyroid carcinoma WDTC welldifferentiated thyroid carcinoma FNAC fine needle aspiration cytologynodular goiterPTCQuality of included studiesThe quality assessment of these studies is assessed bythe NOS and the resultis shown in SupplementalTable Five or more scores are determined as highquality Two studies conducted by Cao G in [] and L Kayser in [] only get two scoresshowing a poor quality in methodology The rest studies are assessed as high qualityTNFα and thyroid carcinomaTwelve studies reported the expression of TNFα bothin patients with thyroid carcinoma and control subjects[ ] Among these sevenstudies [ ] had tested the level ofserum TNFα two studies [ ] had tested the expression of TNFα in tissues and the ratio of TNFα immunoreactivity was tested in four studies [ ] Firstly fixedeffect model is used to merge the SMDvalues of serum TNFα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test Chi2 P I2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors Then randomeffect model to merge the SMD isused and pooled effect size in favor of control group is CI to P Fig 2a SMDvalues of the expression of TNFα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testChi2 P I2 The different unitsand limited numbers of research may be the original ofheterogeneity So the pooled SMD with randomeffectmodel of the expression of TNFα in tissues is CI to P Fig 2b The pooled ORwith fixedeffect model of the ratio of TNFα immunoreactivity in thyroid carcinoma tissues is CI to P However a significant heterogeneity is detected heterogeneity test Chi2 P I2 The published by L Kayser in with a poor quality in methodology may attributeto this high heterogeneity Then randomeffect model ofpooled OR is used and pooled effect size in favor of 0cZhao BMC Cancer Page of Fig Forest plot of the TNFα level and the ratio of TNFα immunoreactivity in tissues in patients with thyroid carcinoma a Level of serum TNFα b Expression of TNFα in tissue c Ratio of TNFα immunoreactivity in tissuecontrol group is CI to P Fig 2c In level of serum TNFα and theratio of TNFα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomaIL6 and thyroid carcinomaAmong the included studies reported the level ofserum IL6 in patients with thyroid carcinoma and control subjects [ ] Due to thelarge heterogeneity of the merged SMD values of serumIL6 level by the heterogeneity analysis heterogeneitytest Chi2 P I2 randomeffectmodel was used to pooled the SMD values and thepooled effect size in favor of control subjects is CI to P Fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum IL6 than control subjects Twostudies reported the ratio of IL6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] The pooled OR of the limited two studies donot show an increased ratio of IL6 immunoreactivity inthyroid carcinoma tissues OR CI to P and a large heterogeneity always existsheterogeneity test Chi2 P I2 Fig3b Thus the level of serum IL6 is higher in patientswith thyroid carcinoma However it needs more clinicaldata to verify the relationship between the expression ofIL6 and thyroid carcinoma tissueLeptin and thyroid carcinomaTwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] Because ofthe considerable heterogeneity of the pooled WMD ofserum leptin level heterogeneity test Chi2 P I2 and pooled OR of the ratio of leptinimmunoreactivity in tissues heterogeneity test Chi2 P I2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis Howeverthere is no association of higher level of serum leptin 0cZhao BMC Cancer Page of Fig Forest plot of the IL6 level and ratio of IL6 immunoreactivity in tissue in patients with thyroid carcinoma a Level of serum IL6 b Ratio ofIL6 immunoreactivity in tissueFig Forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a Level of serum leptin bRatio of leptin immunoreactivity in tissue 0cZhao BMC Cancer Page of with risk of thyroid carcinoma WMD 95CI to Fig 4a Moreover the pooled OR of theratio ofleptin immunoreactivity in tissues from fivestudies is 95CI to Fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaAdiponectin and thyroid carcinomaThree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in Table It could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects P Interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity However there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed Thus itneeds more clinical studies to confirm in the futurePublication biasThe funnel plot was applied for assessing publicationbias of studies included in the three results includingTNFα Fig 5a IL6 Fig 5b and leptin Fig 5c InFig 5a and Fig 5b almost all studies lies inside the95CIs with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot Howevera potential publication bias was found in Fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisDiscussionCurrently obesity affects one third of population amongUS adults [] and China has become a big country ofobesity with the incidence ranking first worldwide in theyear of [] Nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonHodgkins lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [] Becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma It was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [] Furthermore obesity is an independent risk factor for thyroid carcinoma[] Increased insulin resistance elevated serum cholesterol level and upregulated COX2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma [] It is reported that people with higherbody mass index have a higher concentration of adipokines [] Adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping So adipokines may be one of the targetslinking obesity with thyroid cancer The metaanalysiswas based on previous published studies In previousstudies the analysis of adiponectin and thyroid cancermostly focused on TNF IL6 Leptin and AdiponectinWhile few studies focused on other molecules includingIL1 and IL8 and we failed to combine statisticsTherefore in this metaanalysis only TNF IL6 Leptinand Adiponectin which are the most published adiponectin were analyzedTNFα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability Meanwhile TNFα plays animportant role in the process of inflammation insulinresistance diabetes and obesity A moderate amount ofTNFα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to TNFassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedTNFα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts [] which may play an important role in thyroidcancer Moreover TNFα can stimulate the vasoactivemediators such as interleukin and prostaglandin []and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function TNFα can also induce an increased expression ofvascular endothelial growth factor VEGF [] the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasisTable Summary of adiponectin expression in thyroid carcinomaserum adiponectin []ratio of adiponectin immunoreactivity []Effect sizeWMD OR adiponectin in tissue [] CI confidence interval WMD weighted mean differences OR odds ratiosWMD 95CI PI2Not applicable 0cZhao BMC Cancer Page of Fig Funnel plots of a TNFα b IL6 and c leptin revealed no significant publication bias SE SMD standard error of standardizedmean differenceIn surprisingly the results of clinical studies provide evidence for basic research Simonovic SZ [] evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with DTC before and days after radioactiveiodine 131Itherapy and control subjects andfound that the expression of TNFα in DTC patients ishigher than control subjects and it showed a decreasedlevel after 131I therapy than those before therapy However no statistical difference found for the limited sample size Another study conducted by Kobawala TP et al[] with more patients patients with benign thyroiddisease PTC patients and healthy individuals determined the circulating levels of TNFα and it wasfound that the serum level of TNFα was significantlyhigher in PTC patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals Furthermore serum TNFα was reported to be a significant prognosticator for overall survival in PTC patients It is a pity thatopposite result wasreported in a casecontrol study that included DTCcases and matched cancerfree cohort participantswhich found that TNFa was not associated with thyroidrisk in either gender []Based on current evidence our metaanalysis suggeststhat TNFα exhibit a strong association with thyroid carcinoma It may because that elevated TNFα may involved in the tumorigenesis and development of thyroidcancer Another possible reason is that the TNFα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomaThereforemore clincal studies and basic reseaches should be conducted in the futureIL6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells It is reported that elevated serum IL6 level is closely related tothe tumorigenesis and development of a variety of tumors [] A strong positive association between theserum IL6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [] Serum IL6 level in thyroid cancer has been evaluated in numerous studies including 0cZhao BMC Cancer Page of in vivo and in vitro studies Provatopoulou X []found that serum IL6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols However other studies show a different resultthat no significance different of IL6 was found betweenthyroid cancer and nonthyroid cancer [ ] A limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference For in vitro research IL6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof IL6 in PTC was confirmed indirectly []The underlying mechanism may be the followingsbelow Tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine IL6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors [] Also IL6R was found associated with the characterization of thyroid nodules malignancy and tumor aggressivenessIn additionIliopoulos D [] found that Src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating NFκB and thistransformation contributes to tumorigenesis IL6 is considered as an important regulatory factor in this processAnother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level[]In general the data above support that IL6 is important for thyroid cancer but the detail mechanism remainto be further studyLeptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus Meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney It is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin Moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway RhoALIMK1Cofilinpathway and MAPKERK pathway [] Kim WG et al[] evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer Thrb PVPV Pten mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byJAKSTAT3increased activation of the JAK2 signaling transducerand activator of STAT3 signaling pathway and inductionof STAT3 target gene expression Leptin is always reported a high expression on solid tumors [] and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly PTC while otherstudies showed a same results in cancer tissues [ ] Yu Xiao [] conducted a clinical studycomparing the level of serum leptin in PTC patientsincluding patients with lymph node metastasis and thyroid adenoma patients in Dalian China and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisLeptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue [] In addition it can also inhibit the apoptosisthrough Bcl2 dependent mechanism Meanwhile leptinreceptor exists in all thyroid cancer cells It is overexpressed in PTC and is involved in tumor invasion andlymph node metastasis [ ] Thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance Due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma The cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiesCompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance [] In addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia [] renal carcinoma [] gastric carcinoma [] and colon cancer[] Moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed []Otvos L Jr [] tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells Mitsiades N [] measured circulatingadiponectin levels in ptaients with PTC and found thatit is independently and inversely associated with the riskof thyroid cancer As the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof PTC Adiponectin receptor1 and are higher expression in PTC tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis []However other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0cZhao BMC Cancer Page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsFrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded And this may explain why increased incidence of obesity and thyroidcancer are consistent Thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefutureHowever some limitations in our metaana | 2 |
properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest chronic ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The hosts ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein traï¬cking viral transformation and oncogenesis []Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe signiï¬cance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ ]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inï¬ammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRPammationNowadays CRP is considered a true marker for assessingammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and speciï¬city [] In addition inmelanoma elevated levels of CRP may reï¬ect the amountand activity of circulating proammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ammatory stimuli Melanoma cells have been reportedto express IL8 and this uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reï¬ecting the stage ofthe disease []Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases Victor BabesDermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of puriï¬ed antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpuriï¬ed antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coeï¬cientp was considered with statistical signiï¬cance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological proï¬les compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following proï¬le antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation toammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical signiï¬cance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM AntiGD1b IgGAntiGD1a IgM AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups p have been considered as targets for cancer immunotherapy[] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ ]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the hosts protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehosts ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp signiï¬cancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psigniï¬cancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psigniï¬canceIL8pgml ± ± ± psigniï¬canceour ï¬ndings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justiï¬ed our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ammation [ ] The presentstudy is the ï¬rst one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic signiï¬cance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identiï¬ed increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was deï¬ned as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic signiï¬cance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the ï¬rst study which otherresearchers and clinicians can use and analyze in order toevaluate the uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideproï¬le characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ¤ r p r p r p ¤ r p ¤ r p ¤ r p IL8r p ¤ r p r p r p ¤ r p ¤ r p ¤ r p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ammatory markersIL8 and CRP The hosts ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the ï¬ndings of this study areincluded within the articleConflicts of InterestThe authors declare no conï¬icts of interestAuthors ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski Multisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp [] I Horwacik and H Rokita Targeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp [] J L Daniotti R D Lardone and A A Vilcaes Dysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor Gnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp [] Y Ohmi M Kambe Y Ohkawa Diï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton Ganglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal ï¬uid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae Gangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau Bindingactivities and antitumor properties of a new mousehumanchimeric antibody speciï¬c for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s5620s [] K Bennaceur I Popa J A Chapman Diï¬erent 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poljar Melanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp [] M Costache A V Dumitru O M PÄtraÅcu A challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp [] R Ancuceanu and M Neagu Immune based therapy for melanoma Indian Journal of Medical Research vol no pp [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton Serum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp [] B Mondal and S Sahal Inhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp [] S Sahal and S Mondal Supression of Ehrlich subcutaneoussolid tumor growth by immunization with 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breast cancer bc is the most common malignancy among women emerging studies have demonstrated that circular rna circrna zinc finger rna binding protein circzfr serves as a crucial regulator in many human cancers however the role and mechanism of circzfr in bc tumorigenesis remain unclearmethods the levels of circzfr mir578 and hypoxiainducible factor 1α hif1a were detected by quantitative realtime polymerase chain reaction qrtpcr or western blot cell viability colony formation apoptosis migration and invasion capacities in vitro were determined by using the cell counting kit8 cck8 standard colony formation flow cytometry and transwell assays respectively glucose uptake lactate product and adenosine triphosphate atp levels of cells in vitro were measured using the commercial human assay kits targeted relationships among circzfr mir and hif1a in bc cell lines were verified by dualluciferase reporter and rna pulldown assays animal studies were performed to assess the effect of circzfr on tumor growth in vivoresults our data indicated that circzfr was overexpressed in bc tissues and cells and the increased circzfr level predicted poor prognosis of bc patients circzfr silencing or mir578 overexpression repressed bc cell viability colony formation migration invasion and glycolysis and enhanced cell apoptosis in vitro circzfr silencing also hampered tumor growth in vivo mechanistically circzfr acted as a sponge of mir578 and circzfr silencing hindered bc cell malignant behaviors by mir578 hif1a was a functional target of mir578 in regulating bc cell viability colony formation migration invasion glycolysis and apoptosis in vitro furthermore circzfr modulated hif1a expression through sponging mir578 our findings first identified that the silencing of circzfr suppressed bc malignant progression in vitro via the regulation of the mir578hif1a axis providing evidence for the crucial involvement of circzfr in bc pathogenesiskeywords breast cancer bc circzfr mir578 hif1a malignant progressioncorrespondence qiuxinguang2020163com department of thyroid surgery the first affiliated hospital of zhengzhou university no1 jianshe east road erqi district zhengzhou henan chinafull list of author information is available at the end of the breast cancer bc remains the most commonly diagnosed cancer and the leading cause of cancerassociated death among females in although the therapeutic methods have greatly improved over the past two decades effective treatment against metastatic bc is still limited [ ] therefore a deeper understanding of what drives this disease is the first step to design innovative interventions the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchen a0et a0al cancer cell int page of circular rnas circrnas are covalently closed endogenous rnas that have crucial noncoding functions in human physiologic and pathologic processes work in biological functions has demonstrated the roles of circrnas as microrna mirna sponges dysregulation of circrnas has recently implicated in the pathogenesis of bc for example yuan et a0al uncovered that hsa_circ_0068033 a downregulated circrna exerted a repressive impact on bc malignant progression via sequestering mir659 cao and colleagues demonstrated that hsa_circ_0087784 functioned as a potential promoter in bc development through sponging mir487a xu et a0al reported that circtada2as attenuated bc progression and metastasis by the regulation of mir203a3p moreover hsa_circ_001783 and circabcb10 were reported as oncogenic regulators in bc through functioning as specific mirna sponges [ ] as for circrna zinc finger rna binding protein circzfr hsa_circ_0072088 it has been identified as an oncogenic modulator in many human cancers such as renal carcinoma bladder cancer and nonsmall cell lung cancer [] nevertheless the biological roles of circzfr in bc tumorigenesis remain largely unknownmirnas modulate gene expression but are frequently dysregulated in human cancers including bc danza et a0al reported that mir578 was underexpressed in brcabc and it regulated tumor angiogenesis however the precise function of mir578 in bc progression is still undefinedhere we undertook to examine the biological effect of circzfr in the malignant progression of bc in a0 vitro and in a0 vivo we identified that circzfr a prominently upregulated circrna in bc controlled bc progression in a0vitro via targeting the mir578hypoxiainducible factor 1α hif1a axismaterials and a0methodsclinical tissues and a0cellsin this study we enrolled bc patients admitted to the first affiliated hospital of zhengzhou university from april to june the clinicopathological features of these patients were provided in table a0 seventy pairs of primary tumor tissues and matched healthy breast tissues were collected and stored at a0°c to detect the expression levels of circzfr mir578 and hif1a accession nm_1810543 these patients were followedup for at least a0 months and the followup information was obtained by telephone calls every a0 months the study was approved by the ethics committee of the first affiliated hospital of zhengzhou university and written informed consent was provided by all participantstable correlation and a0the a0clinicopathological features of a0bc patientsbetween a0circzfr expression characteristicsnumbercirczfr expressionphighlowage years ¥ distant metastasis present absenttumor size cm tnm stage i ii iii ivher2 status positive negativepr status positive negativeer status positive negativeher2 human epidermal growth factor receptor2 pr progesterone receptor er estrogen receptorp p bc cell lines mcf7 atcc ®htb22 bt549 atcc ®htb122 mdamb231 atcc ®htb26 and mdamb453 atcc ®htb131 american type collection culture atcc manassas va usa were cultured in rpmi1640 medium supplemented with fetal calf serum fcs and antibiotic solution all from cell line atcc ®crl10317 atcc was maintained in hyclone logan ut usa the immortalized mcf10a dulbeccos modified eagles mediumnutrient mixture f12 dmemf12 with fcs a0ngml epidermal growth factor a0 μgml hydrocortisone and a0 μgml insulin all from hyclone all cells were cultured in a co2 incubator at a0°cquantitative realtime polymerase chain reaction qrtpcrthe expression levels of circzfr hif1a and mirnas were gauged by qrtpcr complementary dna cdna synthesis was done using total rna a0 ng 0cchen a0et a0al cancer cell int page of isolated by isogen nippon gene tokyo japan from tissues and cell lines the levels of circzfr and hif1a were quantified using the taqman gene expression assays with the indicated primers and mature mirnas were assayed using the taqman microrna assays with taqmanspecific primer probes as recommended by the manufacturers applied biosystems rotkreuz switzerland qrtpcr was run in triplicate on the icycler iq5 device biorad munich germany using the pcr conditions as previously reported results were normalized to the expression of glyceraldehyde3phosphate dehydrogenase gapdh or u6 internal control using the δδct method primer sequences for circzfr forward ²atg gtc tgc agt cct gtg tg3² were and reverse ²tgg tgg cat gtt ttg tca tt3² for hif1a were forward ²ttc ccg act agg ccc att c3² and reverse ²cag gta ttc aag gtc cca tttca3² for mir578 were forward ²gtg cag ggt gtt agga3² and reverse ²gaa gaa cac gtc tggt3² for mir944 were forward ²gag tag gct aca tgt tat taaa3² and reverse ²gtg cag ggt ccg aggt3² for mir5323p were forward ²atc ctc cca cac cca agg ² and reverse ²gtg cag ggt ccg aggt3² for gapdh and u6 were described previously lentivirus transduction and a0transient transfectioncirczfr knockdown in mcf7 and bt549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shrnas specific to circzfr shcirczfr1 shcirczfr shcirczfr2 and shcirczfr3 fulengen guangzhou china and nontarget shrna lentiviruses shnc were used as the negative control vectortransduced cells were selected by puromycin solarbio beijing china at a final concentration of a0μgml at least a0h mir578 overexpression and knockdown cell lines were generated using the synthetic mir578 mimic a0nm ribobio guangzhou china and inhibitor antimir578 a0nm ribobio respectively with a corresponding nontarget oligonucleotide mirnc mimic or antinc ribobio as the negative control to elevate hif1a expression in bc cell lines a recombinant overexpressing plasmid for hif1a hif1a a0 ng ribobio or negative control plasmid vector ribobio was transiently transfected into cell lines using lipofectamine reagent invitrogen breda the netherlands as per the manufacturers protocols each experiment was performed in triplicatecell viability colony formation and a0apoptosis assaysshcirczfrinfected or shnctransduced cell lines were transfected with or without antinc or antimir578 and mcf7 and bt549 cell lines were carried out the indicated transfections followed by the incubation for a0h at a0°c cell viability assay was done using the cell counting kit8 cck8 abcam cambridge uk assay as per the manufacturers guidance cell colony formation was tested using standard colony formation protocols as previously reported cell apoptosis measurement was performed by flow cytometry using doublestaining with fluorescein isothiocyanate fitclabeled annexin v and propidium iodide pi based on the directions of manufacturers invitrogen events were analyzed by a flow cytometer and the apoptotic cells were determined by calculating the sum of early annexin vpi and late annexin vpi apoptotic cells all experiments were done in triplicatetranswell migration and a0invasion assayscell migration and invasion were detected by the transwell assay using modified boyden chambers in 24transwell plates a0 μm pores corning amsterdam the netherlands chambers of cell invasion assays consisted of matrigelprecoated membrane inserts corning after transfection bc cell lines were seeded into the top chamber of a 24transwell insert and medium containing fcs was used as a chemoattractant in the lower chamber a0h later the well was stained with crystal violet solarbio and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope nikon shinagawa tokyo japan at à magnification each experiment was performed in triplicatemeasurement of a0glucose uptake lactate product and a0adenosine triphosphate atp levelthe colorimetric glucose uptake assay kit llactate assay kit and atp assay kit all from abcam were used to determine the levels of glucose uptake lactate product and atp according to the recommendations of manufacturers briefly the lysates of transfected cells were prepared using the assay buffer and incubated with standard protocols for the indicated time point followed by the measurement of the absorbance with a microplate reader invitrogen at od a0nm for glucose uptake a0nm for lactate product and a0nm for atp level all assays were done in triplicate 0cchen a0et a0al cancer cell int page of animal studiesthe xenograft models were constructed to assess the role of circzfr on tumor growth in a0vivo animal studies were implemented in accordance with a protocol approved by the ethics committee on animal use and care of the first affiliated hospital of zhengzhou university twelve a0weeks balbc female mice shanghai animal laboratory center shanghai china were used and randomly divided into two groups n per group shnc and shcirczfr approximately à shncinfected or shcirczfrtransduced bt549 cell line was subcutaneously injected into the dorsal flank of the mice one week later tumor size was measured every week with callipers and tumor volume was calculated using the following formula volume à length à width2 all mice were sacrificed at a0days after implantation and tumor tissues were collected for weightbioinformatics dualluciferase reporter and a0rna pulldown assaysanalysis for the targeted mirnas of circzfr was performed using the online web circinteractome https circi ntera ctome nianihgovindex html and circbank httpwwwcircb ankcnsearc hcirc html the putative targets of mir578 were predicted by targetscan v7 online software httpwwwtarge tscan vert_71targeted relationships among circzfr mir578 and hif1a were confirmed by dualluciferase reporter and rna pulldown assays in dualluciferase assays the fragment of circzfr containing the mir578binding sites and hif1a ²utr were individually cloned into pmirglo vector promega madison wi usa to construct corresponding wildtype reporters circzfrwt and hif1a3²utrwt the takara mutanbest kit was used to construct the corresponding mutations circzfrmut and hif1a3²utrmut as per the insturctions of manufacturers takara dalian china each reporter construct a0ng and a0nm of mir578 mimic or mirnc mimic were cotransfected into bc cell lines cell line extracts were prepared with ripa buffer takara a0h posttransfection and the ratio of renilla to firefly luciferase was detected using the promega dualluciferase assay in rna pulldown assays cell lysates were incubated with the biotinlabeled mir578 mimic biomir578 ribobio or nontarget control sequence bionc ribobio for a0 h at a0 °c before adding to the streptavidin beads sigmaaldrich for a0 h the beads were collected and total rna was extracted for circzfr quantification each experiment was performed in triplicatewestern blot for a0hif1a expressionwestern blot was used to determine the expression of hif1a using standard protocols the preparation of cell line extracts was done using ripa buffer with proteinase inhibitors roche charente france proteins a0 μg were resolved on a sdspolyacrylamide gel electrophoretically blotted onto nitrocellulose membranes ge healthcare little chalfont uk and probed with antibody against hif1a ab51608 a0μgml abcam or gapdh ma515738 a0 μgml invitrogen following the incubation with horseradish peroxidasecoupled igg secondary antibody ab97051 a0 μgml abcam the signals were visualized by cheniluminescence ge healthcare as recommended by the manufacturers all experiments were done in triplicatestatistical analysisdata were shown as the mean ± standard deviation from at least three independent assays pairwise comparisons were done using a twotailed students t test mannwhitney u test or analysis of variance anova with spss version software spss chicago il usa for survival analysis the kaplanmeier survival curve and logrank test were used correlations among circzfr mir578 and hif1a expression levels in bc tissues were determined by the spearman correlation test all tests were considered statistically significant at pvalue resultsoverexpression of a0circzfr predicted poor prognosis of a0bc patientsas demonstrated by qrtpcr circzfr was significantly upregulated in bc tissues and cell lines compared with their counterparts fig a01a b to determine its clinical relevance we preliminarily examined the link between circzfr level and the prognosis of bc patients kaplanmeier survival curves showed that the patients in low circzfr level group had a longer survival time than those in high circzfr group fig a0 1c additionally circzfr expression was remarkably associated with the distant metastasis and tnm stage of these patients table a0silencing of a0circzfr hindered bc cell viability colony formation and a0enhanced apoptosis in a0vitro and a0weakened tumor growth in a0vivoto study the biological role of circzfr in bc progression the lossoffunction experiments were carried out using shrnas against circzfr shcirczfr1 shcirczfr2 and shcirczfr3 in contrast to the negative control the transfection of the three shrnas prominently reduced circzfr expression in both mcf7 and bt549 0cchen a0et a0al cancer cell int page of fig circzfr was overexpressed in bc and associated with poor prognosis circzfr expression by qrtpcr in pairs of tumor tissues and adjacent normal tissues a mcf10a mcf7 bt549 mdamb231 and mdamb453 cell lines b c analysis for the overall survival of bc patients in high n or low n circzfr level group divided by the median of circzfr expression in bc tissues using kaplanmeier survival analysis and logrank test p cell lines fig a02a b notably shcirczfr1 also named shcirczfr caused the most significant downregulation in circzfr expression so we used it for further analyses functional experiments data revealed that the silencing of circzfr led to a striking inhibition in cell viability fig a02c colony formation fig a02d e as well as a strong promotion in cell apoptosis fig a02f gto determine whether circzfr regulated bc tumor development in a0vivo we performed the xenograft model assays when we infected bt549 cell line with shcirczfr tumor growth was remarkably blocked compared with the shnc control fig a02h isilencing of a0circzfr suppressed bc cell migration invasion and a0glycolysiswe also asked whether circzfr regulated bc cell migration invasion and glycolysis in a0 vitro transwell assays showed that in comparison to the control group cell migration fig a0 3a and invasion fig a0 3b were significantly repressed by circzfr knockdown moreover in both cell lines circzfr silencing resulted in decreased glucose uptake fig a0 3c lactate product fig a0 3d and atp level fig a0 3e demonstrating the suppression of circzfr knockdown on cell glycolysiscirczfr directly interacted with a0mir by a0binding to a0mirto further understand the role of circzfr in bc pathogenesis we performed a detailed analysis for its targeted mirnas the two online algorithms circinteractome and circbank collectively revealed that circzfr harbored a putative complementary sequence for mir578 mir944 and mir5323p fig a04a the data of qrtpcr showed that the silencing of circzfr led to a striking overexpression in mir578 and mir5323p levels but mir expression was not affected by circzfr knockdown in the two bc cell lines fig a0 4b c previous work had reported that circzfr regulated colorectal cancer progression through acting as a sponge of mir5323p so we aimed to identify whether mir578 was a molecular mediator of circzfr in bc progression by contrast mir was prominently upregulated in the two bc cell lines transfected with mir578 mimic fig a04d we then cloned circzfr fragment containing the mir578binding sites into a luciferase plasmid and mutated the mir578binding sites fig a0 4e the elevated mir578 expression significantly reduced the activity of circzfr wildtype reporter circzfrwt fig a04f however the mutation of the target sites circzfrmut completely abrogated the effect of mir578 on reporter gene expression fig a04f indicating the validity of the target sequence for interaction additionally rna pulldown assays revealed that the enrichment level of circzfr was remarkably elevated by biomir578 in both cell lines fig a04goverexpression of a0mir restrained bc cell viability colony formation migration invasion and a0glycolysis and a0promoted apoptosis in a0vitroin bc tissues mir578 expression was significantly decreased compared to the normal control fig a05a and it was inversely correlated with circzfr level fig a0 5b moreover mir578 level was lower in bc cell lines than that of control fig a05c subsequently we analyzed the biological effect of mir578 on bc progression in contrast to the control group the increased expression of mir578 0cchen a0et a0al cancer cell int page of fig circzfr silencing suppressed bc progression in vitro and in vivo a b qrtpcr for circzfr expression in mcf7 and bt549 cell lines transduced with shnc shcirczfr1 shcirczfr2 or shcirczfr3 cck8 assay for cell viability c colony formation assay for cell colony formation d e flow cytometry for cell apoptosis f g in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines h i shncinfected or shcirczfrtransduced bt549 cell line was subcutaneously injected into the nude mice n per group followed by the measurement of tumor volume and weight and the capture of representative pictures p 0cchen a0et a0al cancer cell int page of fig circzfr silencing suppressed bc cell migration invasion and glycolysis transwell assay for cell migration and invasion a b corresponding assay kits for glucose uptake lactate product and atp level ce in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines p induced a distinct repression in cell viability fig a05d and colony formation fig a0 5e and a strong promotion in cell apoptosis fig a0 5f as well as a striking reduction in cell migration fig a05g invasion fig a05h and glycolysis fig a05iksilencing of a0circzfr regulated bc cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitro by a0upregulating mira crucial question was whether circzfr regulated bc progression by mir578 to address this we reduced mir578 expression in shcirczfrtransduced mcf7 and bt549 cell lines as expected in comparison to the negative control the reduced level of mir578 significantly reversed circzfr knockdowninduced antiviability fig a0 6a anticolony formation fig a0 6b proapoptosis fig a0 6c antimigration fig a0 6d antiinvasion fig a0 6e and antiglycolysis fig a06fhaca aga a was circzfr modulated hif1a expression via a0acting as a0a a0sponge of a0mirusing the software targetscan a predicted mir578binding sequence identified within the ²utr of hif1a fig a0 6a when we cloned the ²utr fragment containing the putative mir578binding sites downstream of a luciferase coding sequence the cotransfection of the luciferase reporter hif1a3²utrwt and mir578 mimic into the two bc cell lines produced lower luciferase activity than in cell lines cotransfected with the mirnc control fig a07b c however the mutation of the target sequence hif1a²utrmut prominently abolished the suppression of mir578 fig a07b c by contrast hif1a mrna and protein levels were significantly reduced by mir578 overexpression in the two bc cell lines fig a07d e these data together pointed that hif1a in bc cell lines was directly targeted and inhibited by mir578 0cchen a0et a0al cancer cell int page of fig circzfr directly interacted with mir578 by binding to mir578 a venn diagrams representing the putative mirnas that bind to circzfr identified by circinteractome and circbank online algorithms b c the levels of mir578 mir944 and mir5323p by qrtpcr in shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines d qrtpcr for mir578 expression in the two bc cell lines transfected with mirnc mimic or mir578 mimic e schematic of the mir578binding sites within circzfr and the mutation of the seed sequence f relative luciferase activity in mcf7 and bt549 cell lines cotransfected with circzfrwt or circzfrmut and mirnc mimic or mir578 mimic g qrtpcr for circzfr level in cell lysates incubated with bionc or biomir578 and streptavidin beads p we then examine whether circzfr influenced hif1a expression in bc cell lines as expected in comparison to their counterparts hif1a expression was remarkably downregulated by circzfr silencing at both mrna and protein levels in the two bc cell lines and this effect was significantly abrogated by antimir578 introduction fig a0 7f g additionally qrtpcr data showed a striking upregulation of hif1a mrna level in bc tissues fig a0 7h furthermore in bc tissues hif1a mrna expression was positively correlated with circzfr expression and negatively correlated with mir578 level fig a07ihif1a was a0a a0functional target of a0mir in a0regulating bc cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitroto provide further insight into the link between mir and hif1a in bc progression we elevated hif1a expression using a recombinant overexpressing plasmid in mir578 mimictransfected bc cell lines as a result hif1a protein level was prominently increased by the overexpressing plasmid in the two cell lines fig a0 8a functional experiments results revealed that the upregulation of hif1a dramatically abolished mir578 overexpressionmediated antiviability fig a0 8b proapoptosis 0cchen a0et a0al cancer cell int page of fig mir578 overexpression hindered bc progression in vitro a qrtpcr for mir578 expression in pairs of tumor tissues and adjacent normal tissues b correlation between mir578 expression and circzfr level in bc tissues using the spearman test c mir578 expression by qrtpcr in mcf10a mcf7 bt549 mdamb231 and mdamb453 cell lines mcf7 and bt549 cell lines were transfected with mirnc mimic or mir578 mimic followed by the determination of cell viability by cck8 assay d colony formation using a standard colony formation assay e cell apoptosis by flow cytometry f cell migration g and invasion h by transwell assay glucose uptake lactate product and atp level using assay kits ik p fig a08c antimigration fig a08d antiinvasion fig a08e and antiglycolysis fig a08fhdiscussionto date the emerging links between circrnas and bc progression have opened up a novel field for cancer diagnosis and treatment [ ] in the meantime understanding the molecular basis underlying the actions of circrnas has been still challenging in this study we identified the biological role of circzfr in bc progression in a0 vitro and in a0 vivo and investigated the mechanisms governing ithere we firstly demonstrated that circzfr was overexpressed in bc and the elevated expression of circzfr was associated with the poor prognosis of these patients by using lossoffunction in a0vitro and in a0vivo analyses we were first to uncover that the silencing of circzfr performed a suppressive effect in bc progression previous reports had highlighted the potential oncogenic role of circzfr in several other malignancies such as hepatocellular carcinoma papillary thyroid cancer and nonsmall cell lung cancer [ ] conversely circzfr was reported as a tumor suppressor in colorectal cancer and gastric cancer [ ] these contradictory findings may attribute to different type tumor or complex tumor microenvironmentusing the online algorithms we first identified that circzfr sequestered mir578 through sponging mir in bc cell lines ji et a0al showed that the increased mir578 level weakened osteosarcoma progression via directly interacting with circ_001621 farhana et a0al unraveled that in pancreatic cancer mir578 was 0cchen a0et a0al cancer cell int page of fig circzfr knockdown repressed bc malignant progression by mir578 in vitro shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines were transiently transfected with antinc or antimir578 a cck8 assay for cell viability b a standard colony formation assay for cell colony formation c flow cytometry for cell apoptosis d e transwell assay for cell migration and invasion fh glucose uptake lactate product and atp level using assay kits p associated with the tumor cell apoptosis as it has been reported our data validated the downregulation of mir578 expression in bc tissues and cells moreover we first identified that mir578 overexpression restrained bc cell malignant behaviors in a0 vitro more importantly for the first time we substantiated that circzfr knockdown hampered bc progression in a0vitro by mir578using targetscan software we identified that mir in bc cell lines directly targeted and inhibited hif1a hif1a a transcriptional regulator in response to intratumoral hypoxia [ ] contributes to bc metastasis and malignant progression [] we also uncovered that the upregulation of mir578 hampered bc malignant progression via downregulating hif1a in a0 vitro previous studies had reported that several 0cchen a0et a0al cancer cell int page of fig circzfr modulated hif1a expression via sponging mir578 a schematic of the putative mir578binding sequence and mutated the target sequence b c relative luciferase activity in mcf7 and bt549 cell lines cotransfected with hif1a3²utrwt or hif1a3²utrmut and mir578 mimic or mirnc mimic hif1a mrna and protein levels by qrtpcr and western blot in mcf7 and bt549 cell lines transfected with mir578 mimic or mirnc mimic d e shncinfected or shcirczfrtransduced mcf7 and bt549 cell lines transfected with antinc or antimir578 f g h relative hif1a mrna expression by qrtpcr in pairs of tumor tissues and adjacent normal tissues i correlation between hif1a expression with circzfr or mir578 level in bc tissues using spearman test p other mirnas such as mir18a and mir497 exerted an antitumor activity in bc through targeting hif1a [ ] furthermore our data first illuminated the role of circzfr as a sponge of mir578 to mediate hif1a expression in bc cell lines the findings by liang et a0al underscored that circrna circdennd4c contributed to bc cell proliferation under hypoxia via regulating hif1a in our present study demonstrated that circzfr was overexpressed in bc and the silencing of 0cchen a0et a0al cancer cell int page of fig the repression of mir578 upregulation on bc progression in vitro was mediated by hif1a a hif1a protein level by western blot in mcf7 and bt549 cell lines transfected with vector or hif1a mcf7 and bt549 cell lines were transfected with mirnc mimic vector mir578 mimic vector or mir578 mimic hif1a followed by the determination of cell viability by cck8 assay b cell apoptosis by flow cytometry c cell migration and invasion by transwell assay d e glucose uptake lactate product and atp level using assay kits fh vector negative control plasmid hif1a recombinant hif1a overexpressing plasmid p circzfr suppressed bc malignant progression via the regulation of the mir578hif1a axis this is the first report of circzfr in bc pathogenesis providing evidence for the crucial involvement of circzfr in bc progressionsupplementary informationsupplementary information accompanies this paper at https doi101186s1293 additional file a0 supplement material the str authentication of mcf7 a and bt549 b cellsadditional file a0 supplement material the detailed quantification of the western blot analysisabbreviationsbc breast cancer hif1a hypoxiainducible factor 1α cck8 cell counting kit8 atp adenosine triphosphate anova analysis of varianceacknowledgementsnoneauthors contributionszc and xq designed and performed the research fw yx nw and yg analyzed the data zc wrote the manuscript all authors read and approved the final manuscriptfundingno | 0 |
test the hypothesis that levobupivacaine has antitumour effects on breast cancer cellsResults Colony formation and transwell assay were used to determine breast cancer cells proliferation Flow Cytometry annexin V and PI staining was used to investigate breast cancer cells apoptosis The effects of levobupivacaine on cellular signalling and molecular response were studied with Quantitative Polymerase Chain Reaction and western blot Induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates The results of the western blot and Quantitative Polymerase Chain Reaction indicated activation of active caspase and inhibition of FOXO1 The results of the flow Cytometry confirmed that levobupivacaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells Quantitative Polymerase Chain Reaction and Western blot analysis showed increased p21 and decreased cyclin D Quantitative Polymerase Chain Reaction and western blot analysis showed that levobupivacaine significantly increased Bax expression accompanied by a significant decreased Bcl expression and inhibition of PI3KAktmTOR signalling pathway These findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitroKeywords Levobupivacaine Proliferation Invasion Apoptosis Breast cancerIntroductionBreast cancer is one of the most recorded cancer illness among women [] In the United States it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments []Correspondence yanqiu63126com wqp89163com Department of Anaesthesiology Dalian Medical University Dalian China Department of Biochemistry and Molecular Biology Dalian Medical University Dalian ChinaFull list of author information is available at the end of the Molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment [] The mechanisms of the PI3KAktmTOR signalling pathway have present some promising targets for cancer treatments This signalling pathway hinders the functions of several tumour suppressor genes such as Bad GSK3 FOXO transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [] Suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deathThe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited [] The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cKwakye a0et a0al BMC Res Notes Page of At the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor EGFR which is a potential target for antiproliferation in cancer cells [] Evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [] To the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined The present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsMain textMaterials and a0methodsEthics statementThe ethical committee of the Dalian Medical University First Affiliated Hospital approved for this study to be carried outCell cultureWe purchased MCF7 and MDAMB231 breast cancer cells from the ATCC Beijing Zhongyuan limited China We maintained the MCF7 and MDAMB231 cells with highglucose DMEM or DMEMF12 Gibco USA medium The medium was supplemented with fetal bovine serum FBS Gibco USA penicillin a0unitsml and streptomycin a0µgml TransGen Biotech China to maintain the cells The MCF7 and MDAMB231 cells were then maintained in an incubator at a0ºC humidified air with CO2 atmospheric condition The cells were routinely subcultured subsequentlyAntibodies and a0reagentsEPR17671 Akt monoclonal Antibody Abcam China Y391 mTOR Polyclonal Antibody Abcam China A2845 Bcl2 Polyclonal Antibody ABclonal Technology A11550 Bax Polyclonal Antibody ABclonal Technology A0265 PIK3CA Polyclonal Antibody ABclonal Technology A2934 FOXO1 Polyclonal Antibody ABclonal Technology EPR21032 Active caspase monoclonal Antibody Abcam China AFO931 Cyclin D1 Polyclonal Antibody Affbiotech China AF6290 p21 Polyclonal Antibody Affbiotech China AntimTOR phospho S2448 Antibody Abcam China PA517387 PhosphoPI3K p85p55 Tyr458 Tyr199 Polyclonal Antibody ThemoFisher Scientific PosphopanAKT123 Ser473 Antibody Affbiotech China Peroxidaseconjugated goat antirabbit IgG Proteintech China PRAP antibodies Proteintech China and GAPDH antibodies Proteintech ChinaCell viability assay and a0IC50We determined the MCF7 and MDAMB cells viability using CCK8 assay Levobupivacaine at a concentration of or a0mM was used to treat MCF7 and MDAMB cells plated in 96well plates a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °C with CO2 The rest of the procedures used for the CCK8 assay were the same as described elsewhere []Flow cytometryAnnexin V and propidium iodide PI staining assay were used to investigate the apoptosis of MCF7 and MDAMB cells following levobupivacaine treatment After treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min The MCF7 and MDAMB treated cells were again suspended with Binding Buffer and then a0 µl of fluorochromeconjugated annexin V SigmaAldrich Saint Louis USA was added into a0µl of the cell suspension to stain intracellular phosphatidylserine PS The cells were then incubation in a dark under room temperature The cells were again suspended and a0 µl propidium iodide staining solution SigmaAldrich Saint Louis USA added into a0µl of the cell suspension We detected the percentage of the apoptotic cells via FlowJo software Treestar Ashland USA and Flow cytometry FACS Calibur Becton Dickinson and Sunnyvale CA USAQuantitative polymerase chain reaction qPCRThe procedures used for the qPCR were the same as previously described [] The primers sequences were BAX 5TGG CAG CTG ACA TGT TTT CTG3 F 5TCC CGG AGG AAG TCC AAT G3 BCL2 5ACG GTG GTG GAG GAG CTC TT3 F 5GCC GGT TCA GGT ACT CAG TCAT3 R p21 5GCG ACT GTG ATG CGC TAA TG3 F 5GAA GGT AGA GCT TGG GCA GG3 R GAPDH ²CAT GTT CGT CAT GGG TGT GAA² F ²GGC ATG GAC TGT GGT CAT GAG3² RR Western blotAt the log phase of treated MCF7 and MDAMB cells growth we harvested the cells and then washed twice with icecold PBS The rest of the procedures used for the western blot were the same as described elsewhere []Colony formation assayThe procedures used for the colony formation assay were the same as previously described [] 0cKwakye a0et a0al BMC Res Notes Page of Transwell assayThe MCF7 and MDAMBA231 cells that were pretreated with different dose of Levobupivacaine a0mM for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores Merck KGaA P18P01250 China The procedures used for the Transwell assay were the same as previously describe []Data analysisValues were expressed as the mean ± SD Statistical analysis was performed with GraphPad Prism version 501GraphPad Software La Jolla CA US Oneway ANOVA was used to measure significance p Dunnetts post hoc tests were used to test the difference between groupsResultsLevobupivacaine decreases breast cancer cell invasionTranswell assay analysis showed significantly decreased in the invasion ability of MCF7 and MDAMB231 cells in a dosedependent manner compared with the untreated cells Additional file a0 Fig S1a b Levobupivacaine inhibits proliferation in a0breast cancer cellsThe MCF7 and MDAMBA231 cell viability decreased as the concentrations of levobupivacaine or a0mM increased The MCF7 cells showed a cytotoxic effect while the MDAMB231 cells showed a similar cytotoxic effect of Fig a01a Under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates Additional file a0 Fig S2a b The viability of breast cancer cells decreased in a dosedependent manner The results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells Fig a01b c The data showed that the mRNA level of p21 significantly increased following levobupivacaine treatment Fig a0 1d e Western blot analysis showed a similar increased in p21 and decreased in FOXO1 and cyclin D1 expressions in a dosedependent manner compared with the untreated cells Fig a01f g Additional file a03f gLevobupivacaine promote apoptosis in a0breast cancer cellsLevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells Fig a0 2a b The qPCR data showed a decreased in Bcl2 and increased in Bax expressions in MCF7 and MDAMB231 cells compared with the untreated cells Fig a0 2c d Western blot analysis also showed a similar decreased in Bcl2 and increased expressions of active caspase and Bax compared with the untreated cells Fig a02e f Additional file a03e fLevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0PI3KAktmTOR signalling pathwayWestern blot analysis showed a significant decreased in the expression of the nuclear localization of pPI3K pAkt and pmTOR compared with the untreated cells Fig a03a b Additional file a03a bDiscussionBreast cancer remains a common cause of mortality among women worldwide Though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited These therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed Several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [] Recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition [] A study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells HCC by increasing the Caspase activity whereas ropivacaine inhibits the growth of HCC cells by stopping the cell cycle in G2 phase [] Lee et a0al demonstrated that local anaesthetics potentiate TNFα mediated apoptosis in HK2 cells [] The cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local See figure on next pageFig Levobupivacaine inhibits proliferation in breast cancer cells MCF and MDAMB cells were treated with different concentrations of levobupivacaine a Cell viability was measured by CCK assay IC50 results of levobupivacaine on MCF and MDAMB cells b c Colony formation of MCF and MDAMB cells treated with various concentrations of Levobupivacaine and stained with crystal violet d e The mRNA expression levels of p21 and GAPDH were analysed by qPCR f g Protein expression assessment of MCF and MDAMB cells by western blot against antibodies FOXO1 p21 Cyclin D1 and GAPDH used as control The data was statistically significant at indicates P indicates P indicates P compared with untreated cells This data corresponds to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of 0cKwakye a0et a0al BMC Res Notes Page of Fig Effects of levobupivacaine on apoptosis of breast cancer cells a b MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h The cells were then stained with fluoresceinconjugated annexin V and PI and analysed by flow cytometry Error bars represent standard error of the mean P versus the control c d Relative gene expression of Bax and Bcl following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qPCR e f MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h and the activities of Bax Bcl and Active caspase were examined by Western blot analysis using specific antibodies GAPDH was used as internal controls The data was statistically significant at indicates P indicates P compared with control The data correspond to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of Fig MCF and MDAMB cells were treated with different concentrations of levobupivacaine for h a b The cells were lysed and subjected to SDSPAGE and analysed by western blotting and probed with specific antibodies pPI3K pAkt and pmTOR The results showed a decrease in the expressions of pPI3K pAkt and pmTOR proteins GAPDH was used as internal controls The data represent the mean ± SD of three independent experimentsanaesthetic [] In this study we employed MCF and MDAMB231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro The antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerPI3KAktmTOR signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response Breast cancer cell resistance to therapies can result from the activation of PI3KAktmTOR signalling pathway [] This has made the PI3KAktmTOR signalling pathway an important object of study for understanding the development and progression of breast cancer In patients with breast cancer PI3KAktmTOR signalling pathway can be a target for diagnostic prognostic and treatment purposes [] Akt plays a role in the activation and inactivation of many transcription factors Activation of Akt correlated with the activation of mTOR Phosphorylation of the FOXO proteins by Akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes Cyclin D1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers [] Reports show that overexpression of cyclin D1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin D1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages [] Datta et a0al reported that Akt can phosphorylate the proapoptotic Bcl2 family member Bad causing its isolation from the mitochondrial membrane by other proteins [] Local anaesthetics modify the protein levels of key members of the Bcl2 family in a manner that presents an increase in the ratio of BaxBcl2 which may contribute to the response of cancer cells to apoptosis In the present study the role of levobupivacaine on the expression of PI3K Akt and mTOR was investigated to illustrate the potential molecular mechanism We observed a significantly decreased expression of pAkt pPI3K pmTOR and subsequent decreased expression of FOXO Cyclin D1 and Bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis These emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing PI3KAktmTOR signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyConclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the PI3KAktmTOR signalling pathway These findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0cKwakye a0et a0al BMC Res Notes Page of LimitationsNumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [] However our work is not without limitations In a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededBiology Dalian Medical University Dalian China Department of Anaesthesia and Critical Care School of Medicine University of Health and Allied Sciences Ho Ghana Department of Biochemistry and Molecular Medicine School of Medicine and Health Sciences University for Development Studies Tamale Ghana Departments of Anaesthesia and Critical Care Ridge Hospital Accra Ghana Department of Medicine Princefied University Ho Ghana Received June Accepted July Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 Additional file a0 Figure S1 Levobupivacaine decreases breast cancer cell invasionAdditional file a0 Figure S2 Effect of levobupivacaine on the morphology of MCF and MDAMB cellsAdditional file a0 Original gelsblots scan used in Fig 1f g Fig 2e f and Fig 3a b for MCF and MDAMB cellsAbbreviationsEGFR Epidermal growth factor receptor HCC Hepatocellular carcinoma cells NC Nitrocellulose PI Propidium iodide PS Phosphatidylserine qPCR quantitativepolymerase chain reactionAcknowledgementsWe thank the First Affiliated Hospital and The Department of Biochemistry of Dalian Medical University for making available all the necessary materials needed for this work We also thank the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO of China for supporting this work Our thanks also go to the China Scholarship Council and the Government of the Republic of Ghana for giving financial aid to some of the authors to study at Dalian Medical UniversityAuthors contributionsAKK SK QY and QPW conceived and designed this study QPW and QY were responsible for the supervision and coordination of this study AKK SK JL MNR QY and QPW conducted the data collections SK led the data analysis with inputs from AKK QY and QPW AKK and SK wrote the first draft of the manuscript and JL MNR SAR AAF JA and EAN contributed to revising and reviewing the manuscript All authors read and approved the final manuscriptFundingThis study was supported by the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO Availability of data and materialsThe data used andor analysed in this study are available from the corresponding author upon reasonable requestEthics approval and consent to participateThe ethical committee of the First Affiliated Hospital of Dalian Medical University approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyConsent for publicationsNot applicableCompeting interestsAuthors declare that they have no competing interestsAuthor details Department of Anaesthesiology Dalian Medical University Dalian China Department of Anaesthesiology First Affiliated Hospital of Dalian Medical University Dalian China Department of Biochemistry and Molecular References American Cancer Society Breast Cancer Facts and Figures Atlanta American Cancer Society American Cancer Society Cancer Facts and Figures Atlanta Ameri can Cancer Society Siegel R Naishadham D Jemal A Cancer statistics CA Cancer J Clin Chang YC Hsu YC Liu CL Huang SY Hu MC Cheng SP Local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogenactivated protein kinase pathway PLoS ONE 20149e89563 GomezGutierrez JG Souza V Hao HY de Montes OcaLuna R Dong YB Zhou HS McMasters KM Adenovirusmediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells Cancer Biol Ther Sunters A de Fern¡ndez Mattos S Stahl M Brosens JJ 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1i32i3838 Li K Yang J Han X Lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the upregulation of RARβ2 and RASSF1A demethylation Int J Mol Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c' | 2 |
"As differential LysoTracker retention may reflect differences in basal lysosomal mass and/or lysosomal pH we therefore next examined phenothiazine-induced changes in LysoTracker retention (?LysoTracker) as an indicator of perturbation in lysosomal pH. Exposure to TFP for 24?h was associated with varying degrees of loss in LysoTracker retention in almost all of the LC cell lines tested (b). Furthermore there was a highly significant positive correlation between the extent of TFP-induced loss in LysoTracker retention and the magnitude of cytotoxicity (b left). Importantly the loss of LysoTracker retention became irreversible in SCLC at lower TFP concentrations than in NSCLC cells (10??M in H82 and 20??M in U-1810) (c) and the afflicted cells underwent clear mitochondrial depolarization (d). By contrast cells exposed to non-cytotoxic concentrations of TFP recovered LysoTracker Green staining to various degrees over time (c). These data showed that prolonged disruption of lysosomal functions was detrimental to LC cell viability and that SCLC were more sensitive to phenothiazines as result of lower intrinsic lysosomal buffer capacity. In line with these results TFP-induced cell death was significantly antagonized by the protease inhibitor E-64d (e). Moreover preventing the accumulation of TFP within lysosomes by BafA1 pre-treatment also largely abolished its negative impact on cell viability (f). Thus our data suggest that the more prominent cytotoxic effect of phenothiazines in SCLC is related to a higher degree of lysosomal pH neutralization (?LysoTracker) that is more persistent in SCLC than in NSCLC. Moreover although phenothiazines initially induced LC3-II in both NSCLC and SCLC only SCLC failed to adapt to phenothiazine-induced lysosomal dysfunction and this led to increased cell death preferentially in SCLC. Together these data reinforce the notion that persistent lysosomal perturbation induced upon single-agent phenothiazine treatment is a major mediator of cytotoxicity in SCLC cells. SCLC cells show increased sensitivity toward lysosome-disrupting agents On the basis of the findings presented above we reasoned that the increased susceptibility of SCLC cells to phenothiazines may be due to lower basal lysosomal mass and/or lysosomal pH buffer capacity leading to intrinsically lower tolerance toward compounds that disrupt lysosomal functions. Consistent with this idea we found that SCLC cells were typically more sensitive than NSCLC counterparts toward a number of lysosomotropic agents (Supplementary Figure S3) including the clinically approved drugs tamoxifen (TMX) and chloroquine (CQ) in addition to the phenothiazine compounds TFP and CPZ. Notably TFP CPZ TMX and CQ all dissipated LysoTracker staining to a larger extent in SCLC (H69 H82 H592 and U-1285) than in NSCLC (A549 and U-1810) cell lines (Figures 6a and b). Moreover the increased sensitivity of SCLC cells toward lysosomotropic agents was not recapitulated with the CaM antagonist W7 suggesting that phenothiazine-induced lysosomal perturbation does not involve modulation of Ca2+/CaM signaling a well-known process inhibited by phenothiazines (c Supplementary Figure S3A). Overall our findings highlight not only the potential for treating SCLC with FDA-approved drugs that target lysosomal acidification in general but also the use of phenothiazines against this tumor type in particular. Discussion Phenothiazines are most well-known for their dopamine antagonistic activity in the central nervous system (CNS) which has been utilized clinically for the management of psychiatric disorders such as schizophrenia.1 2 In addition these compounds affect the fate of non-CNS cells in a variety of ways and depending on the experimental context may result in cellular differentiation cell death or protection from toxic injuries.34 9 One well-recognized property of phenothiazines is their ability to induce apoptosis in certain cell types.34 9 The mechanism(s) behind this is not clearly understood although inhibition of CaM-regulated processes and increases in membrane fluidity/permeability are thought to be at least partially responsible. However the cell-intrinsic determinants that govern which of these effects that will be elicited in a particular model system are not known and the underlining molecular pathways remain poorly defined. Elucidating the contextual utilities of phenothiazines in in vitro system response in tumor xenografts and animal models will enable them to be harnessed appropriately for treating different human ailments including tumors as illustrated here with SCLC. Although targeted agents have been introduced for the clinical management of LC cases the majority of SCLC and NSCLC patients with advanced disseminated diseases are still treated with conventional CT agents such as cisplatin. For SCLC the initial response is often good but most cases relapse and present high degrees of chemoresistance while for NSCLC a less CT-sensitive phenotype is usually found already at start of treatment.14 Consequently there is an urgent need for the development of new treatment regimen to combat both subtypes of LC. In this study we evaluated a novel strategy involving the use of phenothiazines as single treatment agents in LC. Our data demonstrate that phenothiazines are generally more cytotoxic in SCLC than in NSCLC cell lines despite comparable responses to cisplatin etoposide and gemcitabine which are standard chemotherapeutic agents for the treatment of LC. Importantly we show that normal lung fibroblasts are less affected by phenothiazines at concentrations which were toxic for SCLC indicating a favorable therapeutic window that would allow its use in SCLC without incurring significant adverse effects on healthy tissues. Although it needs to be confirmed by further in vivo toxicity analysis; several earlier reports have shown that phenothiazines are in general well-tolerated by cancer patients.10 15 To uncover responsible mechanisms for the preferential susceptibility of SCLC to phenothiazines we dissected the molecular details of phenothiazine-induced cell death using multiple SCLC and NSCLC cell lines and with TFP as a model compound. We found that TFP treatment led to a rapid neutralization of lysosomal pH as judged by decreased retention of the lysosomotropic dye LysoTracker Green accompanied by accumulation of LC3-II in SCLC cells. Our data thus corroborated a previous study that identified TFP as an inducer of autophagy at low doses in H4 human neuroglioma cells.16 However we found that TFP at cytotoxic concentrations irreversibly disrupted lysosomal homeostasis especially in SCLC cells driving LC3 conversion while blocking its degradation through autophagy. This was logical given that protonation of the weakly basic phenothiazines within lysosomes is expected to increase lumenal pH and could thereby adversely affect the activities of acid hydrolases.13 17 Prolonged exposure to cytotoxic concentrations of TFP (10??M in H82 and 20??M in U-1810) was associated with lysosomal membrane permeabilization followed by mitochondrial depolarization." | 1 |
"incidence of thyroid carcinoma is increasing all over the world Some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma However other studies have come to theopposite Therefore we studied the relationship between adipokines and thyroid carcinomaMethods DatabasesPubMed Cochrane Library SinoMed CNKI Wanfang and clinical trial registries weresearched A metaanalysis was then performed through a fixed or randomeffects model to calculate I values forheterogeneity analysisResults Twentynine s were finally included for analysis The level of serum tumor necrosis factoralpha TNFα [standardized mean difference SMD confidence interval CI to I2 P ]and the ratio of TNFα immunoreactivity in tissues [odds ratios OR CI to I2 P ]in thyroid carcinoma are significantly higher than those in control The serum interleukin6 IL6 in patients withthyroid carcinoma is higher than that in control SMD CI to I2 P There is nosignificant difference of the ratio of IL6 immunoreactivity in tissues between carcinoma and control OR CI to I2 P The ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma OR CI to I2 P However after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control P Conclusions Adipokines TNFα IL6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma However the association between adiponectin and thyroid carcinomaneeds further researchKeywords Thyroid carcinoma Adipokines TNFα IL6 Leptin Metaanalysis Correspondence liaolinsdueducn cwc_llsdueducn Junyu Zhao and Jing Wen contributed equally to this work1Department of Endocrinology and Metabology The First Affiliated Hospitalof Shandong First Medical University Shandong Provincial QianfoshanHospital Jinan China5Department of Endocrinology and Metabology Qilu Hospital of ShandongUniversity Cheeloo College of Medicine Shandong University Jinan ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhao BMC Cancer Page of BackgroundThyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis During the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] Someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason [] But this may be only partial andcan not totally explain the increased incidence of microcarcinoma Changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore Recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [] Various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic low grade inflammation altered immune response and DNA damage caused byoxidative stress [] Furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [] Adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphaTNFα interleukin6 IL6 and complement factor Dalso known as adipsin Adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin The concentration of adipokines such as TNFαIL6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index []It is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development At present many kindsof adipokines have been reported to be associated withthyroid carcinoma Rehem RA [] suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery Another envidence showed that adiponectin related with tumor size [] However the opposite resultswere also found in other studies [] Some researchesreported the expression of adipokines is lower in tumortissue than normal control [] It is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomaThese confunding factors above may be the cause of inconsistency results from different researches Additionaly the association between adipokines and thyroidcarcinoma are still not well documented Therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomaMethodsSearching progressWe conducted a search of all studies published until27th July regarding the association between adipokine and thyroid carcinoma Eligible casecontrol studieswere found by searching the database of PubMedCochrane library Sinomed CNKI and Wanfang and restricted to published results Clinical trial register centers httpwwwclinicaltrialsgov were also searchedThe following search terms Adipokine or Leptin oradiponectin or resistin or tumor necrosis factoralpha or Interleukin6 or Complement factor D orAdipocytokines or tumor necrosis factorα or TNFα or IL6 or adipsin and thyroid cancer or thyroid neoplasm or thyroid tumor or thyroid carcinoma or differentiated thyroid carcinoma or DTC orPapillary thyroid carcinoma or Thyroid carcinomapapillary or PTC or Thyroid cancer follicular orFTC or Thyroid Carcinoma Anaplastic or ATC orThyroid cancer medullary or MTC Hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews Eligible studies met the following criteria published in English or Chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue Studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate OR and95CI the type of study was not a casecontrol designhave not fulltext animal trialsStudy selection and data extractionTwo reviewers screened the studies and extracted dataindependently Any disagreement was resolved by discussion or consensus with a third senior reviewer Dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0cZhao BMC Cancer Page of scores were considered to be of high quality Disagreements were resolved by reevaluating and discussing between two reviewersinSearchingthis metaanalysisResultsSearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites Afterscreening the title and abstracts s were selected for fulltext review Finally studies were eligibleprogressincluded and excluded details are all shown in Fig Eighteen of these studies are published in Chinese[ ] and the rest are published in English[] Nineteen studies were conducted in Chinatwo in India and two in Turkey Brazil Greece IranItaly Denmark and Serbia each had one study Totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group The sample size ranges from to in the case group while to in the controlgroup All the thyroid carcinoma patients were confirmed by pathologically Among these studiesfourteen studies reported papillary thyroid carcinomaPTC eight studies reported differentiated thyroidcarcinoma DTCreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma MTC and the restfour studies did not show the pathological detailsThe detailed characteristics ofincluded studies aresummarized in Table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups Thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesStatistical analysisFor metaanalysis dichotomous outcomes were analyzedby using the odds ratios OR computed using the MantelHaenszel method fixed or random models Continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences WMD Otherwise standardized mean difference SMD were used for different scaleAll results were reported with confidence interval CI I2 was used to assess heterogeneity between studies and I2 values of and representing no lowmoderate and high heterogeneity respectively Visual inspection of the funnel plot was done to assess publicationbias The analyses were performed by Review Manager Cochrane Collaboration United Kingdom httpwwwcochraneQuality assessment and risk of biasThe methodological quality of casecontrol study wasassessed by the NewcastleOttawa Scale NOS Supplement Table which consists of the three parameterseight questions with nine possible scores Selection Exposure and Comparability A study can be awarded amaximum of one score for each numbered item withinthe Selection an Exposure categories A maximum oftwo scores can be got for Comparability A higher scoremeans better quality in methodology and five or moreFig Flow chart of the systematic search process 0cZhao BMC Cancer Page of Zhao Jianqiang []ChinaPTC FTC ATCand MTCthyroid adenoma andnormal healthUnknownUnknownTable Characteristic of included studiesFirst authorYearCountryPathologicaltype of thyroidcancerSource of controlsL Kayser []Denmark PTC and FTCCao Guangyao []ChinaUnknownMTrovato []ItalyDTC andundifferentiatedcarcinomamultinodular goitersadenomas Hashimotosthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesMelih Akinci []Wang Jingxia []ZhuangXiaoming []Yu Xiao []Hou Sen []SnezanaZivancevicSimonovic []Xu Xiaocheng []XeniProvatopoulou []TurkeyPTChealthy volunteersChinaPTC and FTCnormal thyroid tissuesChinaPTC FTC andMTCthyroid adenoma andnormal healthChinaPTCthyroid adenoma andnormal thyroid tissue nearcarcinomaChinaPTCthyroid adenomaSerbiaWDTChealthy subjectsChinathyroidcarcinomaGreecePTCthyroid adenomabenign thyroid disease andhealthy controlsSun Qinnuan []ChinaPTCnormal thyroid tissue nearcarcinoma and healthycontrolsChinaPTCthyroid adenomaChinaPTCthyroid adenomaMean age yearFemale Outcome indexNumber ofparticipants ncases control casesUnknowncontrolcontrolcasesUnknownUnknownUnknownUnknownUnknownTNFα tissueTNFα tissueIL6 tissueIL6ãTNFαblood ± ±Unknown leptinblood Unknown TNFα tissue Unknown IL6ãTNFαUnknownUnknownbloodleptintissueUnknown Unknown leptin ± ± tissueTNFαblood ± ± ± ± ± ± ± IL6blood IL6blood TNFαbloodandtissue ±Unknown Unknown leptinUnknownUnknowntissueadiponectintissueUnknown Unknown adiponectintissue IL6ãTNFαblood ± ± Zhang Zijie []Zhong Xiuxiu []Zhang Bo []Hu Jinhua []SnezanaZivancevicSimonovic []YanLan Fan []ChinaDTCChinaDTCnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±SerbiaPTCcontrol subjectsUnknownUnknownIL6bloodChinathyroidcarcinomanodular goitre Hashimotosthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesUnknownUnknownleptintissue 0cZhao BMC Cancer Page of Table Characteristic of included studies ContinuedFirst authorSource of controlsYearCountryPathologicaltype of thyroidcancerChinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseaseChinaPTCthyroid adenomaTurkeyPTChealthy volunteersIndiaPTCIndiaPTCbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsNumber ofparticipants ncases control cases ±Mean age yearFemale Outcome indexcontrol ±casescontrol TNFαtissue ± ±TNFα tissue IL6bloodUnknown Unknown TNFαbloodUnknown Unknown IL6bloodWangXinzheng []Song Runbo []Kemal Beksac []Toral PKobawala []Toral PKobawala []RaziyehAbooshahab []Zhang Bo []ZhouXiaodong []Ma Xiaokai []MarianaBonjiornoMartins []IranMTChealthy subjects ± ± leptinãadiponectinbloodChinaDTCnormal thyroid tissue nearcarcinomaUnknown Unknown leptintissueChinaDTChealthy subjects ± ±IL6ãTNFαbloodChinaPTCthyroid adenomaUnknown Unknown leptinBrazilDTCbenign thyroid nodules andhealthy controls ±tissue IL6blood ± ±ChinaIL6 Sun Zhenhua []tissueTNFα tumor necrosis factora DTC differentiated thyroid carcinoma IL6 interleukin6 PTC papillary thyroid carcinoma FTC follicular thyroid carcinoma ATCanaplastic thyroid carcinoma MTC medullary thyroid carcinoma WDTC welldifferentiated thyroid carcinoma FNAC fine needle aspiration cytologynodular goiterPTCQuality of included studiesThe quality assessment of these studies is assessed bythe NOS and the resultis shown in SupplementalTable Five or more scores are determined as highquality Two studies conducted by Cao G in [] and L Kayser in [] only get two scoresshowing a poor quality in methodology The rest studies are assessed as high qualityTNFα and thyroid carcinomaTwelve studies reported the expression of TNFα bothin patients with thyroid carcinoma and control subjects[ ] Among these sevenstudies [ ] had tested the level ofserum TNFα two studies [ ] had tested the expression of TNFα in tissues and the ratio of TNFα immunoreactivity was tested in four studies [ ] Firstly fixedeffect model is used to merge the SMDvalues of serum TNFα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test Chi2 P I2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors Then randomeffect model to merge the SMD isused and pooled effect size in favor of control group is CI to P Fig 2a SMDvalues of the expression of TNFα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testChi2 P I2 The different unitsand limited numbers of research may be the original ofheterogeneity So the pooled SMD with randomeffectmodel of the expression of TNFα in tissues is CI to P Fig 2b The pooled ORwith fixedeffect model of the ratio of TNFα immunoreactivity in thyroid carcinoma tissues is CI to P However a significant heterogeneity is detected heterogeneity test Chi2 P I2 The published by L Kayser in with a poor quality in methodology may attributeto this high heterogeneity Then randomeffect model ofpooled OR is used and pooled effect size in favor of 0cZhao BMC Cancer Page of Fig Forest plot of the TNFα level and the ratio of TNFα immunoreactivity in tissues in patients with thyroid carcinoma a Level of serum TNFα b Expression of TNFα in tissue c Ratio of TNFα immunoreactivity in tissuecontrol group is CI to P Fig 2c In level of serum TNFα and theratio of TNFα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomaIL6 and thyroid carcinomaAmong the included studies reported the level ofserum IL6 in patients with thyroid carcinoma and control subjects [ ] Due to thelarge heterogeneity of the merged SMD values of serumIL6 level by the heterogeneity analysis heterogeneitytest Chi2 P I2 randomeffectmodel was used to pooled the SMD values and thepooled effect size in favor of control subjects is CI to P Fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum IL6 than control subjects Twostudies reported the ratio of IL6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] The pooled OR of the limited two studies donot show an increased ratio of IL6 immunoreactivity inthyroid carcinoma tissues OR CI to P and a large heterogeneity always existsheterogeneity test Chi2 P I2 Fig3b Thus the level of serum IL6 is higher in patientswith thyroid carcinoma However it needs more clinicaldata to verify the relationship between the expression ofIL6 and thyroid carcinoma tissueLeptin and thyroid carcinomaTwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] Because ofthe considerable heterogeneity of the pooled WMD ofserum leptin level heterogeneity test Chi2 P I2 and pooled OR of the ratio of leptinimmunoreactivity in tissues heterogeneity test Chi2 P I2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis Howeverthere is no association of higher level of serum leptin 0cZhao BMC Cancer Page of Fig Forest plot of the IL6 level and ratio of IL6 immunoreactivity in tissue in patients with thyroid carcinoma a Level of serum IL6 b Ratio ofIL6 immunoreactivity in tissueFig Forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a Level of serum leptin bRatio of leptin immunoreactivity in tissue 0cZhao BMC Cancer Page of with risk of thyroid carcinoma WMD 95CI to Fig 4a Moreover the pooled OR of theratio ofleptin immunoreactivity in tissues from fivestudies is 95CI to Fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaAdiponectin and thyroid carcinomaThree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in Table It could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects P Interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity However there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed Thus itneeds more clinical studies to confirm in the futurePublication biasThe funnel plot was applied for assessing publicationbias of studies included in the three results includingTNFα Fig 5a IL6 Fig 5b and leptin Fig 5c InFig 5a and Fig 5b almost all studies lies inside the95CIs with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot Howevera potential publication bias was found in Fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisDiscussionCurrently obesity affects one third of population amongUS adults [] and China has become a big country ofobesity with the incidence ranking first worldwide in theyear of [] Nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonHodgkins lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [] Becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma It was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [] Furthermore obesity is an independent risk factor for thyroid carcinoma[] Increased insulin resistance elevated serum cholesterol level and upregulated COX2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma [] It is reported that people with higherbody mass index have a higher concentration of adipokines [] Adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping So adipokines may be one of the targetslinking obesity with thyroid cancer The metaanalysiswas based on previous published studies In previousstudies the analysis of adiponectin and thyroid cancermostly focused on TNF IL6 Leptin and AdiponectinWhile few studies focused on other molecules includingIL1 and IL8 and we failed to combine statisticsTherefore in this metaanalysis only TNF IL6 Leptinand Adiponectin which are the most published adiponectin were analyzedTNFα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability Meanwhile TNFα plays animportant role in the process of inflammation insulinresistance diabetes and obesity A moderate amount ofTNFα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to TNFassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedTNFα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts [] which may play an important role in thyroidcancer Moreover TNFα can stimulate the vasoactivemediators such as interleukin and prostaglandin []and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function TNFα can also induce an increased expression ofvascular endothelial growth factor VEGF [] the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasisTable Summary of adiponectin expression in thyroid carcinomaserum adiponectin []ratio of adiponectin immunoreactivity []Effect sizeWMD OR adiponectin in tissue [] CI confidence interval WMD weighted mean differences OR odds ratiosWMD 95CI PI2Not applicable 0cZhao BMC Cancer Page of Fig Funnel plots of a TNFα b IL6 and c leptin revealed no significant publication bias SE SMD standard error of standardizedmean differenceIn surprisingly the results of clinical studies provide evidence for basic research Simonovic SZ [] evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with DTC before and days after radioactiveiodine 131Itherapy and control subjects andfound that the expression of TNFα in DTC patients ishigher than control subjects and it showed a decreasedlevel after 131I therapy than those before therapy However no statistical difference found for the limited sample size Another study conducted by Kobawala TP et al[] with more patients patients with benign thyroiddisease PTC patients and healthy individuals determined the circulating levels of TNFα and it wasfound that the serum level of TNFα was significantlyhigher in PTC patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals Furthermore serum TNFα was reported to be a significant prognosticator for overall survival in PTC patients It is a pity thatopposite result wasreported in a casecontrol study that included DTCcases and matched cancerfree cohort participantswhich found that TNFa was not associated with thyroidrisk in either gender []Based on current evidence our metaanalysis suggeststhat TNFα exhibit a strong association with thyroid carcinoma It may because that elevated TNFα may involved in the tumorigenesis and development of thyroidcancer Another possible reason is that the TNFα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomaThereforemore clincal studies and basic reseaches should be conducted in the futureIL6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells It is reported that elevated serum IL6 level is closely related tothe tumorigenesis and development of a variety of tumors [] A strong positive association between theserum IL6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [] Serum IL6 level in thyroid cancer has been evaluated in numerous studies including 0cZhao BMC Cancer Page of in vivo and in vitro studies Provatopoulou X []found that serum IL6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols However other studies show a different resultthat no significance different of IL6 was found betweenthyroid cancer and nonthyroid cancer [ ] A limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference For in vitro research IL6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof IL6 in PTC was confirmed indirectly []The underlying mechanism may be the followingsbelow Tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine IL6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors [] Also IL6R was found associated with the characterization of thyroid nodules malignancy and tumor aggressivenessIn additionIliopoulos D [] found that Src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating NFκB and thistransformation contributes to tumorigenesis IL6 is considered as an important regulatory factor in this processAnother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level[]In general the data above support that IL6 is important for thyroid cancer but the detail mechanism remainto be further studyLeptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus Meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney It is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin Moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway RhoALIMK1Cofilinpathway and MAPKERK pathway [] Kim WG et al[] evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer Thrb PVPV Pten mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byJAKSTAT3increased activation of the JAK2 signaling transducerand activator of STAT3 signaling pathway and inductionof STAT3 target gene expression Leptin is always reported a high expression on solid tumors [] and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly PTC while otherstudies showed a same results in cancer tissues [ ] Yu Xiao [] conducted a clinical studycomparing the level of serum leptin in PTC patientsincluding patients with lymph node metastasis and thyroid adenoma patients in Dalian China and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisLeptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue [] In addition it can also inhibit the apoptosisthrough Bcl2 dependent mechanism Meanwhile leptinreceptor exists in all thyroid cancer cells It is overexpressed in PTC and is involved in tumor invasion andlymph node metastasis [ ] Thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance Due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma The cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiesCompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance [] In addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia [] renal carcinoma [] gastric carcinoma [] and colon cancer[] Moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed []Otvos L Jr [] tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells Mitsiades N [] measured circulatingadiponectin levels in ptaients with PTC and found thatit is independently and inversely associated with the riskof thyroid cancer As the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof PTC Adiponectin receptor1 and are higher expression in PTC tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis []However other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0cZhao BMC Cancer Page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsFrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded And this may explain why increased incidence of obesity and thyroidcancer are consistent Thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefutureHowever some limitations in our metaana | 2 |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o | 0 |
" tumor mutational burden tmb has both prognostic value in resected nonsmall cell lung cancernsclc patients and predictive value for immunotherapy response however tmb evaluation by wholeexomesequencing wes is expensive and timeconsuming hampering its application in clinical practice in our study weaimed to construct a mutational burden estimation model with a small set of genes that could precisely estimatewestmb and at the same time has prognostic and predictive value for nsclc patientsmethods tmb estimation model was trained based on genomic data from nsclc samples from the cancergenome atlas tcga validation was performed using three independent cohorts including rizvi cohort and ourown asian cohorts including earlystage and n latestage asian nsclc patients respectively tcga data wereobtained on september the two asian cohort studies were performed from september to march pearsons correlation coefficient was used to assess the performance of estimated tmb with westmb thekaplanmeier survival analysis was applied to evaluate the association of estimated tmb with diseasefree survivaldfs overall survival os and response to antiprogrammed death1 pd1 and antiprogrammed deathligand pdl1 therapyresults the estimation model consisted of only genes correlated well with westmb both in the training setof tcga cohort and validation set of rizvi cohort and our own asian cohort estimated tmb by the 23gene panelwas significantly associated with dfs and os in patients with earlystage nsclc and could serve as a predictivebiomarker for antipd1 and antipdl1 treatment responsecontinued on next page correspondence zhangli6mailsysueducn jie_969163comzlhuxi163com yanhua tian jiachen xu and qian chu contributed equally to this work5state key laboratory of oncology in south china collaborative innovationcenter for cancer medicine sun yatsen university cancer center eastdong feng road guangzhou guangdong china1state key laboratory of molecular oncology department of medicaloncology national cancer centernational clinical research center forcancercancer hospital chinese academy of medical sciences and pekingunion medical college panjiayuan south lane chaoyang districtbeijing chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctian bmc medicine page of continued from previous pages the 23gene panel instead of wes or the currently used panelbased methods could be used toassess the westmb with a high relevance this customized targeted sequencing panel could be easily applied intoclinical practice to predict the immunotherapy response and prognosis of nsclckeywords tmb estimation 23gene panel prognostic and predictive value nonsmall cell lung cancertoimmuneinhibitorscheckpoint tumor mutational burden tmb commonly defined asthe number of nonsynonymous mutations has been proposed as a promising predictive biomarker for the responseicisimportantly this metric tightly correlates with overallsurvival os in resected nonsmall celllung cancernsclc patients in rizvi demonstratedthat an increased number of nonsynonymous mutationswere associated with improved objective response durable clinical benefit dcb and progressionfree survivalpfs in nsclc patients who received antiprogrameddeath pd1 therapy clinical studies have also revealed a significant correlation between tmb and objective response rate orr to icis in multiple tumortypes [] in addition devarakonda recently reported that high tmb was associated with a better survival prognosis in patients with resected nsclc andthe benefit of adjuvant chemotherapy was more pronounced in patients with low tmb the gold standards for tmb calculation are throughwholegenome sequencing wgs or wholeexome sequencing wes however several obstacles such as thehigh demand for quality and quantity of tissue samplesthe cost and time consumption and the unavailabilityfor translation to tmb evaluation by circulating tumordna ctdna in blood btmb hinder the clinicalapplication of these techniques as a result targetednext generation sequencing ngs of cancerrelatedgene panels cgp has been developed serving as surrogates for wes for tmb estimation to date the foodand drug administration fda has approved severalngs panels for tmb estimation eg foundationonecdx f1cdx and memorial sloan kettering cancercenters integrated mutation profiling of actionablecancer targets mskimpact which include about genes and cover over one megabase of codingdna [ ] recently many new ngs panels consistingof different numbers of genes have been developed andvalidated most of which were designed initially for guiding the use of target therapies these panels mainly include cancerrelated oncogenes and tumor suppressenes many of which do not contribute to or even negatively correlate with tmb thus are not accurate fortmb evaluation besides inclusion of these genes in anngs panel enlarges the panel size used for tmbis importantestimation and can lead to an inferior costeffective consequence itto note that cancer typespecific mutation load estimation models have proven tobe necessary because of the different mutation landscapes among varying tumor types although dnadamage repair ddr genes negatively predictive genesstk11 and keap1 and tmbassociated genes such asmuc16 pole pold1 and ttn have been included inthe ngs panels for tmb evaluation [] with theburgeoning developments in immunotherapy there is aneed for more specific panels that focus on tmb estimation for nsclcherein by using the cancer genome atlas tcgadatabase as a training set and multiple realworld cohorts as a validation set we constructed an optimizedtmb estimation model with the smallest number ofcarefully selected tmbassociated genes that could beused as both predictive markers for immunotherapy andprognosis biomarkers for resected nsclc patientsmethodspatient cohortsgenomic and clinical data for nsclc samples including lung adenocarcinoma luad and lungsquamous cell carcinoma lusc samples were downloaded from tcga database for the model constructionfor the validation of the model three independent cohorts were used including a previously published studythe rizvi cohort a surgery cohort composing of earlystage nsclc patients who underwent surgicaltreatment and a zs immunotherapy cohort composingof advanced nsclc patients who received ici treatment all the patients in the zs immunotherapy coreceived either antipd1 nivolumab n hortpembrolizumab n shr1210 n or antipdl1 atezolizumab n monotherapy agents there are patients who received durable clinical benefit dcbantipd1 n antipdl1 n and patientswith no durable benefit ndb antipd1 n antipdl1 n all three validation cohorts were used toevaluate the performance of the tmb estimation modeladditionally the surgery cohort was also used for survival validation in resected nsclc patients both therizvi and immunotherapy cohorts were also used forvalidation of ici outcome predictability in advancednsclc patients the clinical details for all enrolled 0ctian bmc medicine page of patients were collected the treatment efficacy for thosetreated with immunotherapy was assessed using response evaluation criteria in solid tumors recistversion with durable clinical benefit dcb definedas partial or stable disease lasting over months allprocedures were approved by the ethics committees ofthe national cancer center all patients provided written informed consentwholeexome sequencing and data processingwe performed wholeexome sequencing of samplesfrom two cohorts in the validation setincluding earlystage nsclc patients who underwent surgicaltreatment and advanced nsclc patients who received ici treatment for those earlystage nsclcpatients both tumor and matched normal samples wereobtained and subjected to wes briefly dna librarieswere prepared using the mgieasy exome capture v4probe set capture kit cat no with a capture region size of mb bgiseq instruments wereused for pairend sequencing à bp the data wereprocessed according to the manufacturers protocol the mean coverage was à and à in tumor andnormal samples respectivelyfor those advanced nsclc patients biopsy specimens were available for wes the genomic dna wasextracted using the qiaamp dna ffpe tissue kit andquantified using the dsdna hs assay kit thermofisher scientific usa libraries were constructed withthe kapa hyper prep kit kapa biosystems usa anillumina hiseq4000 platform was used for sequencingwith pe150 sequencing chemistry illumina usa the average coverage depth was Ãcandidate gene selectiongenomic data for nsclc samples from tcgawere used for candidate gene selection which were usedto construct the mutation load estimation model thecandidate genes were selected based on two criteria mutation frequency higher than or equal to and significant association with mutation load the mutationfrequency of a gene was calculated as the percentage ofpatients with mutation in the gene mutation loadassociated genes were defined as where the westmbwas significantly different between the patients with themutated gene and those with wildtype counterpartsadditional file table s1mutation estimation model constructionthe mutation estimation model construction was basedon tcga data in the training set in detail the first stepwas to build a mutation estimation model using thefewest genes which tightly associated with westmbin our study we constructed the estimation model bysimply randomly selecting a specified number of genesfrom allthe genes or tmbassociated genes andsummed the mutational number as the estimated tmbunder every given number of genes the procedure wasrepeated times resulting in random modelswe then calculated the pearson correlation coefficientr between the estimated and actual mutation load ofwestmb the results allowed us to select the modelwith highest r under the specified number of genes thenext step was to identify which of those best modelsunder the specified number of genes correlated with theclinical outcomes of overall survival os and diseasefree survival dfs the final step was to select a modelusing the fewest genes that tightly associate with thewestmb and have both prognostic value for thoseearlystage nsclc patients and predictive value forthose latestage nsclc patients who received icitreatmentrna expression difference between tmb high and lowgroupsto compare gene expression patterns we downloadedan mrna data set of nsclc patients from tcgadatabase mrna expression was analyzed using gene setenrichment analysis gsea httpsoftwarebroadinstitutegseaindexjsp we divided these patientsinto estimated high ¥ mutational counts and lowtmb groups mutational counts and identifiedwhether immunerelated gene signatures associated withtumor mutation status the genes found to be on theleading edge of the enrichment profile were subjected topathway analysis genes with expression over in morethan of the samples were included in the gseathe normalized enrichment score nes is generally theprimary statistic for examining gene set enrichmentresultsstatistical analysisthe mannwhitney u test was used to assess thedifferences in the mutation load between the twogroups the genes with kruskalwalliscorrected pvalues lower than were identified as the mutationloadassociated genes and selected as potential candidate genes survival analysis was performed using thekaplanmeier curves with a p value determined by alogrank test and the statistical tests were twosidedand considered statistically significant at p unless otherwise stated the analyses were performedusing graphpad prism version graphpad prismcorrelations between estimated mutation burden andwholeexome sequencingcalculated tmb were determinedcorrelation coefficient theanalyses were performed using r353by pearsons 0ctian bmc medicine page of resultscandidate gene selection for model constructionthe flowchart of the construction of estimation model isshown in fig s1 in additional file the somatic mutation data of cases of nsclc were downloaded fromtcga database as the training set tcga cohort including adenocarcinoma and squamous cell carcinoma subtypes of nsclc additional file table s2subsequently a mutation matrix including screened nonsynonymous mutations in genes was generatedfurthermore we identified genetic alterations in genes with mutation frequency ¥ in general nsclcpatients and significantly correlating with westmb pvalue range 695e to 452e these genes werethen used as candidate genes for the construction of thetmb estimation model additional file table s3construction of the tmb estimation modelgenes used for the tmb estimation model were randomly selected from the candidate genes and theserialrandom models theestimated tmb was defined as the sum of all nonsynonymous mutation counts of the selected genes undereach specified number of abstracted genes the procedure was repeated times thus resulting in separatecorrelations ofestimated tmb by these random models and westmbwere evaluated using the pearson correlation coefficientr as expected the correlations between the estimationmodels and westmb increased with the number ofgenes fig 1a b additional file fig s2a b compared with unselected genes in the range of genomicgenes the estimated tmb based on selected geneswas significantly more closely associated with westmb in terms of either the mean or the maximum rfig 1c d additional file fig s2c d the maximumr increased from with one gene included to greaterthan with genes included and then reached aplateau when the included gene number exceeded the r values were comparable though increased slowlyas the number increased fig 1b we asserted that rfig the correlation of westmb and tmb as estimated by different gene panels a b correlation is represented by the pearson correlationcoefficient r genes used for the mutation model construction were either from unselected genes a or from selected genes b that correlatewith westmb c d comparisons of mean c and maximum d r of estimated tmb and westmb using unselected genes or selected genes 0ctian bmc medicine page of greater than in the estimation models was acceptable as such we considered a model with this effectbut including the least number of genes an ideal modelfor clinical applicationin reference to previous reports that tmb is associated with prognosis in patients with resected nsclcsthe optimal tmb estimation model was further evaluated based on the correlation of estimated tmb with osand dfs in models with r over ultimately we constructed an estimated tmb model with only genesand r of p fig 2a additional file which was significantly associated with both os anddfs fig 2b c the cutoff value of the estimated tmbby the 23gene panel was defined as mutational countsthe median value of estimated tmb based on tcgadatabase additional file fig s3a b that were equalor over mutational counts as tmbhigh cases and lessthan mutational counts as tmblow ones these genesincluded unc13c hmcn1 znf536 kmt2d ush2axirp2 pcdh15 ahnak2 adgrl3 reln nf1 ttnadgrg4 cubn cacna1e mrc1 col11a1 nav3csmd1 apob csmd3 col22a1and epha5additional file table s4 the model yielded goodperformances in both subtypes of nsclc with correlations of for luad additional file fig s4aand for lusc additional file fig s4b theaverage cds length of these genes was 12k nucleotides 3k80k additional file table s4 and the totallength was 028m nucleotides which was considered tobe a great reduction of sequencing cost for mutationload estimation we concluded that the 23gene panel isthe ideal model based on tcga training setanalytic validation of the 23gene panel in asian resectednsclc patientsto validate the performance of the estimation model weconducted wes on chinese stage iaiiia nsclcpatients after radical pneumonectomy surgery cohortadditional file table s1 the correlation of 23genetmb with wes was r p fig 3a asshown in fig 3b tmbhigh ¥ mutational counts according to the 23gene panel associated with a betterdfs compared with those with tmblow logrank p besides a tendency towards improved os wasobserved in the patients with higher estimated tmbthough a statistical difference was not reached due tothe fact that most patients were still alive fig 3cperformance verification by comparing the 23gene panelwith other commercial panelsnext we compared the 23gene panel with two commercial panels based the earlystage nsclc data including f1cdx genes and mskimpact genes there are two overlap genes between the gene panel with f1cdx and mskimpact namelynf1 and epha5 the 23gene tmb has a tight correlation with the tmb estimated by f1cdx f1cdxtmbor mskimpact msktmb r and respectively both p fig 4a b in additionwhen the genes were added to the two commercialpanels the correlation of the incorporated panels withwestmb significantly increased from ci to ci p for f1cdx fig 4c d and from ci to ci p for mskimpact fig 4e f to further verify thespecificity of these 23gene panels we compared themwith other randomly selected gene panels from the genes the procedure was repeated timesresulting in the random pearson correlation coefficientsfrom to of f1cdx plus random genesand from to of msk plus random genes the performance of our 23gene model was better than of random models which indicated the irreplaceability of these genesfig tmb estimation model construction based on tcga data in the training set a the correlation of 23gene tmb with westmb is with an empirical p value of r of p b the overall survival is significantly higher in the tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the diseasefree survival is significantly higher in thetmbhigh group than in the tmblow group with logrank test p 0ctian bmc medicine page of fig validation of the tmb estimation model based on the earlystage nsclc patients in the validation set a the pearson correlationcoefficient of estimated tmb by the 23gene panel and westmb is with an empirical p value of r of p b the diseasefree survivalis higher in the estimated tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the overall survival is comparable in the two groups with logrank test p fig performance evaluation of the 23gene panel against commercially used gene panels a b the pearson correlation coefficient of 23genetmb with f1cdxtmb a and msktmb b c d the pearson correlation coefficient of westmb with f1cdxtmb c and incorporated panel of cancerassociated genes in f1cdx with 23gene panel f1cdx genetmb d e f the pearson correlation coefficient of westmb withmsktmb e and incorporated panel of cancerassociated genes in mskimpact with genepanel msk genetmb f 0ctian bmc medicine page of f1cdxbased on the survival data from our earlystagensclc patients significant correlations were observedbetween survival outcomes dfs and the tmb levelstratified withor mskimpact paneladditional file fig s5a c interestingly the genescould improve the association of these two commercialpanels with dfs additional file fig s5b d if the incorporated panels were used for analysis tmbhigh estimated by both of the two new panels f1cdx 23genepanel or mskimpact 23gene panel demonstratedimproved dfs compared with those of estimated tmblow under the cutoff values indicated in fig s3c and s5of additional file immuneregulatory gene expression signatures stratifiedby tmb level based on the 23gene panelto investigate the difference in immune status betweentmbhigh and tmblow estimated by the 23genepanel we analyzed immuneregulatory gene expressionsignatures based on the rnaseq data of nsclccases from tcga the gsea revealed a prominent enrichment of mrna signatures involved in the inflammainterferonα γ ifnα γtoryresponse tnfαresponse il6jakstat3 signaling and allograft rejection fig immunotherapy response prediction by the established23gene panelfinally we analyzed the performance of tmb estimatedby the 23gene panel in the prediction of response toicis using two independent nsclc cohorts in therizvi cohort the correlation between the tmb estimatedby the 23gene panel and wes was empirical pvalue of r fig 6a the estimated tmb was significantly different between the patients with durableclinical benefit dcb a partial or stable response lastingover months and no durable benefit ndb mannwhitney p fig 6b survival analysis was thenapplied for the comparison of the pfs between the patients n with tmbhigh ¥ counts and tmblow counts by the 23gene panel patients withtmbhigh demonstrated significantly improved pfscompared with those with tmblow vs months logrank p fig 6cto further validate the performance of the estimationmodel for response to icis we performed wes of fig gene expression differences between the estimated high tmb and low tmb groups af tmbassociated pathways such as inflammatoryresponse tnfα signaling via nfκb interferon α response il6jakstat3 signaling interferon γ response and allograft rejection nes normalizedenrichment score fdr false discovery rate 0ctian bmc medicine page of fig immunotherapy response estimation by the 23gene panel a the correlation of the estimated tmb with westmb using the rizvi datan b estimated tmb in tumors from patients with dcb n or with ndb n mannwhitney p c pfs in tumors withestimated tmbhigh n compared to tumors with tmblow n in patients in the rizvi cohort hr ci to logrank p d the correlation of estimated tmb with westmb using the latestage nsclc patient cohort n e estimated tmb in tumors frompatients with dcb n or with ndb n mannwhitney p f pfs in tumors with estimated tmbhigh n compared totumors with tmblow n in patients in the latestage nsclc patient cohort hr ci to logrank p advanced stage iiibiv nsclcs in another asian cohort zs immunotherapy cohort all of these patients received with antipd1 or antipdl1 treatmentthe r between the estimated and actual mutation burden was calculated to be empirical p value of r fig 6d the estimated tmb was significantlydifferent between the patients with dcb and ndbmannwhitney p fig 6e the pfs was associated with estimated tmb logrank p fig 6fdemonstrating that the estimated mutation burden derived from caucasian nsclcs from tcga could predict the immunotherapy treatment response quite wellin asian patients we further calculated the hr at different cutoff values in the zs immunotherapy cohort andfound the mutational counts in this cohort resultedthe best hr value additional file fig s6 as a resultwhen applied in clinical practice the cutoff value stillneeds to be further evaluated accordinglycomparison of the 23gene panel with previouslyreported tmbrelated genesmutations in ttn muc16 pole and pold1 havebeen previously reported to correlate with elevated tmblevels [] the frequencies ofthese genes innsclc based on cases from tcga were and respectively westmb was significantly different between the patients with these mutatedandthose with wildtypegenescounterpartsadditional file fig s7 however only muc16 mutations exhibit significant correlation with os and dfs intcga cohort additional file fig s7ac while theyfailed to confirm the results in our surgery cohortadditional file fig s8 notably none of these genemutations could predict the response or pfs in eitherthe rizvicohortadditional file fig s9cohort or ourimmunotherapydiscussionin the present study we developed a novel and optimaltmb estimation model composed of only geneswhich allowed precise estimation ofthe wesbasedtmb both in earlystage and latestage nsclc patientsimportantly our established 23gene panel can successfully predict the survival outcomes in both resectednsclcs and patients receiving icis in multiple validation cohorts to the best of our knowledge our tmbestimation model is both the first and the smallest paneldescribed to date which can be used as a biomarker tostratify patients not only after radical pneumonectomybut also with advanced nsclc receiving icisthe total cds length of the 23gene panel was 028mnucleotides with an average of 12k 3k80k the ttnis also included in our panel although it has the longestcds length of 81k the total length was acceptable when 0ctian bmc medicine page of ttn is included besides in a recent study ttn mutation was reported to be associated with tmb in solid tumors including nsclc and correlated with response toicis as a result the 23gene panel was consideredto be a great reduction of sequencing cost for mutationload estimationseveral cancerrelated genes have been previously reported to be associated with westmb in some cancertypes for example melanoma patients with lrp1b mutations exhibited a higher mutationalload than thosewith the wildtype gene li reported that mutations in muc16 are associated with tmb and survivaloutcomes in patients with gastric cancer twoddrrelated genes pole and pold1 were also shownto correlate well with westmb in pancancer types undoubtedly it would be ideal to utilize a singlegene to estimate tmb and effectively predict responseto immunotherapy however we found that singly allthese genes failed to correlate well with the efficacy oficis or survival outcomes after resection the correlationof any individual gene with westmb was moderatemean r these results indicate thatusing a single gene to estimate tmb is insufficienttheoretically the larger a ngs gene panel the closerthe estimated tmb is to the actual amount howeverthe costeffective balance for clinical usage must be considered in particular when tmb is detected using peripheral blood super sequencing depth eg à due to the low abundance of circulating tumordna will significantly drive up the cost to datetwo commercial gene panels f1cdx and mskimpact have been widely used for tmb estimation thesetwo panels demonstrate good performance in correlationwith westmb our established gene panel whichincludes a very limited number of genes demonstratedcomparable correlation coefficients with these two largepanelsindicating the promising reliability of a smallpanel as a surrogate for westmb notably the majority of genes used in our model were not included in thecurrently used commercial gene panels if the genes inour panel were incorporated into the big commercialgene panels the correlation coefficients with westmbincreased these results demonstrate that the geneswe have selected here may be used independently or ascomplement to the currently used gene panels specificfor nsclc inclusion of the genes should be considered in future ngs gene panelsrecently lyu developed a small gene panel with genes to estimate actual tmb derived from luads in tcga database the construction and validation cohorts used for lyu s 24gene panel weremainly from caucasian patients however our 23genepanel though also derived from tcga database wassuccessfully validated in multiple asian patient cohortsthese results suggest that our 23gene panel may bemore suitable to nsclc and applicably potent regardless of race and subtypes additional file fig s10similar with the findings of devarakonda weobserved that high tmb associated with improved os inresected nsclc patients in colon cancer patients withresected stage ii mismatch repair deficiency high tmbhas been utilized as a good prognostic biomarker indeed these results possess internal rationality both highneoepitope burden and intense til infiltration have been associated with favorable survival outcomes inearlystage lung cancer high tmb may reflect the immunogenicity in some degree which could mediate theshaping of tumorhost immune interactions taken together these and our findings suggest that quantifyinggenomic instability through tmb estimation can be usedto stratify patients so as to guide adjuvant treatmentowing to the lack of information on hlai it is difficultto judge whether the predictive value of our gene panel isdue to neoantigen generation derived from the includedgene mutations or if the estimated tmb based on the gene panel is simply a representative reflection of genomicinstability as an accompanying passenger the otherlimitation of our study is the small number of patientswho received the immunotherapy treatment thus a larger number of cases from a multicenter study are requiredfor the validation of the performance of the treatment response prediction in addition our validation cohorts wereretrospective a prospective study is necessary to translateour estimation model into clinical practice in addition totmb other features such as pdl1 expression microsatellite instability and neoantigen burden have emerged aspotential predictive biomarkers for icis [] howeverchallenges in defining cutoff valuesintertumoral andintratumoral heterogeneity and test platform uniformitieshave limited their clinical applications therefore future strategies that combine different predictive featuresmay be more effective biomarkers for the accurate prediction of cancer immunotherapy response but need tobe carefully integratedsin summary we have successfully constructed a noveltmb estimation model using only genes that can beused to estimate the westmb and stratify survivalprognosis after radical surgery and clinical outcomes ofici therapy in nsclc patients thus a customized panelfor the targeted sequencing of these selected genes instead of wholeexome sequencing can be designed or utilized as complementary genes included in the currentngs panels consequently by using our model the costeffectiveness may be considerably improved makingrealization of cancer immunotherapy response more accessible in standard clinical settings 0ctian bmc medicine page of supplementary informationsupplementary information accompanies this paper at httpsdoi101186s12916020016948competing interestsno potential conflicts of interest were disclosed by the authorsadditional file table s1 data sets used to calculate westmb forthe study cohorts table s2 characteristics of the patients included inthis study table s3 candidate genes and related information tables4 genes and the corresponding cds length fig s1 flowchart ofthe construction of estimation model fig s2 the correlation of westmb and tmb as estimated by different gene panels fig s3 forestplots of hrs for os and dfs in the tcga and earlystage nsclcpatients study cohort fig s4 the performance of 23gene based tmbestimation model for the luad and lusc subtypes of nsclc tcgadata fig s5 forest plots of hrs for dfs in the earlystage nsclcpatients study cohort fig s6 forest plots of hrs for pfs of the nsclc patients in zs immunotherapy cohort fig s7 westmb is shownbased on muc16 a ttn b and pole c mutation status fig s8 thecorrelation of muc16 mutation status with overall survival a anddiseasefree survival b based on the earlystage nsclc patients figs9 the correlation of muc16 ttn and pold1 mutation status withprogressionfree survival pfs based on the rizvi cohort and ourimmunotherapy cohort fig s10 comparison of predictive performanceof response to icis by our 23gene panel with lyus 24gene paneladditional file the correlation of estimation models with genenumber to with os and dfs in the training setacknowledgementswe thank all patients that were involved in this study we also thankguoqiang wang jing zhao and shangli cai the medical department 3dmedicines inc shanghai peoples republic of china for their contribution tothe st | 0 |
this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples a blanka control a blank the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisb ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa \uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at °c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aba where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlichs hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturers instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ct ct is the difference between the mean ctsample and mean ctcontrol where ctsample is the difference between the mean ctsample and the mean ctβactin and ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferronis posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a also it displays intact cancer cells arrow and giant cells arrow figure 3b and c the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d also displays a notable necrosis of cancer cells n figure 3e the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m and a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f and 4g however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h and 3i the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101a ± 217ab ± 201b ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1 | 0 |
since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus sarscov2 was identified as the pathogen to cause pneumonia as a member of the betacoronavirus genus sarscov2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus sarscov and shares amino acid sequence identity with sarscov the world health anization who named the disease caused by sarscov2 as coronavirus disease covid19 until april the virus has swept through countries more than million cases with covid19 have been confirmed and more than cases died which has been posing significant threats to public health sarscov2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ards multiple an failure and even death in severe cases in addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and abbreviations ace2 angiotensin converting enzyme aki acute kidney injury ali acute liver injury alp alkaline phosphatase als artificial liver system alt alanine aminotransferase ami acute myocardial infarction ards acute respiratory distress syndrome ast aspartate aminotransferase at2 alveolar cells bun blood urea nitrogen ccle cancer cell line encyclopedia cns central nervous system covid19 coronavirus disease geo gene expression omnibus ggt gammaglutamyltransferase gi gastrointestinal injury gtex genotypetissue expression icu intensive care unit mcs mechanical circulatory support np nucleoprotein pci percutaneous coronary intervention sarscov2 severe acute respiratory syndrome coronavirus scr serum creatinine scrnaseq single cell rna sequencing stemi stelevation myocardial infarction tbil total bilirubin tem transmission electronic microscope tmprss2 transmembrane protease serine vv venousvenous who world health anization corresponding authors at department of infectious disease and institute of hepatology qingdao municipal hospital qingdao university digestive disease key laboratory of qingdao qingdao china email addresses xinyongning9812163com y xin zlk0823163com l zhuang 101016jbiopha2020110678 received june received in revised form august accepted august biomedicinepharmacotherapy1312020110678availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [] fig in this section the expression levels of ace2 and tmprss2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed heart m dong nervous system in the early stages of covid19 which brings more challenges to the timely diagnosis of patients angiotensin converting enzyme ace2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase the primary physiological role of ace2 is involved in the regulation of vasoconstriction and blood pressure [] transmembrane protease serine type2 tmprss2 belonging to the type ii transmembrane serine protease family could cleave the coronavirus spike s protein [] it was demonstrated that ace2 and tmprss2 were crucial for the entry of sarscov and sarscov2 into the host cells cell entry of sarscov2 depends on binding of the s protein to the specific cellular receptor and s protein priming by host cell proteases as shown in fig each s protein of sarscov2 consists of two subunits a globular s1 domain at the nterminal region and the membraneproximal s2 domain sarscov2 utilizes receptorbinding domain within the s1 domain to bind to the cellular receptor ace2 which could trigger the effects of tmprss2 on the cleavage of protein s at the s1 and s2 sites and priming cell membrane fusion for viral entry as receptors and mediators of virus entry are important for determining viral host and an the route of sarscov2 infection and the infected an may depend on the expression and distribution of ace2 and tmprss2 studies have shown that ace2 and tmprss2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that sarscov2 may also affect extrapulmonary ans [] in this review the distributions of ace2 and tmprss2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by sarscov2 were summarized we believe that this will be important in understanding on the infection of extrapulmonary ans in patients with covid19 the mrna expressions of ace2 and tmprss2 in extrapulmonary ans the mrna expressions of ace2 in different human ans were analyzed and the results showed that ace2 was expressed in the heart furthermore chen analyzed the feature of ace2 expressions among cardiac cell types and found that ace2 was specifically expressed in pericyte moreover rna sequencing from patients with failing hearts and normal donors revealed that myocardial ace2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors another study also showed that the expression of ace2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues these two studies suggested that the expression of ace2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue guo et al analyzed the mrna expression of tmprss2 from the genotypetissue expression gtex database and the results showed that tmprss2 is also expressed in the heart by singlecell rna sequencing scrnaseq to profile the gene expression landscapes of cardiac cells from human embryos qi revealed that the cardiomyocytes from the heart contain ace2expressed cells and tmprss2expressed cells and the cardiovascular progenitor cells and cells tmprss2expressed cells respectively these data showed that both ace2 and tmprss2 were expressed in the heart contain ace2expressed kidney studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of sarscov infection studies have shown that ace2 and tmprss2 were expression analysis from the gtex database showed that kidney displayed the fifth high expression of ace2 to investigate the expression of ace2 in kidney lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the fig entry of sarscov2 into host cells sarscov2 infected the host cells by the spike protein of the virus and the functions of ace2 and tmprss2 in host cells biomedicinepharmacotherapy13120201106782 0cm dong fig tissue distributions of ace2 and tmprss2 in human ab the schematic diagram of the expressions of ace2 a and tmprss2 b in multiple human tissues the colour strength is corresponding to the gene expression level ace2 and tmprss2 were expressed in the brain and heart ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in the hepatocytes and cholangiocytes ace2 and tmprss2 were highly expressed in kidney and intestinal epithelial cells both ace2 and tmprss2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ace2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells fan et al confirmed the specific ace2 expression in tubular cells from the gene expression omnibus geo dataset while it was not observed in immune cells and glomerular parietal epithelial cells rna and protein expression data of ace2 in different human tissues and cancer cell lines were obtained from three online datasets including the cancer cell line encyclopedia ccle gtex database and the human protein atlas dataset and the results indicated that both mrna and protein expression levels of ace2 were relatively high in kidney cells especially in renal tubular cells meanwhile suryawanshi analyzed the data of kidney tissues in scrnaseq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ace2 and tmprss2 the data of the scrnaseq from geo dataset gse134355 showed that ace2 and tmprss2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney recently pan also found that the tmprss2 gene was coexpressed with ace2 in kidney podocytes these data showed that both ace2 and tmprss2 were highly expressed in tissues and cells of kidney liver chai et al analyzed the scrnaseq data from geo database gse124395 to evaluate ace2 gene expression in liver the results showed that ace2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes the gtex database also showed that both ace2 and tmprss2 were expressed in the liver zhou identified that tmprss2 is highly expressed in hepatocytes from human cell atlas database recently wen indicated that ace2 and tmprss2 are specifically coexpression in trop2 liver progenitors of human liver tissue using scrna sequencing these data indicate that ace2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while tmprss2 is expressed in hepatocytes digestive tract a previous study showed that ace2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon quantitative mrna expression profiling of ace2 across human tissues by harmer showed that ace2 was expressed at a high level in gastrointestinal tissues zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ace2 was not only highly expressed in the type ii alveolar cells at2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ace2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells besides the scrnaseq data showed that ace2 was significantly elevated in the proximal and distal enterocytes guo et al suggested that tmprss2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus using published scrnaseq data and seven inhouse normal colon samples lee reported that the coexpressions of ace2 and tmprss2 transcripts were mainly observed in the small intestine and colon the highest expressions of tmprss2 and ace2 were found in enterocytes among the intestinal cell types these data showed that tmprss2 and aec2 are highly expressed in the digestive tract nervous system analysis using the gtex database showed that both tmprss2 and ace2 are expressed at relatively low levels in the brain cortex chen found that ace2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases ace2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex qi analyzed the scrnaseq data of substantia nigra biomedicinepharmacotherapy13120201106783 0cclinical classification of acute cardiac injury nonicucases icu cases nonicucases icucases nonsevere cases severecases1965 recoveredcases died cases nonicucases icucases survivor cases nonsurvivor cases chen hong zhou china korea china m dong and cortex of brain from geo database the results showed that both ace2 and tmprss2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex there are limited reports on the expressions of ace2 and tmprss2 in peripheral nervous system brann analyzed the ace2 and tmprss2 expression in different cell type from human scrnaseq dataset gse139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ace2 and tmprss2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes these data showed that ace2 and tmprss2 could also be coexpressed in the nervous system other ans or tissues table characteristics of acute cardiac injury after sarscid0 cov2 infection study basic heart disease acute cardiac injury country subject china wang china na huang li china moreover ace2 and tmprss2 were also reported to be coexpressed in some other ans it has been revealed that both ace2 and tmprss2 are expressed in testis by scrna sequencing and expression profile analysis indicating that testicular cells might be the potential targets of sarscov2 another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ace2expressed and tmprss2expressed cell clusters moreover ace2 and tmprss2 were also expressed in pancreas breast prostate and thyroid and these ans might also be the targets of sarscov2 infection of sarscov2 and extrapulmonary an injury of patients with covid19 sarscov2 infection and cardiac injury recently autopsy analysis by fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis sarscov2 rna was detected in the cardiac tissues of the patients with covid19 these data suggested that sarscov2 may directly infect heart the epidemiology of covid19 reported that cardiac injury was one of the most severe an damages the clinical manifestations of cardiac injury in covid19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction ami [ ] inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for sarscov2 using nucleic acid test cardiac injury is a common symptom in patients with covid19 shi reported that patients with covid19 had cardiac injury moreover there were patients with acute cardiac injury in a cohort including covid19 patients and of patients with acute cardiac injury in the intensive care unit icu furthermore a study by wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of covid19 patients while acute cardiac injury was observed in of patients with civid19 in the icu these cases suggested that sarscov2 may cause serious heart damage which should be widespreadly concerned furthermore acute cardiac injury is more prevalent in severe cases with covid19 [] table and it has been reported that covid19 patients with cardiac injury had higher mortality than those without cardiac injury in this review we also summarized the possible relationship between basic heart disease and further cardiac injury [] table in a cohort of covid19 patients from renmin hospital of wuhan university china shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure and only patients with basic heart disease of covid19 patients without cardiac injury similarly liu suggested that patients with basic heart disease in covid19 patients had table comorbidity with cardiac injury in covid19 patients with basic heart disease subjects with covid19 proportion of basic heart disease patients with cardiacinjury study shi liu xu ma guo patients with basic heart disease with cardiac injury without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of covid19 patients without cardiac injury other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in covid19 patients [ ] in view of the points above covid19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury sarscov2 infection and kidney injury recently autopsy analysis on six covid19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens nucleoprotein np antigens and np positive inclusion body of sarscov2 could be seen in kidney tissues from all the samples moreover viruslike ps were seen in kidney tissues by transmission electronic microscope tem su analyzed kidney abnormalities in autopsies of patients with covid19 and found that diffuse proximal tubular damage with the loss of brush border were biomedicinepharmacotherapy13120201106784 0c sarscov2 infection and liver injury m dong observed further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes it was reported that both np antigens and rna of sarscov2 were detected in urine of covid19 patients these data coincide with the finding of the sarscov2 invasion in kidney collectively sarscov2 could directly infect human renal tubules and lead to kidney damage recent studies have shown that the incidence of acute kidney injury aki in covid19 patients ranged from and higher frequency of renal function damage with elevated blood urea nitrogen bun or serum creatinine scr was observed in covid19 patients [ ] table a study of patients with covid19 indicated that levels of bun and scr were increased in and patients with covid19 respectively and routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria another study also showed that about patients with covid19 had abnormal renal function moreover covid19 patients with more severe disease progression have higher rates of aki huang and colleagues reported that of patients with aki in the icu were observed and none of the patients who did not require care in the icu suffered aki xu found that the fatality rate was obviously higher in covid19 patients with aki than those without renal injury furthermore in another study investigating patients with covid19 at hospital admission more severe patients had higher rates of aki and the cox regression analysis also suggested that covid19 patients who developed aki had a significantly higher mortality risk therefore aki is more prevalent in severe cases with covid19 an autopsy report of a 50yearold patient with covid19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues moreover zhao used human liver ductal anoids as a tool to investigate the sarscov2 infection and the tissue damage induced by sarscov2 ex vivo and the results showed that the expression of sarscov2 np was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to sarscov2 infection in addition sarscov2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that sarscov2 might directly induce cholangiocyte injury and consequently bile acid accumulation in view of the points above liver damage in the covid19 patients might be directly caused by the viral infection abnormal liver functions were frequently reported in covid19 patients epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [ ] table chen reported that out of patients had elevated alanine aminotransferase alt patients had elevated aspartate aminotransferase ast and had elevated total bilirubin tbil in wuhan jinyintan hospital wuhan china similarly a nationwide study involving patients with covid19 in china showed that more than of patients had elevated alt and ast and of patients had elevated tbil it was revealed that the levels of direct bilirubin indirect bilirubin alt alkaline phosphatase alp and gammaglutamyltransferase ggt were significantly higher in males than that in females with covid19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ali in covid19 patients these data indicated that male patients with covid19 may be more susceptible to liver injury furthermore table characteristics of acute kidney injury after sarscid0 cov2 infection study chen wang huang guan xu preexisting kidney conditions na na na country china china china china china subject li chen hong cheng xiao richardson wan li qian pei china china korea china china america china china china china na na na na na na scr serum creatinine bun blood urea nitrogen aki acute kidney injury abnormal renal functional indices scr bun na scr scr scr scr bun na scr bun scr na na na scr bun scr na aki na clinical classification of aki na nonicu cases icu cases icu cases nonsevere cases severe cases mild cases severe cases critical ill cases nonsevere cases severe cases recovered cases died cases nonicu cases icu cases na nonsevere cases severe cases cured cases in hospital cases died cases mild cases severe cases nonsevere cases severe cases na moderate cases severe cases critically ill cases biomedicinepharmacotherapy13120201106785 0cm dong table characteristics of liver injury after sarscid0 cov2 infection study country subject china china china china china china china korea america china china chen wang huang guan xu li chen hong richardson et wan li al qian china na patients with preexisting liverconditions na na na patients with abnormal liver functional indices alt ast tbil na ast ast alt tbil alt ast alt ast tbil alt ast alt ast tbil ast alt ast alt ast tbil alt ast abnormal liver functional indices in the nonsevere patients ast alt na na tbil na abnormal liver functional indices in the severe patients alt na tbil na ast na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na na nonsevere patients include patients without icu care and recovered patients severe patients include patients with icu care and death alt alanine aminotransferase ast aspartate aminotransferase tbil total bilirubin multiple studies found that ast alt and tbil were significantly higher in patients treated in the icu than that in nonicu patients li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively fu analyzed the relationship between ali and mortality risk in covid19 patients and the results showed that ali is more common in the critically ill patients and ali at the early stage increased death risk of covid19 patients together abnormal liver functions might be associated with the severity of patients with covid19 sarscov2 infection and digestive tract injury epithelial cells of the esophagus stomach duodenum and rectum in one covid19 patient tested positive for sarscov2 rna and the staining of viral np was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum moreover minimally invasive autopsies were performed on three patients died of covid19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached these studies strongly supported that sarscov2 may directly infect the epithelial cells of digestive tract table sarscid0 cov2 detection in gastrointestinal specimens study subject xiao zhang tan xing young holshue lescure tang wang xu gastrointestinal samples stool anal swabs rectal swab stool stool stool stool stool stool rectal swabs tested positive in gastrointestinal specimens the positive time in gastrointestinal specimens days na 6cid0 1cid0 5cid0 na 3cid0 positive time for gastrointestinal samples after respiratory samples were negative days na na 8cid0 na na na na na 2cid0 biomedicinepharmacotherapy13120201106786 0cm dong multiple studies have identified that the sarscov2 rna was detected in anal swabs rectal swabs and stool specimens [ ] of covid19 patients it has been demonstrated that sarscov2 rna could be detected in feces from more than half of covid19 patients in another study xing reported that sarscov2 rna was detected in the feces of three pediatric cases with covid19 in qingdao china and the persistence of sarscov2 in the digestive tract lasted for 6cid0 days the possibility of fecaloral transmission of sarscov2 infection needs to be taken into account furthermore as shown in table long duration of sarscov2 detection in digestive tract by rtpcr has been reported and viral rna remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [] the studies suggested that sarscov2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus some covid19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain holshue reported the first case of covid19 patient in the usa which had nausea and vomiting before admission multiple studies found that gastrointestinal symptoms including diarrhea nausea and vomiting and abdominal pain were common at presentation in covid19 patients [ ] table in a cohort of patients with covid19 in wuhan china gastrointestinal symptoms were described in up to moreover sun showed that critically ill patients with covid19 had gastrointestinal injury gi during hospital stay and the survival curves showed that the mortalities of patients with gi was greater than that of patients without gi jin also found that the rate of the severe type was markedly higher in covid19 patients with gi symptoms than that in those without gi symptoms these data suggested that gi is one of the common extrapulmonary an injuries in covid19 patients and may be related to the severity of the disease on the other hand many studies showed that patients with covid19 could present initially with the typical gastrointestinal symptoms and diarrhea may even occur earlier than pyrexia or respiratory symptom in some cases with covid19 [] luo reported that of covid19 cases presented initially only with gastrointestinal symptoms the covid19 patients initially only with gastrointestinal symptoms are more difficult to diagnose and might be overlooked which could lead to potentially serious consequences together digestive tract symptoms especially diarrhea are the main complications of covd19 patients which should be noticed during the outbreak of covid19 sarscov2 infection and nervous system injury transmission electron microscopy of autopsy sections showed the presence of sarscov2 virallike ps in frontal lobe brain and neural cell bodies moreover researchers confirmed the presence of sarscov2 in cerebrospinal fluid by genome sequencing the pathological mechanism may be the invasion of sarscov2 into the nervons system the virallike ps in brain capillary endothelium was also observed which suggested that hematogenous route might act as the pathway for sarscov2 to the brain in addition study using the mouse model have shown that sarscov can lead to neuroinvasion via disruption of the nasal epithelium and subsequent neuronal dissemination which suggested that coronavirus may use the olfactory nerve to enter the brain the symptoms from nervous system of covid19 patients including headache dizziness anosmia and dysgeusia have been observed in the clinic [] table disturbance of consciousness and seizures can occur as complications in the cases with severe covid19 it was convincing enough that the neurological deficits of patients with covid19 could be ongoing if it did not get noticed it was indicated that sarscov2 can cause nervous system damage the neurological deficits meningoencephalitis and acute myelitis in covid19 patients have been reported in the usa switzerland and china [] according to a recent study out of covid19 patients had central nervous system cns symptoms including dizziness headache impaired consciousness ataxia and epilepsy up to of patients with covid19 have headache and clinical manifestations of dizziness were found from of the patients with covid19 [ ] olfactory and gustatory disorders are prevalent peripheral nervous system pns symptoms in covid19 patients in patients with mild and moderate covid19 a high proportion of patients presented olfactory and gustatory dysfunctions and olfactory dysfunction appeared prior to the other symptoms in some cases [ ] these studies showed that the damage of neurological system may also act as a significant feature of covid19 table gastrointestinal symptoms after sarscid0 cov2 infection study chen wang liu xiao huang guan li chen zhou wan li [ | 0 |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
Overexpressed EphB4 conduce to tumor development and is regarded as a potential anticancer target HomoharringtonineHHT has been approved for hematologic malignancies treatment but its effect on hepatocellular carcinoma HCC has notbeen studied This study elucidated HHT could restrain the proliferation and migration of HCC via an EphB4catenindependent manner We found that the antiproliferative activity of HHT in HCC cells and tumor xenograft was closelyrelated to EphB4 expression In HepG2 Hep3B and SMMC7721 cells EphB4 overexpression or EphrinB2 Fcstimulation augmented HHTinduced inhibitory effect on cell growth and migration ability and such effect wasabrogated when EphB4 was knocked down The similar growth inhibitory effect of HHT was observed in SMMC and EphB4SMMC7721 cells xenograft in vivo Preliminary mechanistic investigation indicated that HHTdirectly bound to EphB4 and suppressed its expression Data obtained from HCC patients revealed increasedcatenin expression and a positive correlation between EphB4 expression and catenin levels HHTinducedEphB4 suppression promoted the phosphorylation and loss of catenin which triggered regulation of catenindownstream signaling related to migration resulting in the reversion of EMT in TGFinduced HepG2 cellsCollectively this study provided a groundwork for HHT as an effective antitumor agent for HCC in an EphB4catenindependent mannerIntroductionGlobally hepatocellular carcinoma HCC is one of themost fatal malignancies with poor prognosis and anincreasing incidence1 Although the major therapeuticapproaches such as surgical resection radiation therapyand chemotherapy have advanced clinical applicationsthe 5year survival rate of HCC remains less than Most patients still suffer from tumor recur invasivenessand metastasis At present sorafenib a multiple tyrosinekinase inhibitor is one of the most representative optionsfor advanced HCC butlimited andaccompanied with reduced sensitivity and severe adversesometimesisCorrespondence Yanmin Zhang zhang2008mailxjtueducn1School of Pharmacy Health Science Center Xian Jiaotong University No Yanta Weststreet Xian Shaanxi PR ChinaEdited by B Zhivotovskyevents34 Therefore much effort is needed on this frontto uncover new antiHCC therapeutic strategies5Erythropoietinproducing hepatocytereceptor B4EphB4 is a member of the tyrosine kinase family andplays a pivotal role in tumor progression6 Activatedby its corresponding ligand EphrinB2 EphB4 controlscellcell interactions angiogenesis tumor growth andmetastasis910 Studies on the expression of EphB4 innumerous cancer types have shown overexpressed levelin breast colorectal lung and blood cancers correlatingwith poor prognosis11 It has been reported that highEphB4 expression enhanced the growth and migrationof pancreatic colorectal and papillary thyroid carcinoma and such effect could be reversed by EphB4knockdown making EphB4 a promising target for cancer treatment14 Our previous study has conï¬rmed The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT exhibited a growth inhibitory effect on HCC cells in vitro and in vivo a The chemical structure of HHT b Effects of HHT on cellproliferation in Hep3B HepG2 SMMC7721 Bel7402 and Bel7404 cells were determined by MTT assay p comparedto the IC50 of HepG2 cells Cells were treated with increased gradients of HHT for h n cultures for each dose c Protein expression of EphB4 inHep3B HepG2 and cells d Quantiï¬cation of c n independent experiments e Effects of HHT on colony formation in HepG2cells The upper row the colony formation picture the lower row the individual colony picture magniï¬cation f Photographs of control andHHTtreated group tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor inhibitory rate n mice g h data represent mean ± SEM p p compared to vehicle controls i Inhibitory rate of HHT on tumor mass n micethe high expression of EphB4 in HCC17 and its functionin HCC migration remains poorly understoodHomoharringtonine HHT Fig 1a is a compoundextracted from traditional Chinese medicine and has beenapproved for the treatment of leukemia by Food and DrugAdministration18 Previous studies indicated that HHTcould suppress protein synthesis essentialfor cancersurvival and induce apoptosis by upregulating the proapoptotic protein Bax and inducing caspase3mediatedcleavage of PARP19 In addition to hematologic tumorsHHT also demonstrated its effectiveness in renal cellcarcinoma colon rectal cancer and nonsmall cell lungcancer20 However the effect of HHT on HCC and theunderlying EphB4related mechanism of action have notbeen studied In this study HHT was found to suppressthe proliferation and migration of HCC cells through anEphB4catenin dependent mannerResultsHHT exhibited a growth inhibitory effect on HCC cellsin vitro and in vivoTo determine the effect of HHT on the cell viability ofHCC cells several different HCC cells HepG2 Bel7402Hep3B and SMMC7721 were treated with an increasedgradient of HHT for h The results showed that HepG2cells were most sensitive to HHT treatment with an IC50value of μM while the IC50 values of Bel7402Hep3B Bel7404 and SMMC7721 cells were and μM respectively Fig 1b Immunoblotting analysis showed that HepG2 cells exhibitedhigher EphB4 expression Fig 1c d suggesting thepositive correlation between the inhibitory effect of HHTand EphB4 expression Similar results were obtained fromthe colony formation assay HHT significantly reduced thecolony size and the number of HepG2 cells at a doseOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig The inhibitory effect of HHT on HCC cells was associated with EphB4 expression a EphB4 expression analysis of EphB4siRNA or EphB4overexpression OE HepG2 cells b Effects of HHT on cell proliferation in wildtype EphB4siRNA EphB4OE or EphrinB2 Fc stimulated HepG2 cellsn cultures for each dose p compared to the IC50 of HepG2 cells c EphB4 expression analysis of EphB4OE Hep3B cells d Effects of HHTon cell proliferation in wildtype and EphB4OE Hep3B cells n cultures for each dose p compared to the IC50 of Hep3B cells e EphB4expression analysis of wildtype and EphB47721 cells f Photographs of control and HHTtreated group of tumors and EphB47721tumors n mice g Tumor volume change throughout the study n mice h Effect of HHT on tumor mass n mice i Body weight ofcontrol and HHTtreated group mice n gi data represent mean ± SEM p compared to vehicle controlsdependent manner in comparison to the control groupFigs 1e and S1a Moreover xenografts model of HepG2cells conï¬rmed the antitumor effect of HHT in vivo HHTgavage groups showed remarkable reduction in tumrowth Fig 1fi and the inhibitory rate reached and at the mgkg mgkg and mgkg inHHT gavage groups respectivelyThe inhibitory effect of HHT on HCC cells was associatedwith EphB4 expressionTo evaluate whether the proliferation inhibitory effectof HHT on HCC cells was related to EphB4 expressionEphB4 siRNA or plasmid was utilized to transfect theHCC cells Figs 2a and S1b and EphrinB2 Fc was used tostimulate the HCC cells As is shown in Fig 2a b HepG2cells with EphB4 knockdown were less sensitive to HHTwhereas HepG2 cells with EphB4 overexpression EphB4OE demonstrated elevated sensitivity to HHT treatmentcompared with wild type HepG2 cells HepG2 cells following EphrinB2 Fc stimulation showed a drug responsecurve that was similar to that of EphB4 OE subline Fig2b Meanwhile following transfection with EphB4 plasmid Hep3B cells harboring high expression ofEphB4 showed less cell viability after HHT treatmentcompared with wild type Hep3B cells Fig 2c d Forin vivo test an EphB4overexpressing SMMC7721EphB47721 cell line was established Figs 2e and S1cand the antitumor effect of HHT on xenograft model ofOfï¬cial journal of the Cell Death Differentiation Associationcellsand EphB4wild type SMMC7721 cells was investigated HHT has an enhanced inhibitory effect on EphB47721 tumor growth comparedwith that on wild type tumor Fig 2fh And therewas no obvious body weight and spleen index reductionduring the test Figs 2i and S1dThe suppression of HHT on SMMC7721 cells migrationwas associated with EphB4Migration assay and wound healing assay were conducted to investigate the effect of HHT on HCC cellmigration The results showed that HHTtreated wide typeSMMC7721 cells had decreased migration as comparedwith controls whereas both of EphB4 overexpression andEphrinB2 Fc stimulation in SMMC7721 cells strikinglyenhanced migration restraint effect of HHT Fig 3a cSimilar result was observed in wound healing assay whichdemonstrated that both transfection with EphB4 andexogenous stimulation with soluble EphrinB2 Fc inSMMC7721 cells delayed the closure of wound gapsfollowing HHT treatment Fig 3b d These results indicated that the suppression of HHT on HCC cells migration was closely associated with EphB4 expressionHHT suppressed HepG2 cell migration induced by TGFstimulationTGF stimulation could induce EMT and increase themigration of tumor cells We next investigated the effect 0cZhu Cell Death and Disease Page of Fig The suppression of HHT on SMMC7721 cell migration was associated with EphB4 a Transwell assays were conducted to observe themigratory cells in HHTtreated wide type EphB4OE or EphrinB2 Fc stimulated cells Scale bars μm b The migration rate of HHTtreated EphB4OE or EphrinB2 Fc stimulated cells observed through woundhealing assays Scale bars μm c Quantiï¬cation of a n Leftp compared to the migrated cell number of cells Right p and p compared to the migration rate of cells atindicated concentration of HHT d Quantiï¬cation of b n p compared to HHTtreated cells All data represent mean ± SEMof HHT on HCC cells migration after TGF stimulationby transwell migration assay and wound healing assay Asshown in Fig 4a c although the higher number ofmigration cells was observed in the TGF inducedHepG2 cells as compared to controls the addition ofHHT reduced the migrated cells Importantly concurrenttreatment with HHT and NVPBHG712 a small molecule EphB4 kinasespeciï¬c inhibitor had a greaterrestraint effect on the migration of TGF inducedHepG2 cells Wound healing assay showed similar resultsthat HHT could delay the closure of wound gaps in TGF induced HepG2 cells whereasthe addition ofEphB4 siRNA impaired such effect Fig 4b d Theseresults indicated that TGF induced the migration abilityin HepG2abrogated byEphB4 suppression of HHTcells which could beHHT bound to EphB4 and suppressed its expressionWe further evaluated the regulation of HHT on EphB4expression The results showed decreased EphB4 proteinexpression after HHT treatment both in HepG2 cells andtumor tissues Figs 5a c and S2a b Exogenous stimulation with soluble EphrinB2 Fc increased EphB4 proteinexpression while in HepG2 cells treated with EphrinB2 Fcand HHT the protein levels of EphB4 were strikinglydecreased Figs 5b and S2c HHT treatment resulted in aremarkably reduced EphB4 mRNA level at a dosedependent manner Figs 5d and S2d We treatedHepG2 cells with NVPBHG712 HHT or both to evaluate the change of EphB4 expression The results indicated that coadministration of HHT and NVPBHG712produced an even greater decrease in the expression levelof EphB4 in HepG2 cells than by either alone Figs 5e andS2e Given these ï¬ndings a molecular docking assay wasconducted to conï¬rm the afï¬nity of HHT bound to theactive site of EphB4 The results revealed that HHToccupied in the active site of EphB4 through ï¬ve hydrogen bonds which were associated with amino acid residues LYS647 GLU664 TYR736 ASP758 and THR Fig 5f The molecular docking results indicated thatHHT ï¬t well with EphB4EphB4 was positively correlated with catenin in HCCpatients and HHT inhibited the phosphorylation andnuclear translocation of cateninEpithelial to mesenchymal transition is a prerequisitefor cell migration and lies downstream of catenin23Although previousstudies have reported that EphOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT suppressed HepG2 cell migration induced by TGF stimulation a Transwell assays were conducted to observe the migratory cellsin control and TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 treated HepG2 cells Scale bars μm b The migrationrate of control and TGF TGF HHT or TGF HHT EphB4 siRNA treated HepG2 cells observed through woundhealing assays Scale bars μm c Quantiï¬cation of a n d Quantiï¬cation of b n All data represent mean ± SEM p p and p compared tothe indicated groupsreceptor is conducive to EMT progression in hepatomacells24 the relationship between EphB4 and catenin hasnever been shown before To investigate the role ofcatenin in HCC we analyzed the mRNA level ofcatenin in HCC patients using The Cancer GenomeAtlas TCGA database RNASeq data from this databaseshowed that catenin expression was significantly higherin carcinoma tissue compared with paracarcinoma tissueFig 6aImmunohistochemistry was used to detectcatenin expression in pairs of HCC and noncarcinoma tissues The results showed that cateninexpression was remarkably increased in carcinoma tissuescompared with noncarcinoma tissues Fig 6b c whichwas consistent with the ï¬ndings in TCGA database Todelineate the possible relationship between EphB4 andcatenin Spearmans correlation analysis was conductedand the results revealed that catenin expression waspositively correlated with EphB4 levels in HCC tumortissues Fig 6dOfï¬cial journal of the Cell Death Differentiation AssociationWe next analyzed the regulation of catenin in HepG2cells exposed to HHT Western blot analysis indicatedthat HHT could downregulate catenin expression andupregulate the phosphorylation of catenin level both inHepG2 cells and xenograft tumors Figs 6e f and S2f gThese results were also observed in immunohistochemicalassay for xenograft tumors Fig 6g And a remarkablyreduced catenin mRNA level was also observed inHHTtreated HepG2Immunoï¬uorescence staining was used to examine the distribution of catenin in HepG2 cells exposed to HHT Theresults in Fig 6h demonstrated that HHT restrained thelevel of catenin in the nucleus as well as in the cytoplasm Figure 6eshowed that phosphorylation ofcatenin was obviously increased at and nM ofHHT which has been reported to contribute to process ofcatenin degradation25 These data indicated that HHTsuppressed nuclear translocation of catenin and promoted its phosphorylationS2hcellsFig 0cZhu Cell Death and Disease Page of Fig HHT bound to EphB4 and suppressed its expression a Western blot analysis of HepG2 cells EphB4 expression after HHT treatmentb Western blot analysis of EphB4 expression in HepG2 cells treated with HHT orand EphrinB2 Fc c Immunochemistry analysis of EphB4 expression inHepG2 tumors after HHT treatment n magniï¬cation d RTPCR analysis of HepG2 cells EphB4 expression after HHT treatment n Alldata represent mean ± SEM p compared to vehicle controls e Western blot analysis of HepG2 cells EphB4 expression after HHT NVPBHG712or both treatments f Molecular docking analysis of the EphB4 protein and HHTEcadherin was overexpressed in HCC patients and HHTregulated EMTrelated moleculesGiven the positive correlation of EphB4 and cateninin HCC patients the Ecadherin expression in HCCpatients was examined As shown in Fig 7a b lowerEcadherin protein was observed in carcinoma tissueswith higher expression in the noncarcinoma tissue groupBased on the result that Ecadherin was reduced in HCCpatients and HHT could restrain the migration of HCCcells we next analyzed the effect of HHT on EMTrelatedmolecules by western blotting and immunohistochemistryassay Promotion of Ecadherin and inhibition of Snailwere observed in HHTtreated HepG2 cells and tumorsFigs 7c d g and S3a b Furthermore the results in Figs7eg and S3c d indicated that the essential members ofMMPs family MMP2 MMP3 and MMP9 were suppressed by HHT both in HepG2 cells and in the tumortissues of xenograft models And the mRNA level ofMMP2 and MMP9 were reduced in a dose depensentmanner in HepG2 cells exposed to HHT Fig S3eHHT repressed catenin and EMTrelated moleculesthrough EphB4 suppressionNext the expression changes of EphB4 catenin andEMTrelated molecules after HHT administration for thedifferent time points were evaluated by western blottingThe results in Figs 8a and S4ac demonstrated that theprotein level of EphB4 was significantly decreased afterHHT treatment within h and the expression of othermolecules was unchanged attime point ThethisOfï¬cial journal of the Cell Death Differentiation Associationexpression and phosphorylation of catenin wereremarkably changed within h of HHT administrationIncreased Ecadherin expression and decreased SnailMMP2 and MMP9 expression were observed within hof HHT treatment These ï¬ndings indicated that HHTmight regulate the expression of catenin and EMTrelated molecules by targeting EphB4 receptor NVPBHG712 was utilized to investigate the changes in thesemolecules after EphB4 suppression The results in Figs 8band S4e demonstrated that both HHT and NVPBHG712could suppress catenin expression and promote itsphosphorylation level Furthermore the upregulation ofEcadherin and downregulation of Snail MMP2 andMMP9 were also seen in HHT or NVPBHG712 monotherapy These effects exerted by a single administrationof HHT or NVPBHG712 were significantly augmentedby the combination of the two moleculesEMTrelated molecules in HepG2 cells following TGF stimulation was also investigated by western blot assayand the results were shown in Figs 8c and S5a b Theexpression of Ecadherin was downregulated and theprotein levels of Snail MMP2 and MMP9 were upregulated by TGF and these effects could be reversed by theaddition of both HHT and NVPBHG712 And concurrent addition of HHT and NVPBHG712 furtheraugmented the effect of monotherapyDiscussionContinuous stimulation of proliferative signals andmaladjustment of related monitoring mechanisms are 0cZhu Cell Death and Disease Page of Fig EphB4 was positively correlated with catenin in HCC patients and HHT inhibited the phosphorylation and nuclear translocation ofcatenin a mRNA expression of EphB4 in HCC carcinoma tissue and paracarcinoma tissue in the TCGA database p b Representativeï¬gures of immunohistochemical analysis of catenin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCC patients respectively magniï¬cation c Quantiï¬cation of b n p d The positive correlation between the expression ofcatenin and EphB4 e Protein expression of catenin and pcatenin in HepG2 cells treated with HHT for h f Protein expression of cateninand pcatenin in HepG2 tumor EphB4 expression after HHT treatment g Immunochemistry assay of catenin and pcatenin in HepG2 tumortissues magniï¬cation h Immunoï¬uorescence analysis of the catenin protein in HepG2 cells treated with HHT catenin green DAPI bluestaining and merged images indicate the nuclear translocation and expression of catenin Scale bars μm All data represent mean ± SEMimportant causes of tumor formation The growth factorreceptor can be activated by growth factors to generateintracellular cascade signals to regulate the proliferationof tumor cells EphB4 is an important member of thereceptor tyrosine kinase family which is overexpressedand conduces to tumor growth and migration in variouscancers61315 Our previous study has conï¬rmed theoverexpression of EphB4 in the tumor tissues of HCCpatients emphasizing EphB4 a potential target for HCCtreatment17 However there is no drugs targeting EphB4on the market In this study we found the inhibitory effectof HHT on HCC cell proliferation and migration in anEphB4 dependent manner and the underlying preliminarily mechanism was clariï¬edHHT has been proved effective in the treatment ofleukemia butin HCC inhibition wasunknown We revealed the antiproliferative ability ofits potentialHHT on several HCC cell lines In particular HepG2cells with the highest EphB4 protein expression were themost sensitive to HHT treatment demonstrating thatthe inhibitory effect of HHT on HCC cells might berelated to EphB4 expression Xenograft models in nudemice conï¬rmed the inhibitory effect of HHT on HepG2cell growth in vivo For in vitro experiments EphB4overexpression and EphrinB2 Fc stimulation increasedthe sensitivity of wild type HepG2 or Hep3B cells toHHT while transient transfection with EphB4 siRNAdecreased such effect in HepG2 cells Similar resultswere drawn from in vivo experimentsthat HHTexhibited enhanced inhibitory effect in xenograft ofEphB47721 cells compared to xenograft of wild type cells The results above indicated that EphB4might play an indispensable role in the suppression ofHCC cell proliferation by HHTOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Ecadherin was overexpressed in HCC patients and HHT regulates EMTrelated molecules a Representative ï¬gures ofimmunohistochemical analysis of Ecadherin expression in carcinoma and noncarcinoma tissues derived from HCC patients and nonHCCpatients respectively magniï¬cation b Quantiï¬cation of a n p All data represent mean ± SEM c Protein expression of Ecadherin and Snail in HepG2 cells treated with HHT for h d Protein expression of Ecadherin and Snail in HepG2 tumor EphB4 expression after HHTtreatment e Protein expression of MMP2 MMP3 and MMP9 in HepG2 cells treated with HHT for h f Protein expression of MMP2 MMP3 andMMP9 in HepG2 tumor tissues after HHT treatment g Immunochemistry assay of Ecadherin MMP2 and MMP9 in HepG2 tumor tissues magniï¬cationInvasion and migration are the main causes of tumormetastasis and the critical juncture of tumor staging inHCC2627 Since EphB4 has been reported with promotingcell migration potentialin both normal and malignantcells7 we investigate the role of EphB4 in cell migrationsuppression in HHTtreated HCC cells Our results indicated that both EphB4 overexpression and exogenous stimulation with soluble EphrinB2 Fc exacerbated theantimigratory ability of HHT on SMMC7721 cells both inwound healing and transwell migration assay FurthermoreTGF a multifunctional cytokine was used to stimulatethe migration ability of HepG2 cells28 The obtained resultsdemonstrated that HHT restrained the migration of HepG2cells stimulated by TGF while EphB4 knockdown bysiRNA impaired such inhibitory effect Combined HHT andNVPBHG712 treatment significantly augmented the antiin TGFstimulated HepG2 cells asmigratory effectcompared to either agent alone Our further investigationconï¬rmed that HHT was able to bind to EphB4 withhydrogen bonds and suppress its expression both in vitroand in vivo These results indicated that HHT could inhibitcell migration by regulating EphB4 in HCCOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig HHT repressed catenin and EMTrelated molecules through EphB4 suppression a Protein expression of catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either HHT nM NVPBHG712 μM or the combination of both b Proteinexpression of EphB4 catenin pcatenin Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with HHT nM for the indicated durationc Protein expression of EphB4 Ecadherin Snail MMP2 and MMP9 in HepG2 cells treated with either vehicle TGF TGF HHT TGF NVPBHG712 or TGF HHT NVPBHG712 d Schematic diagram of HHT inhibited the migration of HCCIt has been reported that Eph receptor could mediateEMT progression and adhesion to conduce migratory andmetastatic processes in hepatoma cells24 There is a wideacceptance that EMT is a prerequisite for cell migrationand catenin can trigger EMT2329 yet whether EphB4could regulate catenin remains unknown catenin wasthe key molecule of the Wntcatenin pathway and thenuclear translocation of which could not only promotethe expression of matrix metalloproteinases MMPs butalso suppress Ecadherin expression3031 In this studyboth TCGA database and our own HCC patient samplesanalysis demonstrated that catenin was significantlyoverexpressed in HCC patients at protein and mRNAlevels We also analyzed the expression data of EphB4 andcatenin in TCGA database and the results indicated thatthe mRNA level of the two molecules in HCC was significantly correlated suggesting that catenin might playa critical role in HCC migration suppression by HHT Weexamined the regulation of HHT on catenin and theresults showed that HHT strikingly inhibited cateninexpression at protein and mRNA level and promoted itsphosphorylation in vitro and in vivo Moreover the resultof immunoï¬uorescence assay showed that the nucleartranslocation of catenin was restrained by HHTAs a key molecule of tumor migration Ecadherincould be regulated by catenin and the loss of Ecadherin is the critical marker of EMT2329 Wecompared the protein expression of Ecadherin in thecarcinoma tissues of HCC patients and the noncarcinoma tissues The resultindicated that Ecadherin level was prominently decreased in the carcinoma tissues compared to that in the noncarcinomatissues HHT treatment upregulated the protein levelof Ecadherin both in HepG2 cells and xenografttumors Furthermore Snail is a transcription factorand its expression is increased during the process ofOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of EMT We found that Snail expression wassignificantly downregulated in HHTtreated cells andtumors Most of the primary tumor cells are polarepithelial cells and connected to each other by intercellular adhesion molecules As the tumor progressesthe intercellular adhesion molecules are degraded byMMPs15 Tumor migrationrelated molecules MMPsare the downstream signaling of the Wntcateninpathway and could be regulated by catenin Thisstudy indicated that the expression of MMPs including MMP2 MMP3 and MMP9 was significantlysuppressed by HHT in vitro and in vivoThe obtained data showed that HHT could targetEphB4 and suppress its expression The expression ofEphB4 and catenin in HCC was positively correlatedaccording to TCGA data analysis HHT treatmentregulated the expression of catenin and its downstream signaling such as Ecadherin and MMPs Nextwe focused on the relationship between the effect ofHHT on EphB4 and catenin and the downstreamsignaling We investigated the protein levels in HepG2cells exposed to HHT for different duration and theresults conï¬rmed that catenin might be the downstream signaling of EphB4 receptor EphB4 speciï¬cinhibitor NVPBHG712 could suppress the proteinlevel of catenin and promote its phosphorylationThe expression of Ecadherin Snail and MMPs wasalso significantly changed after EphB4 was suppressedby NVPBHG712 And the regulating effect on EphB4catenin and its downstream cascades was remarkablycoadministration of HHT and NVPBHG712 In addition the increased expression of Snail and MMPs anddecreased expression of Ecadherin conï¬rmed thatTGF induced EMT in HepG2 cells Both HHT andNVPBHG712 could reverse the regulating effect ofTGF and such effect was enhanced by combinedHHT and NVPBHG712 treatment These ï¬ndingsindicated that HHT could reverse the EMT of HepG2cells by restraining EphB4 expression the suppressionof which further inhibited the nucleus translocation ofcatenin and regulated the expression of EMT related molecules including Ecadherin Snail MMP2and MMP9in HepG2augmentedcellsafterIn conclusion we discovered a positive correlation ofEphB4 and catenin in HCC patients and that EphB4 wasinvolved in HCC cell migration progression by regulatingcateninmediated EMT HHT suppressed EphB4expression and further led to catenin loss resulting inthe regulation of Ecadherin Snail and MMPs to preventEMT progression in HCC cells Fig 8d Our researchmay provide new insight into the migration mechanism ofEphB4 in HCC and HHT possesses great potential in thedevelopment of antiHCC drugsOfï¬cial journal of the Cell Death Differentiation AssociationMaterials and methodsReagentsHHT HPLC ¥ was obtained from Baoji Herbest Biotech Co Ltd Shaanxi China NVPBHG712Purity ¥ was purchased from MedChemExpressCo Ltd Dulbeccos modiï¬ed Eagles mediumDMEM RPMI medium and PBS were fromHyClone Logan UT USA Fetal bovine serum FBSwas purchased from ExCell Bio Co Ltd ShanghaiChina MTT powder RNase and propidium iodidewere from SigmaAldrich St Louis MO USADimethyl sulfoxide DMSO was from Tianjin KemiouChemical Reagent Co Ltd Tianjin China OptiMEM medium was purchased from Gibco CaliforniaUSA Trypsin and PenicillinStreptomycin solutionwere obtained from Beijing Solarbio Science Technology Co Ltd Beijing China Lipofectamine reagent was purchased from Invitrogen Carlsbad CA USA RIPA Lysis Buffer and BCA proteinassay reagent kit were purchased from Pioneer Biotechnology Co Ltd Protease and phosphatase inhibitors were obtained from Roche TechBaselSwitzerland Ultra Signal Enhanced Chemiluminescent ECL Reagent kit was purchased from 4A Biotech Co Ltd Beijing China EphB4 catenin andpcatenin rabbit mAb were obtained from CellSignaling Technology Boston MA USA Ecadherin Snail GAPDH rabbit mAbs and goat antirabbitIgG were purchased from Protein technology GroupChicago Illinois USA MMP2 MMP3 and MMP9rabbit mAb were from ABclonal Boston MA USAThe EphB4 bacterial strain was from VectorBuilderPatientsAll the patients who were eligible underwent surgery atthe First Afï¬liated Hospital of Xian Jiaotong UniversityFifteen HCC tissues from HCC patients and ï¬fteenhepatic tissues from nonHCC patients were obtainedfrom consenting patients and used with permission ofbiomedical ethics committee of Xian Jiaotong UniversityHealth Science Center Project No Cell cultureHuman hepatocellularcarcinoma HepG2 Hep3BSMMC7721 Bel7402 and Bel7404 cells werepurchased from the Shanghai Institute of Cell Biology atthe Chinese Academy of Sciences Shanghai Chinawithout mycoplasma contamination The HepG2 andHep3B cells were cultured in DMEM with FBS and Penicillin and Streptomycin solution SMMC7721Bel7402 and Bel7404 cells were cultured in RPMI1640with FBS and Penicillin and Streptomycin solution Allthe cells were incubated in a humidiï¬edatmosphere incubator of CO2 at °C 0cZhu Cell Death and Disease Page of Cell viability assayMTT method was used to analyze cell viability Thegrowing cells were seeded in 96well plates overnightThen the cells were treated with increased concentrationof HHT for h followed by incubation with the mixtureof serum free medium and MTT solution for h Themixture was replaced by DMSO After min shakingthe plates were determined using EPOCH BioTekInstruments Inc USA at a wavelength of nmColonyforming assayThe growing cells were seeded in 12well plates at adensity of cells per well Following h incubationthe cells were exposed to HTT for h followed bycultured in fresh complete medium for weeks Afterwashed twice with PBSthe colonies were ï¬xed bymethanol and stained using crystal violet Imageswere obtained via an inverted ï¬uorescence microscopeMigration assayThe HCC cells were cultured into the upper chamber at adensity of cells per well accompanied by μLcomplete medium in the lower chamber Following incubation for h for EphB4 p | 2 |
hepatocellular carcinoma hcc is a high mortality disease the fifth most general cancer worldwide and the second leading to cancerrelated deaths with more than new patients diagnosed each year first the high expression of centromere m cenpm in mammary gland tissue of bcatenin transformed mice was identifiedmaterials and methods in our study we evaluated the expression of cenpm in hepatocellular carcinoma based on data obtained from an online database multivariate analysis showed that the expression of cenpm and m classification was an independent prognostic factor for patients with hepatocellular carcinomaresults survival analysis showed that patients with high cenpm had a worse prognosis than patients with low cenpm p a multivariate cox regression hazard model showed that b cells cd8 t cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer p using the network the most frequently changed neighbor genes of cenpm were shown and the most common change was rad21 our study found that the expression of cenpm was significantly increased in patients with hepatocellular carcinoma and it was related to a variety of clinical characteristics its correlation with the level of immune infiltration and poor prognosis so cenpm can be used as a useful prognosis for patients markers and hcckeywords hepatocellular carcinoma centromere protein m data mining prognosis hepatocellular carcinoma hcc a high mortality disease which is the fifth most general cancer in the world and the second most common lead to cancerrelated deaths with over new patients diagnosed each year [ ] viral hepatitis and nonalcoholic steatohepatitis are the most common causes of cirrhosis and approximately of cases develop to hcc due to the recurrence of hcc the prognosis of hcc remains discouraging and the 5year overall survival rate which correspondence wawang123soutlookcomdepartment of infectious diseases union hospital tongji medical college huazhong university of science and technology wuhan chinais only to despite the rapid development of advanced medical technology there are still no useful curable strategies for hcc patients byeno et a0al reported that based on longterm survival data serum opn and dkk1 levels in patients with liver cancer can be deemed as novel biomarkers that show prognostic useful for liver cancer other serum markers such as alphafetoprotein afp and alkaline phosphatase alp or akp are proverbially used in clinical but they lack sufficient sensitivity and specificity therefore finding useful biomarkers is indispensable for diagnosis and treatment for hcc patientsposttranscriptional modifications are essential for tumorigenesis and development centromere protein m cenpm otherwise called pane1 cenpm and the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwu a0and yang cancer cell int page of c22orf18 which encodes a kinetic protein binds to spindle microtubules to regulate chromosomal separation during cell division expression of the pane1 gene was found preferentially in immune cells involving tumor tissues and tumor derived cell lines and leukemias and lymphomas brickner et a0 al found highly expressed in b lineage chronic lymphocytic leukemia bcll cells and resting cd19 b cells may be a potential therapeutic target for bcll bierie et a0al also demonstrated that human cenpm transcript crna was detected only in a0vivo or in a0vitro in activated b cells and t cells these studies suggested cenpm may play critical role in tumor immune response and may be deemed to therapeutic target for immunotherapy however the role of cenpm in hcc prognostic remains unclear in our study we evaluated the expression of cenpm in hcc based on data from an online database to further understand the biological pathway of cenpm related to the pathogenesis of hcc in addition we also analyzed the connection between cenpm expression and clinical features as well as the correlation of its expression with immune infiltration level in hcc comes an online tumor infiltrating immune cells analysis toolmaterials and a0methodsdata collectioninformation on rnasequencing data tissues workflow type htseqcounts and comparative clinical data patients data format bcr xml were identified and got from the level standardized fpkm of the tcgahcc cohort use boxplots to imagine expression differences for discrete variables the clinical factors included gender stage age grade tphase mphase nphase survival status and number of days of survival data analysis were checked by r version and r bioconductor software packagesgsea enrichmentgene set enrichment analysis gsea created a list of all gene permutations related to cenpm expression the samples were then divided into a high cenpm group and a low cenpm group as training sets to distinguish potential functions and use gsea to clarify significant survival differences genome replacement is performed multiple times with each exam the degree of expression of cenpm was used as a phenotypic marker normalized enrichment scores nes and nominal pvalues have been used to classify the pathways of enrichment in each phenotypeimmune infiltrates analysistimer is a comprehensive database for the systematic study of immune infiltration in various malignant tumor types the abundance of immune infiltrates cd8 t cells b cells cd4 t cells macrophages neutrophils and dendritic cells was evaluated by our statistical methods and has been estimated using pathology methods evaluated it the network also enables users to explore the clinical relevance of one or more tumor immune subpopulations and has the flexibility to correct multiple covariates in a multivariate cox proportional hazard model meanwhile we contrast the differential level of cenpm between tumors and normal on all tcga tumorsualcan and a0cbioportal analysisualcan is a userfriendly intelligent network asset for analyzing discovering cancer data and indepth analysis of tcga gene expression information one of the highlights of the portal is that it allows users to found between biomarkers or computer approval of potential genes of interest and to evaluate genes in different clinical subgroups such as gender age race tumor grade etc expression cbioportal is an online free asset that can visualize analyze and download largescale cancer transcription datasets the portal included cancer studies the tab biological interaction network of cenpm and its coexpressed genes was got and neighboring genes with altered frequencies were containedtargetscan analysistargetscan is a web for predicting potential biological targets of mirnas targetscanhuman deems that the match to human ²utr and its orthologs is estimate by a ucsc genomewide adjustment as an alternative they are ranked according to their predicted conservative positioning possibilities funrich is a tool designed to process varieties of geneprotein datasets in spite of the anism and used for functional enrichment analysis we used funrich tools for mirna enrichment analysis including analysis of biological pathways biological processes bp cellular components cc and molecular functions mfstatistical analysisscatter plots and paired plots visualize the differences between normal and tumor samples use delete ways to handle disappeared data and if any individual value is disappeared the data will exclude the full sample the relationship between clinical factors and cenpm was used by logistic regression wilcoxon rank sum test and kruskal test multivariate cox analysis was used to assess the effect of cenpm expression on survival and other clinical factors such as age gender stage distant metastasis benjaminihochbergs means of converting p values to fdr 0cwu a0and yang cancer cell int page of resultspatients characteristicsthe tcga database contains patients the clinical and pathological properties of these samples are shown in table a0 the middle age at diagnosis in tcga was a0 years old range a0 years and median finally contact for subjects was a0 months range a0months meanwhile followup for subjects conformed alive and death patients our study cohort included female and table tcga hepatic carcinoma patient characteristicsclinical characteristicsage at diagnosis yearfutime monthgender female malestage i ii iii iv nagrade g1 g2 g3 g4 natclassification t1 t2 t3 t4 tx namclassification m0 m1 mxnclassification n0 n1 nx nastatus alive deathdata express as mean minmaxtotal male patients stage i was located in patients stage ii in stage iii in and stage iv in tumor stage was found t1 in patients t2 in t3 in and t4 in node stage contained n0 in and n1 in of cases had distant metastases all the subjects were adenomas or adenocarcinomascenpm expression and a0clinical factorsscatter plot showing difference in cenpm expression among normal and tumor samples p we then use paired plot to demonstrated the cenpm expression between normal and tumor from the same patients and the results was significant difference p fig a01a b the outcomes suggested that the expression of cenpm was significant difference the expression of cenpm correlated significantly with the patient grade p clinical stage p and tclassification p fig a01df univariate analysis utilizing logistic regression uncovered that cenpm expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors table a0 cenpm expression in hcc as appreciably connected with grade or ci g1 vs g3 stage or ci i vs iii and tclassification or ci t1 vs t3 indicated that patients with low cenpm expression are inclined to advance to a further advanced stage than those with high cenpm expressionsurvival and a0multivariate analysissurvival analysis found that hcc with cenpmhigh had a worse outcome than that with cenpmlow p fig a0 1c the univariate analysis suggested that cenpm linked essentially to stage hr ci p and tclassification hr ci p table a0 multivariate analysis showed that the expression of cenpm hr p and m classification hr p were independent prognostic factors for patients with hcc table a0gsea analysisto identify useful pathways that may be differentially initiated in liver cancer we performed a gsea analysis between low and high cenpm expression datasets we chose the most abundant signaling pathway depending on the standardized enrichment score nes table a0 the results showed that cenpm high expression differentially enriched cell cycle dna replication rna degradation certain cancers phagocytosis p53 signaling pathway and purine metabolism fig a0 0cwu a0and yang cancer cell int page of fig cenpm expression and the association among clinicopathologic factors a the scatter plot showed the difference of cenpm expression between normal and tumor samples p b paired plot to demonstrated the cenpm expression between normal and tumor from the same patients and the results was significant difference p c survival analysis p d grade e stage f tstagetable cenpm expression associated with a0pathological characteristics logistic regressionclinical clinical characteristicstotal nage vs ¤ gender female vs malegrade g1 vs g3stage i vs iiitstage t1 vs t3odds ratio in a0cenpm expression pvaluecategorical dependent variable greater or less than the median expression levelimmune infiltrates related to a0cenpm in a0hccthe correlation between cenpm liver cancer in expression and the abundance of immune infiltrates was statistically significant p fig a0 3a a multivariate cox proportional hazard model showed that bcells cd8 t cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer p indicating that these immune cells significantly affect the prognosis it is worth further research and exploration table a0 at the same time the expression of cenpm was also statistically significant p finally we compared cenpm expression between various tumors and normal tissues the results showed that cenpm was overexpressed in various cancers p fig a03bassociations survival table a and a0 clinicopathologic characteristics in a0 tcga patients using cox regression b multivariate survival model after a0variable selectionwith a0overall clinicopathologic variablehr cipvaluea age continuous gender female vs male stage iiiiiiiv grade g1g2g3g4 tclassification t1t2t3t4 distant metastasis m0m1mx lymph nodes n0n1nx cenpm expression high vs lowb distant metastasis m0m1mx cenpm expression high vs low ualcan and a0cbioportal analysis in a0hccin the age subgroup normal age a0years normal age a0years normal age a0years and normal age a0 years among patients with liver cancer cenpm has substantially higher transcription levels than healthy individuals analysis in the weight subgroup gender subgroup ethnicity subgroup tumor grade subgroups analysis also showed significantly higher cenpm in hcc patients fig a0 in order to determine the 0cwu a0and yang cancer cell int page of table gene sets enriched in a0phenotype highgene set namekegg_cell_cyclekegg_dna_replicationkegg_rna_degradationkegg_bladder_cancerkegg_non_small_cell_lung_cancerkegg_thyroid_cancerkegg_fc_gamma_r_mediated_phagocytosiskegg_p53_signaling_pathway kegg_purine_metabolismsizenesnom pvalfdr qvalnes normalized enrichment score nom nominal fdr false discovery rate gene sets with nom pval and fdr qval are considered as significantbiological interaction network of cenpm in liver cancer we used the network in the network tab in cbioportal showing the most frequently changed neighbor genes in cenpm and the most common change was rad21 fig a0 and table a0mirnas related to a0cenpmaccording to the online database the top of the mirna families are hsamir13075p hsamir449b3p and hsamir67785p related to the gene cenpm the conserved sites of the mirna family that are widely conserved in vertebrates fig a06a using the funrich database to explore the function of the identified mirnas bp are significantly enriched in the regulation of nucleobases signal transduction cell communication transport regulation of gene expression and anogenesis cc are mainly concentrated in the nucleus cytoplasm golgi apparatus endosome actin cytoskeleton and early endosome the mf are mainly transcription factor activity transcription regulation activity protein serine gtpase activity and ubiquitinspecific protease activity rich biological pathways in the erbb receptor signaling network trail signaling pathway glypican pathway and syndecan1 mediated signaling events and signal transduction events mediated by hepatocyte growth factor receptor cmet fig a06bediscussionin this work we performed a detailed assessment of cenpm expression in hepatocellular carcinoma based on the tcga database and explored its relationship with clinicopathological features survival function immune infiltration and expression differences understanding whether higher expression biomarkers in tumors are directly related to hepatocellular carcinoma can help us understand the mechanism of the observed clinical survival patterns in our findings the significant expression of cenpm suggests that cenpm may play an important cenpm is an role in regulating cancer progression this should draw attention to current views on the improvement of liver cancer and may reveal potential biomarkers or indicators to determine prognosisindispensable centromere protein involved in centromere assembly which regulates mitochondrial protein assembly and chromosome segregation huang et a0 al cloned and identified the cdna sequence of porcine pane1 and found that porcine pane1 gene was expressed differently in seven different tissues with the highest expression in lymph nodes and the lowest expression in kidney until now the expression of cenpm and its potential prognostic effect on hepatocellular carcinoma has not yet been investigated our outcomes showed that the expression of cenpm in hepatocellular carcinoma was related to advanced clinical pathologic factors grade clinical stage tclassification survival time and poor prognosis univariate analysis uncovered that cenpm expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors and mclassification may play an indispensable role in the inclined to advance to a further advanced stage the univariate and multivariate analysis also suggested cenpm still remained freely connected with os and recommended that cenpm may act as a potential prognostic biomarker of prognosis and therapeutic target in hepatocellular carcinoma but more researches needed to conduct for further study in addition we further analyzed various clinicopathological features of hcc samples using the ualcan database and all of them showed high transcription of cenpmto identify differential signaling pathways in liver cancer gsea analysis results show that cell cycle dna replication rna degradation some cancers phagocytosis p53 signaling pathway and purine metabolism are differentially enriched in cenpm high expression phenotype cenpm may influence cell cycle dna replication rna degradation then controls the begins and development 0cwu a0and yang cancer cell int page of fig enrichment plots from gene set enrichment analysis gseaof cancer cells kim et a0al was identified cenpm as a key gene that mediates the anticancer effect of garlic and cisplatin on bladder cancer and showed that patients with low cenpm expressed better progressionfree survival than patients without high expression studies also found the cenpm genes encode a human minor histocompatibility antigen expressed by tumor cells [ ] yu et a0al found cenpm could as afprelated diagnostic biomarkers in hcc and validate the results using quantitative realtime pcr our study for the first time investigated the cenpm mrna expression and its prognostic significance in hepatocellular carcinoma chen et a0al demonstrated that lhx6 can inhibit the proliferation invasion and migration p53 signaling pathways during hepatocarcinogenesis qin et a0 al found that p53stabilizing and activating rna can strengthen the interaction between hnrnp k and p53 which ultimately leads to the accumulation and transactivation of p53 so cenpm may play a role via p53 signaling pathway and more researches needed to conduct in the future 0cwu a0and yang cancer cell int page of fig immune infiltrates correlation with cenpm in hcc a correlation between cenpm in hcc expression and abundance of immune infiltrates p b cenpm expression between various tumor and normal tissuetable multivariate survival model analysis based on a0timer online toolclinicopathologic variablecoefhr cipvalue sigagegender malerace blackrace whitestage iistage iiistage ivpurityb cellscd cellcd4 t cellsmacrophages neutrphilsdendriticcenpmpvalue significant codes ¤ ¤ ¤ ¤ · ·previous studies demonstrated that human cenpm transcript crna was only detected in activated b and tcells either in a0vivo or in a0vitro these studies suggested cenpm may play important role in tumor immune response so we used an online tool to analysis immune infiltrates correlation with cenpm in hcc multivariable cox proportional hazard model showed that b cells cd8 t cells macrophages and dendritic cells of immune infiltrates statistically significant p in hcc indicating that these immune cells significantly affecting the prognosis a latest study showed cd8 cd68 and foxp3 immune cells were associated with hcc particularly in the invasive margin macrophages not only promote the proliferation colony formation and migration of hcc cells but also maintain tumor growth and metastasis by secreting hepatocyte growth factor hgf pang et a0 al proposed that fusion of dendritic cells dc with tumor cells can effectively activate antitumor immunity in the body and affect tumor progression these studies indicate that cenpm may play an important role in tumor immune response and can be a good therapeutic target for immunotherapy 0cwu a0and yang cancer cell int page of fig boxplot showing relative expression of cenpm in subgroups of patients with hcc ualcanto determine the biological interaction network of cenpm in liver cancer we applied the most frequently changed neighbor genes of cenpm on the network tab in cbioportal and the most frequent change was rad21 rad21 is a nuclear phosphoprotein which becomes hyperphosphorylated in cell cycle m phase one study found that depletion of rad21 resulted in reduced levels of h3k27me3 at the hoxa7 and hoxa9 promoters resulting in enhanced selfrenewal of hematopoietic stem and progenitor cells hspc recent studies have shown that removing rad21 in a lacking pds5 can rescue the phenotype observed only in the absence of pds5 our study may provide information on adhesion kinetics in replication fork studies in patients with liver cancer our study also used the targetscan online tool to distinguish cenpmrelated mirnas to check the function of the identified mirnas bioenrichment was performed through the funrich database it is rich in erbb receptor signaling network trail signaling pathway glypican pathway syndecan1 mediated signaling events and biological pathways of hepatocyte growth factor receptor cmet signaling events studies have reported that selective cmet inhibitors have antitumor activity in hcc and have acceptable safety and tolerability in childpugh a liver function patients a recent study found that abnormal hgfcmet upregulation and activation are often observed in bladder cancer studies have also found that metastasis associated with colon cancer macc1 regulates pdl1 expression and tumor immunity in gastric cancer gc cells through the cmetaktmtor pathway we hypothesized that cenpm may regulate the expression of cmet leading to the occurrence of hcc and more related research fig the network for cenpm and the most frequently altered neighbor genestable the type and a0frequency of a0cenpm neighbor gene alterations in a0hcc cbioportalgene symbolrad21rps27ahctf1nuf2pmf1amplification homozygous deletionmutation total alteration 0cwu a0and yang cancer cell int page of fig enrichment analysis of the mirna altered in the cenpm in hcc funrich and targetscan a conserved sites for mirna families broadly conserved among vertebrates b cellular components c kegg pathway analysis d biological processes e molecular functionsis needed to date this study demonstrates for the first time the important role of cenpm in the prognosis of hepatocellular carcinoma however future clinical trials are needed to validate these results and promote the use of cenpm in the prognostic evaluation of hepatocellular carcinomasour study found that the expression of cenpm was significantly increased in patients with hepatocellular carcinoma and was related to a variety of clinical features its correlation with the level of immune infiltration and poor prognosis so cenpm may become a useful biomarker for the prognosis of patients with liver cancerkegg kyoto encyclopedia of genes and genomes bp biological processes cc cellular components mf molecular functions os over survivalacknowledgementsnot applicableauthors contributionswzh designed and analyzed the research study wzh wrote and revised the manuscript ydl and wzh collected the data and all authors contributed to final manuscript all authors read and approved the final manuscriptfundingthis work is not supported by grantsavailability of data and materialsrnaseq data and corresponding clinical data were acquired from the data portal for tcga https porta lgdccance rgovethics approval and consent to participatenot applicableabbreviationshcc hepatocellular carcinoma cenpm centromere protein m gsea gene set enrichment analysis tcga cancer genome atlas go gene ontology consent for publicationnot applicable 0cwu a0and yang cancer cell int page of competing intereststhe authors declare that they have no competing interestsreceived january accepted august references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin tang y wang h ma l et al diffusionweighted imaging of hepatocellular carcinomas a retrospective analysis of correlation between apparent diffusion coefficients and histological grade abdominal radiol coskun m hepatocellular carcinoma in the cirrhotic liver evaluation using computed tomography and magnetic resonance imaging exp clin transplant 201715suppl lang h sotiropoulos gc brokalaki ei et al survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers j am coll surg jiao y fu z li y meng l liu y high eif2b5 mrna expression and its prognostic significance in liver cancer a study based on the tcga and geo database cancer manag res byeon h lee sd hong ek et al longterm prognostic impact of osteo pontin and dickkopfrelated protein in patients with hepatocellular carcinoma after hepatectomy pathol res pract shen y bu l li r et al screening effective differential expression genes for hepatic carcinoma with metastasis in the peripheral blood mononuclear cells by rnaseq oncotarget renou jp bierie b miyoshi k cui y djiane j reichenstein m shani m hennighausen l identification of genes differentially expressed in mouse mammary epithelium transformed by an activated betacatenin oncogene bierie b edwin m melenhorst j et al the proliferation associated nuclear element pane1 is conserved between mammals and fish and preferentially expressed in activated lymphoid cells gene expr patterns brickner ag the pane1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in blymphoid cells and bcll blood kruppa j jung k automated multigroup outlier identification in molecular highthroughput data using bagplots and gemplots bmc bioinf li t fan j wang b et al timer a web server for comprehensive analysis of tumorinfiltrating immune cells cancer res 20177721e108e110110 https doi10115800085472can chandrashekar ds bashel b balasubramanya sah creighton cj rodriguez ip chakravarthi bvsk varambally s ualcan a portal for facilitating tumor subgroup gene expression and survival analyses neoplasia https doi101016jneo201705002 gao et al sci signal cerami et al cancer discov when publishing results based on cbioportal https doi1011582159 agarwal v bell gw nam j bartel dp predicting effective microrna target sites in mammalian mrnas elife 20154e05005 https doi107554elife pathan m keerthikumar s ang cs gangoda l quek cy williamson na mouradov d sieber om simpson rj salim a bacic a funrich an open access standalone functional enrichment and interaction network analysis tool proteomics https doi101002pmic20140 foltz dr jansen le black be bailey ao yates jr iii cleveland dw the human cenpa centromeric nucleosomeassociated complex nat cell biol huang h deng h yang y et al molecular characterization and association analysis of porcine pane1 gene mol biol rep kim wt seo sp byun yj et al the anticancer effects of garlic extracts on bladder cancer compared to cisplatin a common mechanism of action via centromere protein m am j chin med yu z wang r chen f et al five novel oncogenic signatures could be utilized as afprelated diagnostic biomarkers for hepatocellular carcinoma based on nextgeneration sequencing dig dis sci chen hq zhao j li y et al epigenetic inactivation of lhx6 mediated microcystinlr induced hepatocarcinogenesis via the wntβcatenin and p53 signaling pathways environ pollut 2019252pt a216 qin g tu x li h et al lncrna pstar promotes p53 signaling by inhibiting hnrnp k desumoylation and suppresses hepatocellular carcinoma hepatology https doi101002hep30793 ihling c naughton b zhang y et al observational study of pdl1 tgfβ and immune cell infiltrates in hepatocellular carcinoma front med lausanne dong n shi x wang s et al m2 macrophages mediate sorafenib resistance by secreting hgf in a feedforward manner in hepatocellular carcinoma br j cancer pang yb he j cui by et al a potential antitumor effect of dendritic cells fused with cancer stem cells in hepatocellular carcinoma stem cells int janco jmt lamichhane p karyampudi l knutson kl tumorinfiltrating dendritic cells in cancer pathogenesis j immunol fisher jb peterson j reimer m et al the cohesin subunit rad21 is a negative regulator of hematopoietic selfrenewal through epigenetic repression of hoxa7 and hoxa9 leukemia carvajalmaldonado d byrum ak jackson j et al perturbing cohesin dynamics drives mre11 nucleasedependent replication fork slowing nucleic acids res bouattour m raymond e qin s et al recent developments of cmet as a therapeutic target in hepatocellular carcinoma hepatology sim wj iyengar pv lama d et al cmet activation leads to the establishment of a tgfβreceptor regulatory network in bladder cancer progression nat commun tong g cheng b li j et al macc1 regulates pdl1 expression and tumor immunity through the cmetaktmtor pathway in gastric cancer cells cancer med publishers notespringer nature remains neutral with regard to jurisdictional claims in published maps and institutional 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Lung cancer has high mortality often accompanied with systemic metabolicdisorders The present study aimed at defining values of transnodules crossclinical phenomic and lipidomic network layers in patients with adenocarcinoma ADC squamous cell carcinomas or small cell lung cancer SCLCWe measured plasma lipidomic profiles of lung cancer patients and found thataltered lipid panels and concentrations varied among lung cancer subtypes genders ages stages metastatic status nutritional status and clinical phenomeseverity It was shown that phosphatidylethanolamine elements and were SCLC specific whereas lysophosphatidylcholine and snposition1 and phosphatidylcholine and were ADCspecific There were statistically more lipids declined in male ages latestage metastasis or body mass index Clinical transomics analyses demonstrated that one phenome in lung cancer subtypes might be generated from multiple metabolic pathways and metabolites whereas a metabolic pathway andmetabolite could contribute to different phenomes among subtypes althoughthose needed to be furthermore confirmed by bigger studies including larger population of patients in multicenters Thus our data suggested that transomic profiles between clinical phenomes and lipidomes might have the value to uncoverthe heterogeneity of lipid metabolism among lung cancer subtypes and to screenout phenomebased lipid panels as subtypespecific biomarkersK E Y WO R D Slipidomics lung cancer phenomes subtypes transomicsINTRODUCTIONLung cancer is a systemic and aggressive disease withhigh morbidity and mortality and it is often accompanied with systemic metabolic disorders for exampleup or downregulated expression of mechanismassociatedgenes or activation of metabolismdependent enzymes Forexample metabolismassociated genes of small cell lungThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e151101002ctm2151wileyonlinelibrarycomjournalctm2 of 0c of ZHU et alcancer SCLC cells altered after mitogenactivated proteinkinase MAPK kinase module MEK5ERK5 was blockedaccompanied by dysfunctions of several lipid metabolismpathways like the mevalonate pathway for cholesterolsynthesis1 Lipids mainly including subclasses of phosphatidic acid PA phosphatidylcholines PC phosphatidylethanolamine PE phosphatidylglycerol PGphosphatidylinositol PI and phosphatidylserine PShave multiple important biological functions such asbiomembrane composition vesicular trafficking adhesion migration apoptosis energy storage neurotransmission signal transduction and posttranslational modification They have alterations under circumstance of lungcancer Circulating levels of PCs and PEs in patients withnonsmall cell lung cancer NSCLC differed from thosewith noncancer lung diseases or health and were suggested as diagnostic biomarkers of early NSCLC2 Theheterogeneity of circulating lipidomic profiles was foundto exist among patients with squamous cell carcinomasSCC adenocarcinoma ADC or SCLC and there was aclear correlation between genomic and lipidomic profilesof lipidassociated proteins and enzymes3 As the part ofclinical transomics the lung cancerspecific and subtypespecific lipidomics in the circulation were defined and evidenced by integrating lipidomic data with genomic expression of lipid proteins among lung cancer subtypesClinical transomics is defined as a new subject tointegrate clinical phenomes with molecular multiomicsfor understanding molecular mechanisms of diseases inmultiple dimensions4 Clinical transomics becomes moreimportant as a new and novel approach for the discovery of diseasespecific biomarkers and therapeutic targetsalthough there are still many obstacles to be overcomefor example specificity and decisive role of transnodulesamong multiomic networks for intra and intercellular communication56 Recent studies applied the transomics among phosphorproteomics transcriptomics genesequencing and genomics for new molecular category ofliver cancer to provide a new therapeutic strategy7 As thepart of clinical transomics clinical lipidomics was considered as one of major metabolic profiles for identificationand validation of lung cancerspecific biomarkers by integrating clinical phenomes with lipidomic profiles89 Clinical lipidomics could demonstrate the complexity of thelipidome in metabolic diseases and lung cancer and presented the variation among diseases and subtypes of lungcancer1012Our previous study demonstrated the difference oflipidomic profiles among patients with different lungcancer subtypes and the potential association betweenlipidomic phenotypes and gene expression oflipidmetabolismassociated proteins and enzymes as a conceptevaluation3 The present study furthermore investigatedthe values of transnodules crossclinical phenomic andlipidomic network layers in the recognition of lung cancersubtypes ADC SCC and SCLC in order to understandclinical phenomeassociated lipid changes or lipidomicphenotypeassociated clinical phenomes We also evaluated the differences of lipidomic profiles between maleand female various ages early and late stages with orwithout metastasis body mass index BMI or and digital evaluation scores less or more than METHODS AND MATERIALSChemical agentsThe internal standard cocktails were subscribed fromAvanti Lipids Polar Alabaster AL USA the acetone acetonitrile ammonium bicarbonate dithiothreitol formicacid iodoacetamide and Tris base Analytical Gradefrom SigmaAldrich St Louis MO USA and ammonium acetate NH4OAc hexane isopropyl alcohol IPAmethanol and highperformance liquid chromatographygrade chloroform CHCl3 from Merck Millipore Billerica MA USAPatient populationThe study designed as a casecontrol approach wasapproved by the Ethical Evaluation Committee of Zhongshan Hospital ethical code B2018187 The subjectsgave informed consent for clinical data collection andlipids analysis before all the other procedures The studyincluded lung cancer patients diagnosed according topathology of whom were ADC SCC SCLC and other healthy people The stage and severity of lung cancerwere defined according to the Eighth Edition of TNMClassification for Lung Cancer13 Patients were recruitedduring October to March Healthy controlsparticipated were blood donors in Zhongshan HospitalSubjects with other respiratory diseases or family historyof lung cancer were excluded Fasting blood was drawnfrom healthy controls and lung cancer patients on theday of entering hospital to harvest plasma All the clinicaldata including symptoms signs laboratory tests imagespathologic information and survival status years laterwere collected and followed upDigital evaluation score systemThe Digital Evaluation Score System DESS is a scoreindex system by which clinical descriptive information of 0cZHU of each phenome can be translated into clinical informatics14When the severity of each component was scored as or of which represented the most severe condition whereas indicated normal physiological range Thegross DESS scores ranged from to points the higherthe score the severer the condition A total of clinical phenomes were collected and scored in each of threelung cancer group including histories symptoms signs laboratory measurements image features and pathologic indexes as listed in Table S1spectrometry analysisLipid extraction for massAbout µL plasma was collected into a glass tubeinto which µL internal standard was added and then mL of methanolchloroformformic acid asreported previously315 This mixture was incubated at ¦C overnight after vigorous shaking Two milliliters ofHajras reagent M H3PO4 M KCl were droppedblended and centrifuged at rpm for min Afterstratification chloroform in the lower layer was pipettedto another glass tube and concentrated to µL withthe nitrogen flow where the liquid with isopropyl alcoholhexane100 mM ammonium acetate at the ratio of was added till mL The sample was then centrifugated at rpm at ¦C for min The normalphaseliquid chromatography coupled TripleQuadrupole massspectrometer QTRAP SCIEX Framingham MAUSA was used for lipid extraction by the positive and negative electrospray ionization mode In the multiple reactionQTrap was utilized to scan the precursorproduct ion andexamine the mode operation Each test was repeated threetimes The peak area of each pair was quantified with multiple reaction monitoring data by the software MultiQuantAB SCIEXPurification of plasma lipidsLipid samples were derived through Ultimate SiO2 mm à mm µm Agilent Technologies Santa ClaraCA USA with mLmin flow rate highpurity heliumIn the meanwhile µL was added with the split ratio of at the ignition chamber temperature of ¦C and theinjection port temperature of ¦C It was started at temperature ¦C which gradually increased ¦Cmin to¦C and kept for min The mass spectrometry was subjected to liquid chromatographymass spectrometer analysis FOCUS DSQTM II Thermo Fisher Scientific mainlyunder the following conditions Electron Ionization EI asionization source ion source temperature at ¦C ionization voltage at eV multiplier voltage at kV minsolvent delaying and amu of scan rangeIdentification of lipidomic profilesLipid extracts were loaded onto an Ultremex silica column mm à mm µm which was fitted with a mmà mm silica guard cartridge Phenomenex TorranceCA USA and then eluted The sample was enriched ata gradient of nLmin In the mins run B phasewas from to min then rose to from to min linearly ramped for min as to return from to min until the end The QTrap was conductedin the multiplereaction monitoring mode and the different precursorproduct ion pairs were scanned in thepositivenegative electrospray ionization mode Up to lipids of plasma samples were carried out to get possiblelipids chemical structureslipidomic profilesComprehensive analyses ofMultiQuant software AB SCIEX was used to process dataafter lipids were identified by mass spectrometry Further MetaboAnalyst software wwwmetaboanalystcawas utilized for conducting multivariate statistical analysis cluster analysis dimensionality reduction and makingheat mapphenome and lipidome network layersTransnodule analyses crossThe typespecific lipids were identified as more than twotimes elevated or declined significantly compared withother lung cancer subtypes fold change and Pvalue whereas the coexpression lipids were identified as those similarly changed in all lung cancer subtypesas compared with healthy controls The expression quantitative trait locus eQTL model was utilized to evaluatetransnodules between lipidomic profiles and clinical phenomesStatistical analysisData were presented as mean ± SE The means of eachgroup were used for calculation and comparison Statistical significance of differences between two groupsor among multiple groups was determined by Studentsttest or oneway ANOVA test respectively Statistical 0c of ZHU et alsignificance was affirmed when Pvalue We alsoseparately calculated mean values of each phenomesDESS score in different lung cancer subtypes which wereranked to obtain top clinical phenomes of those threegroups of patients Volcano maps showed the significantlyelevated or declined lipids in ADC SCC or SCLC patientsA VIP plot was further exploited to sort the lipids according to their importance to differentiate the four groups Toexplore the correlation between lipid elements and clinical phenomes we applied the lipidquantitative trait locimodel modified from eQTL model Besides MatrixlQTL Rpackage was used to acquire the significant phenomelipidpairs and corresponding Pvalues Moreover GraphPadPrism was utilized to make the receiver operating characteristic curve to evaluate the earlydiagnostic value andaccuracy of clinical phenomespecific lipid elements inADC SCC or SCLC The present study furthermore analyzed the significant differences of lipids among differentages eg and between female and maleearly and late stage metastasis and nonmetastasis highand low DESS scores ¤ and and high and lowBMI ¤ and RESULTSwith lung cancer subtypesClinical phenomes of patientsEighteen female and male lung cancer patients wereenrolled in the present study aged from to ± years old including ADC SCC and SCLC The totalscores of DESS were ± ± and ± in patientswith ADC SCC and SCLC respectively The DESS values of SCLC group were significantly higher than those ofhealthy control group P Top clinical phenomesof ADC SCC or SCLC patients as well as patients survivedor nonsurvived during study period were listed in Table Stages at primary diagnosis and recruitment period for thestudy lymphatic metastasis N12 in ipsilateral paratracheal hilum or mediastinum and enhanced images egfocus enhanced in CT or hypermetabolism in PETCTwere shown in all three subtypes of lung cancers In addition thyroid transcription factor1 TTF1 Napsin A keratin and location of tumor were noticed in ADC obscureboundary emphysema tumor size the cycle number offirst line chemotherapy obstructive pneumonia atelectasis and pulmonary nodule in SCC as well as number ofmetastatic lymph nodes in SCLC separately Top clinicalphenomes were similar between survived and nonsurvivedpatients but the total amounts of DESS of nonsurvivedpatients were significantly higher than those of survivedpatients Table Of total clinical phenomes hadthe statistical significance of each two groups inbetweenTable P or lesswith lung cancer subtypesLipidomic profiles of patientsTotal lipid elements of plasma were identified qualitatively and quantitatively mainly including PAs PCs PEs PGs PIs PSs lysophosphatidylcholineslysoPC lysophosphatidylethanolamines lysoPE lysophosphatidylglycerols lysoPG lysophosphatidylinositols lysoPI lysophosphatidylserines lysoPS ninediacylglycerides and triacylglycerols TAG Levels ofsome lipid elements in ADC SCC or SCLC patients weresignificantly higher Table S2 or lower Table S3 as compared with healthy control twofold P The majority of those elevated lipid elements were PC PA and lysoPC in ADC PE PC PS and PG in SCC or PS PE PG lysoPS and lysoPI in SCLC Of those declinedlipid elements were PS in ADC whereas and were PA in SCC and SCLC respectively Table demonstrates lung cancer subtypespecific lipid elements identified by those lipid elements elevated or declined exclusively in each lung cancer subtype for example some oflysoPC and PC in ADC whereas lysoPI lysoPS PE andPA in SCLC By partial least squares discrimination analysis PLSDA analysis top lipid elements were definedon the basis of variable import in project VIP score of eachgroup TAG565 lysoPG182 and PG and increased in ADC lysoPG181 PA140245 PI384180PA and and PE385PE180 increased inSCLC and PI362PI180 and lysoPG182 decreased in SCCdetail in Figure 1A There was a clear distribution oftop lipid elements among lung cancer groups as compared with the healthy control Figure 1B Of those significantly increased lipid elements in patients with lungcancers top six lipids of each group were identified Figure levels of LysoPC sn2 sn1 and sn1 in ADC Figure 2A PS363 in SCC Figure 2B andPA and and PI and inSCLC Figure 2C were significantly higher than in otherthree groups PLSDA component analysis demonstratedthat five principal components selected were and Figure 3A In the atom map whichwas based on the expression of major C atom numbersin various lipid types levels of lipids with carbons and increased whereas those with carbons decreased as compared with healthy control Figure 3BAs compared with the healthy control Figure 4A wenoticed that PI mainly declined in ADC Figure 4B PA 0cZHU of TA B L E carcinoma SCLC as well as lung cancer patients survived or nonsurvivedTop clinical phenomes of patients with adenocarcinoma ADC squamous cell carcinomas SCC or small cell lungPatients with ADCStage at primarydiagnosis ± Stage at recruitmenttime ± TTF1 ± N2 ipsilateralmediastinum ± Napsin A ± Enhanced image ± Location ± N1 ipsilateralparatracheal ± N1 ipsilateral hilum ± CK7Patients with SCCN1 ipsilateral hilum ± Enhanced image ± Stage at recruitmenttime ± Stage at primarydiagnosis ± obscure boundary ± Emphysema ± T tumor ± L1 cycle ± Obstructivepneumoniaatelectasis ± pulmonary nodulePatients with SCLCStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralParatracheal ± T tumor ± N1 ipsilateral hilum ± N2 ipsilateralmediastinum ± Enhanced image ± pulmonary nodule ± N LN ± MaintenancetreatmentPatients survivedStage at primarydiagnosis ± N2 ipsilateralmediastinum ± Enhanced image ± Stage at recruitmenttime ± N1 ipsilateral hilum ± TTF1 ± Napsin A ± Location ± N1 ipsilateralparatracheal ± LobularPatientsnonsurvivedStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralparatracheal ± N2 ipsilateralmediastinum ± N1 ipsilateral hilum ± Enhanced image ± N2 below carina ± TTF1 ± N LN ± T tumor ± ± Abbreviations N degrees of lung cancer metastasis to lymph nodes of TNM category N1 degree that has metastatic lymph nodes near pulmonary center andside of main bronchia N2 degree that has metastatic lymph nodes in the same side of the mediastinum as lung cancer TTF1 thyroid transcription factor1 asan immunohistochemical biomarker for adenocarcinoma CK7 keratin as an immunohistochemical biomarker for epithelial cells L1 cycle number of the firstline chemotherapy cycles ± ± ± in ADC and SCC Figure 4C and lysoPG in SCLC Figure 4D whereas PG and TAG increased in ADC and SCCPE in SCLC and PC in SCC The volcanic map demonstrated the clear patterns of lipid elements significantlyincreased or declined between heathy controls with ADCFigure 4E SCC Figure 4F or SCLC Figure 4G and varied among different subtypes of lung cancerspatient gendersDifferent lipidomics betweenAbout or lipid elements significantly increased and or declined more than twofold in male or femalelung cancer patients as compared with male or femalehealthy controls respectively Tables S4 and S5 Of thosePC and PE mainly elevated in male and female patientswhereas PA declined in both although the number ofPA in male patients was more than in female patientsTable demonstrates genderspecific lipid elements identified by those lipid elements elevated or declined exclusively in either male or female lung cancer patients forexample some of lysoPS PC and PS elevated in malepatients whereas lysoPI and PE in female patients Therewere about or increased or declined lipid elementsdiffered between male and female lung cancer patientsTable 0c of ZHU et alTA B L E adenocarcinoma ADC squamous cell carcinoma SCC or small cell lung cancer SCLC patientsComparisons of clinical phenomes in increased folds and statistical significance Pvalues between each two groups ofTTF1Napsin ABullaeP40HemoptysisEmphysemaSputumCK7HbCoughEGFRVacuole cavityCEAN2 ipsilateral mediastinumNew metastasisP63Cyfra211Obstructive pneumonia atelectasisSmokingPleural pullThirdlineWBCL1 cyclePDL1 tumorPTBronchiectasisPD1 tumorL2 chemo regimenSynMaintenance treatmentNSEN1 ipsilateral ParatrachealCD56CHGT tumorPD1 interstitialSum of all tumors mmN LNKi67Bronchial stenosisN3 opposite sideBurrNeuL2 cyclePulmonary noduleCK56ADC vs SCCFoldsNANANANANANANANAPvaluesNANASCC vs SCLCFoldsNANANANANANANANANAPvaluesNANANAADC vs SCLCFoldsNANANANANANANANAPvaluesNANANANA 0cZHU of Lung cancer subtypeassociated lipid elements significantly elevated or declined alone in patients with adenocarcinomaFoldsPvalues LipidsFolds PvaluesSquamous cell carcinomad181SoC1P240d181S1PPS363TA B L E squamous cell carcinoma or small cell lung cancer more than twofold as compared with healthy control PvaluesSmall cell lung cancerAdenocarcinomaLipidsLipidsElevated twofoldlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPE lysoPG lysoPG lysoPS lysoPS lysoPS PA PA PA PC PC PC 332e PC 161e181PC 352e PC 160e202PC PC lysoPG lysoPI sn1lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PA PA PE PE orFoldsPvaluesPC PC or PC PC PC PC orPE PE Declined twofoldPG PS PS SM240PG PS401PE PE PI PI PI PI PI PI PIP PS PS TAG PA PA PA PA PA PA PA PA PA 0c of ZHU et alF I G U R E Scores of altered lipid elements in variable import in project VIP chart A where top lipid elements were defined amongpatients with adenocarcinoma ADC squamous cell carcinomas SCC small cell lung cancer SCL and healthy controls CON The xaxisrepresents the VIP score and the yaxis represents the lipid elements corresponding to the VIP score The right color grid stands for the relativeconcentration of lipid elements in four groups The degree of altered concentrations increased from green to red The heatmap B describesthe top lipid elements at the high concentration and the degree of lipid elements increased from blue low to brown highF I G U R E less than and respectively as compared with the healthy controlTop six significantly increased lipid elements in patients with ADC A SCC B and SCLC C and stand for the Pvalue 0cZHU of F I G U R E Histography of five component distributions and percentages A measured by partial least squares discrimination analysisPLSDA and the carbon atom map B in healthy controls red and patients with ADC green SCLC orange and SCC blue Each ofselected five principal components represents as the model to interpret that values of abscissa and ordinate represents the distance from thesample nodule to the origin of the center after projecting to a plane in multidimensional space A The atom map describes the expression ofmajor carbon atom number between and in various lipid types BF I G U R E The proportion of main lipid elements of healthy controls A ADC B SCC C and SCLC D and volcanic mapbetween heathy controls with ADC E SCC F or SCLC G respectively The lipid elements were identified on the basis of statistical significance The abscissa represents log values of fold changes where the left side of the first dotted line perpendicular to the abscissa represents fold changes and the right side of the second dotted line represents 2fold changes The vertical coordinate represents log10 Pvalue Theupper side of the dotted line perpendicular to the ordinate stands for Pvalue less than as compared with healthy controls 0c of ZHU et alFoldsPvaluesPvaluesFemale patients with lung cancerLipidsFoldsTA B L E Genderassociated lipid elements significantly elevated or declined alone in male or female patients with lung cancer morethan twofold as compared with healthy control PvaluesMale patients with lung cancerLipidsElevated twofoldlysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PC PC or PC PC PC 375ePC 160e225180e205PC PC PC PC PC PC PC PC PC PC PC PC PC C1P120 MeanC1P160 MeanC1P240 MeanCer120d171Sod180Sa1Pd181S1Pd181SolysoPC sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS PC PC PE 355p PE 160p204PE 356p PE 160p205PE PE PE 376p PE 180p205 orPE PE PG PG PI 311p PI 160p160PS PS PS PS PS PS Declined twofoldlysoPS PA PA PA PA PA PA PA PA PA 181p204 160e226PE 377p PE 160p226PE PE PE PE orPI PI or or PS PS PA PA Continues 0cZHU et alContinuedTA B L E Male patients with lung cancerLipidsPG PG PS PS PS FoldsPvalues of Female patients with lung cancerLipidsFoldsPvaluesDifferent lipidomics among patientages stags metastases and survival statusAbout and lipid elements significantly elevatedor and declined in lung cancer patients at years old respectively as compared with healthycontrols P We noticed that elements of PG andPS mainly increased in lung cancer patients at all agegroups for example and at 60year group and at to 70year group and and at year group lysoPC and PC increased at 60year group and and at 70year group and PEincreased at to 70year group and at 70yeargroup as detailed in Table S6 Elements of PA mainlydeclined in lung cancer patients at all ages Table S7 Ofthose significantly altered lipid elements and appeared only at 60year to 70year and 70yeargroups respectively and considered as agespecific lipidelements Table LysoPC and lysoPI mainly increasedin 60year and to 70year old patients whereas lysoPEdeclined in 60year group We also compared lipidomicprofiles between patients at early and late stages of lungcancer and found and lipid elements significantlyincreased at early and late stages respectively of whichPE PG ad PS increased in both stages lysoPI in earlystage and PC in late stage Table S8 About and elements declined at early and late stages where the majority was PA Table S9 Table demonstrates stagespecificlipid elements identified by those lipid elements elevatedor declined exclusively at early and late stages of lung cancer for example some of lysoPI and PE elevated at earlystage and lysoPC and PE at late stageWe noticed about or lipid elements significantlyincreased in patients without or with metastasis of whichlysoPI mainly elevated in patients without metastasiswhereas PC and PE in patients with metastasis Table S10The declined number of lipid elements especially PA inpatients with metastasis was significantly higher than inpatients without metastasis Table S11 There were about or elevated or declined lipid elements in patients withmetastasis of which PA was majority of declined elementsin patients with metastasis Table Lipidomic panel also differed between survived andnonsurvived patients There were only eight lipids exclusively elevated in nonsurvived patients that is lysoPS140PC PC PE PE or PE PE PS330 PS372 andPS387 However far more lipids31 elevated alone in survived patients mainly elements of lysoPC lysoPG lysoPIlysoPS and PS On the contrary there were no lipidsdeclined alone in survived patients while lipids in nonsurvived patients of which were PA elements Table clinical phenomes and lipidomesTransomic profiles betweenWe also compared the difference of lipidomic profilesbetween general metabolism statuses of patients indicatedby BMI and between degrees of clinical phenomes measured by DESS scores Levels of lysoPC or lysoPI mainlyelevated in patients with BMI ¤ or respectivelyTable S12 whereas the number of declined PA in patientswith BMI ¤ was higher than that in patients withBMI Table S13 About BMIassociated lipid elements significantly elevated or declined exclusively inpatients with BMI ¤ and about in patients withBMI Table Levels of lysoPC and PE or PG and PSmainly increased in patients with DESS ¤ or TableS14 The number of declined PA n in patients withDESS ¤ was lower than that in patients with DESS n Figure demonstrates the variation of transomicprofiles among lung cancer subtypes indicated by transomic nodules cross significant networks of clinical phenome and lipidome layersDISCUSSIONThe present study preliminarily found the differencesof lipidomic profiles among patients of different lungcancer subtypes genders ages stages metastatic statusbody qualities and clinical phenome severities Besides itinitially demonstrated clinical phenomeassociated lipid 0c of ZHU et alFolds Pvalues LipidsPatient age Folds Pvalues LipidsPatient age TA B L E Ageassociated lipid elements significantly elevated or declined alone in each age group of patients with lung cancer morethan twofold as compared with healthy control PvaluesPatient age LipidsElevated twofoldC1P120 MeanlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPG d181S1PlysoPC sn1lysoPE190lysoPE191PC PC PC PE 356p PE160p205PE PE PE PE orlysoPG140lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1Folds PvalueslysoPS lysoPS150lysoPS161lysoPS170lysoPS201lysoPS202lysoPS220PA PE PE orPE PE PE PE PE PE orPI 311p PI160p160PI PI PI PI PI361TAG PA PA PG393lysoPS lysoPS lysoPS PC PE PE PG PG PG PS Declined twofoldlysoPE sn1lysoPE sn1lysoPE sn1lysoPE sn2PA PA PS PS PE PE orPE PE PG351PS354PS372PA elements and lipid elementassociated clinical phenomesusing clinical transomics Studies on lipidomic profilesof lung cancer patients have experienced three phasesto detect the difference of lipidomic profiles betweenhealthy and lung cancer patients16 the association ofmultiomics among lung cancer subtypes3 and themolecular mechanism of clinical lipidomicsbased targetlipid elements17 Of those lipidomicsbased data limitedinformation could be adopted to understand the diseaseoccurrence and development phone progression and 0cZHU of Stageassociated lipid elements significantly elevated or declined alone in patients with lung cancer at the early stage or lateFolds Pvalues LipidsPatients at late stageFolds Pvalues LipidsTA B L E stage more than two folds respectively as compared with healthy control PvaluesPatient at early stageLipidsElevated twofoldlysoPE lysoPG lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPS lysoPS lysoPS lysoPS PC PC orlysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS PC PC PE PE PE PE PE 356p PE 160p205PE PE PE PE PE PE PI PI 311p PI 160p160PI PI or or PS PS PC PC PC 375e PC 160e225 or180e205PC PC PC PC PC PC PC PC PC PC PC PC PC PC orPC PC PC PE 355p PE 160p204PE 376p PE 180p205 or181p204 or 160e226PE 377p PE 160p226PE PE PE PE orPE PE PE PE PG PG PG PG PG PG PG PG Patients at late stageFoldsDeclined twofoldlysoPS PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PG PG PS PS PvaluesContinues 0c of ContinuedTA B L E Patient at early stageLipidsPatients at late stageFolds Pvalues LipidsPG PS PS PS ZHU et alPvaluesPatients at late stageFoldsFolds Pvalues Lipidsresponse to therapy due to the lack of link between omicsdata and clinical phenomes Like other omics investigations most genomic data were not tied with clinicalinformation so that with little values to be understoodand applied for clinical precision medicine18 In orderto face the major challenge that most clinical information was descriptive and unmatched with the digitalquantity of omics data clinical phenomes were scoredby DESS and integrated with genomic and proteomicdata of patients with acute respiratory distress syndromeand chronic obstructive pulmonary disease19 Clinicalphenomes were furthermore integrated with lipidomicprofiles in patients with pulmonary embolism acutepneumonia and acute exacerbation of chronic obstructive pulmonary diseases based on clinical transomicsprinciple15Lipidomic profiles difference between health and lungcancer has been defined and it depends upon methodologies of measurement and analysis sample preparationsand sources and patient populations and status8 Forexample serum levels of lysoPC C260 and C261 and PCC424 and C344 were different between stage I NSCLCand healthy patients22 Some elements and pathwaysof serum PC and PE profiles increased in patients withlung benign disease and earlystage NSCLC as comparedwith healthy whereas few eg PC significantlyelevated in earlystage NSCLC patients alone2 It seemsthat patterns of lipid elements may be associated with thespecificity of lung cancer and stage rather than the intactlipid pathways We performed | 2 |
Scars Burns HealingVolume 1011772059513120940499 reuse guidelinessagepubcomjournalspermissions The Authors journalssagepubcomhomesbhKeloids are pathological scars that grow over time and extend beyond the initial site of injury after impaired wound healing These scars frequently recur and rarely regress They are aesthetically disfiguring can cause pain itching discomfort as well as psychological stress often affecting quality of life Many treatment modalities including surgical and nonsurgical have been explored and have been reported to be beneficial however none have been absolutely satisfactory or optimal for the treatment of all keloid subtypes to date This poses a major challenge to clinicians Often a combinational therapeutic approach appears to offer the best results with higher patient satisfaction compared to monotherapy The aetiopathogenesis of keloids is not fully elucidated however with recent advances in molecular biology and genetics insight is being gained on the complex process of scar formation and hence new therapeutic and management options for keloids In this paper we explore the literature and summarise the general concepts surrounding keloid development and review both current corticosteroids surgical excision siliconebased products pressure therapy radiotherapy cryotherapy laser therapy imiquimod and 5fluorouracil and emerging stem cell therapy mitomycin C verapamil interferons bleomycin botulinum toxin type A and angiotensinconverting enzyme inhibitors treatments Increased knowledge and understanding in this area may potentially lead to the discovery and development of novel therapeutic options that are more efficacious for all keloid typesKeywordsKeloids scar recurrence wound healing treatment managementLay SummaryKeloids are problematic scars that are difficult to treat and manage The aetiopathogenesis of keloids is not clear however recent advances in molecular biology and genetics are beginning to shed light on the underlying mechanisms implicated in keloid scar formation which will hopefully lead to the development of treatment options for all keloid types This review summarises current and emerging therapiesIntroductionWound healing is an intricate and complex series of processes comprising overlapping phases of inflammation granulation tissue formation and tissue remodelling and results in tissue structure integrity and damage being restored1 Abnormal wound healing can give rise to keloids which are benign dermal fibroproliferative nodular lesions that tend to recur after excision Keloid scars Faculty of Medical Sciences The University of the West Indies Cave Hill Campus Bridgetown Barbados West Indies Pine Medical Centre 3rd Avenue Belleville St Michael Barbados West IndiesCorresponding authorNkemcho Ojeh Faculty of Medical Sciences The University of the West Indies Cave Hill Campus PO Box St Michael Bridgetown BB Barbados West Indies Email nkemchoojehcavehilluwieduCreative Commons Non Commercial CC BYNC This is distributed under the terms of the Creative Commons AttributionNonCommercial License creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0c Scars Burns Healinginfluence in keloid aetiology9 Although no one specific gene has been associated with the development of keloids a number of genes and gene loci have been identified610 Genomewide association studies and admixture mapping studies have identified singlenucleotide polymorphisms across certain loci genetically linked to keloid development including the NEDD4 gene which encodes E3 ubiquitin ligase enzyme and the myosin genes MY01E and MYO7A10 Studies have also reported the involvement of several human leucocyte antigen HLA alleles p53 bcl2 and fas genes1014 Furthermore rare genetic disorders have been reported to present with spontaneous keloids including Dubowitz syndrome Bethlem myopathy RubinsteinTaybi syndrome Noonan syndrome Geominne syndrome and others1017 These lines of evidence suggest that genetic factors play a role on keloid predispositionPathophysiology of keloidsKeloid pathology is complex involving both genetic and environmental factors Keloids form as a result of abnormal wound healing and excessive dermal fibrosis Development of keloids has been linked to overproliferation and reduced apoptosis of dermal fibroblasts overproduction of collagen fibres and other extracellular matrix ECM components as well as abnormal ECM production and remodelling1 Various cytokines growth factors and proteolytic enzymes have been implicated in the formation of keloids including transforming growth factor TGF epidermal growth factor EGF vascular endothelial growth factor VEGF plateletderived growth factor PDGF connective tissue growth factor CTGF tumour necrosis factorα TNFα insulinlike growth factor1 IGF1 fibroblast growth factor FGF interleukin6 IL6 and matrix metalloproteinases MMPs31418 Furthermore signalling pathways such as Tolllike receptor signalling SMAD signalling and fibronectin have been reported to be associated with keloid development1819Histopathology of keloidsHistologically keloids comprise an abundance of unordered dermal collagen and vasculature with high inflammatorycell infiltrate and overactive mesenchymal cells1415 In addition to collagen elastin fibronectin and proteoglycans are deposited in excess amounts in keloid scars9 Collagen creates frequent crosslinks in ordinary wounds whereas collagen is irregularly anised in keloids forming nodules in the dermis20 During normal Figure Earlobe keloids as a consequence of ear piercingarise from skin trauma or inflammation and may develop years after the initial insult and rarely regress2 The scar tissue extends beyond the original wound site and can be disfiguring and cause psychosocial issues impairing quality of life3 In addition patients may present with symptoms such as burning pain pruritus movement limitation and hyperaesthesia2AetiologyThe aetiology of keloids is still poorly understood The most common regions of the skin for keloids include upper arms skin overlying joints chest shoulders and headneck regions particularly the ear lobes Figure The anatomical location of a keloid appears to alter its morphological characteristics Some keloids can develop spontaneously however most occur years after local trauma and other events including inflammation surgery burns elective cosmesis foreign body reactions acne insect bites vaccinations or mechanical force45Epidemiology and keloid geneticsThe incidence of keloids is highest among darkerpigmented persons of African Asian and Hispanic descent and is estimated to be in the range of Males and females have an equal risk of developing keloids6 although incidence is slightly increased in females likely attributable to them having more cosmetic procedures like ear piercing7 Persons aged years are also at a higher risk of developing keloids Additional risk factors include having blood type A hyperIgE and hormonal peaks during pregnancy or puberty8Familial keloid case studies and twin studies support the notion that genetic factors have an 0cOjeh for the treatment of keloids2627 Intralesional TAC injections have been shown to reduce scar volume and height improve scar pliability and diminish associated scar pain and itching8 as well as prevent recurrence3 Corticosteroids have antiinflammatory and antimitotic properties1 Several other mechanisms have been reported by which corticosteroids reduce keloid scar including inhibition of fibroblast growth attenuation of procollagen and glycosaminoglycan synthesis reduction of endothelial budding and enhancement of collagen and fibroblast degeneration2829 Corticos teroids inhibit TGF1 expression and induce apoptosis in fibroblasts inhibit VEGF and alphaglobulins which are involved in the wound healing process28 VEGF which promotes angiogenesis was reported to be highly expressed in keloid fibroblasts compared to controls but exogenous addition of the glucocorticoid dexamethasone suppressed its expression in vitro32 Furthermore VEGF expression was overexpressed in keloid tissue which later reduced following intralesional TAC injections in vivo33TAC is typically administered at intervals of weeks until pruritic and painassociated symptoms diminish and the scar flattens34 The dose of TAC is in the range of mgmL depending on the size and anatomical location of the lesion and the age of the patient34 TAC is used either alone as a monotherapy or in combination with other treatment modalities13 The response rates to corticosteroid injections vary clinically with regression rates in the range of reported after one year and recurrence rates in the range of reported after five years3 Combined therapy comprising surgical excision followed by TAC treatment also varied with reported recurrence rates in the range of Previous clinical studies where TAC was used alone reported efficacy and good clinical outcome with this treatment including reduced keloid height length width related pruritus and erythema and improved pliability3637 A recent randomised parallelgroup study that compared the role of intralesional TAC fractional CO2 laser or intralesional verapamil in the treatment of keloid in patients showed reduction in scar height vascularity and pliability in all three groups using the Vancouver Scar Scale score however pigmentation was not completely resolved with any of the treatments The response was fastest with TAC followed by verapamil and laser and this was statistically significant38 However intralesional TAC in combination with other treatment modalities such as 5fluorouracil 5FU pulsed dye laser PDL surgery interferon IFNα2b verapamil and Figure Current and emerging treatment strategieswound healing the early wound immature collagen type III can be modified into mature collagen type I In keloid tissue it mostly comprises disanised collagen types I and III made up of palestaining hypocellular collagen clusters lacking nodules or surplus myofibroblasts21 Furthermore recent research has provided four distinct findings only present in keloid specimens presence of keloidal hyalinised collagen presence of a tonguelike advancing edge underneath normalappearing epidermis and papillary dermis a horizontal cellular fibrous band in the upper reticular dermis and a prominent fascialike band22Treatment of keloidsCurrently various forms of treatment for keloids exist however no single treatment has proven to be the most effective This review will explore and discuss current and emerging treatment modalities Figure Some of the ongoing or completed clinical trials of keloid therapy registered on clinicaltrialsgov and accessed on May are summarised in Table Studies with the status terminated suspended or withdrawn were excludedCurrent treatmentsCorticosteroidsSeveral corticosteroids can be used for the treatment of scars including triamcinolone acetonide TAC hydrocortisone acetate dexamethasone and methyl prednisolone24 However since TAC has been the most widely used corticosteroid 0c Scars Burns HealingTCNVI esahPTCNlebacilppa toN vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoC yaMnodessecca vogslairTacinlil Cnoypareht doek fo slairt lliacinilC elbaTeltit ydutSreifitnediTCNlebacilppa toN ybdewoll iednotecaenoonicmairt llanoiseartnliilsdoeK fo tnemtaerTeht n iof resal OC lanoitcarFidoeKli sdoretS lanoiseartnl iI svyparehTdoretSdetsissA resaL lanoitcarFTCNlebacilppa toNTCNlebacilppa toNTCNVI esahP TRS ypareht noitadar liaicifrepuS iEnmativ enocilis enositrocordyh yparehtyhcarbetaresod ihgh tnavudahtij wnoisicxe lacigruS cihportrepyh idoeKlidoeKlidoeKl TRS yparehTnoitadaRi l laicifrepuS fonoitauavEevitcepsorteRA ilsdoeK fo tnemtaerT rof yparehtyhcarBetaResoDhgHi isracSdoeKdnal dnaytili llbareoTeht gnitauavEydutSevitarapmoCdezimodnaRA TCNlebacilppa toN llamsaphcir teetap suogootuAllidoeKl llllamsaPhciR teetaP suogootuAyb racSdoeK fo tnemtaerTli TCB®tiKnegeRhti wdenatboiTCBtiKnegeRhti wdenatbOi l ieg tcartxenono ro draugracS noitolamredeM xirtaciCracScihportrepyh ildna sdoeK fo tnemtaerTeht rof separehTi l acipoTowT foycaciffE sracScihportrepyHTCNI esahP muiclac fonoitcenj i laruomutartnIidoeKlrefsnartortceeybdewol llof edirohclTCNIII esahP dnaenoonicmairt llanoiseartnlIidoeKl ienodnefriP l ilsdoeK fo tnemtaerTeht rof noitaroportceEmuiclaCl acipoTdnaenoonicmairTl lanoiseartnl I foycaciffEienodnefrip lacipotnoitcenji UFidoeKllygooisyhpohtaPdna tnemtaerT gnirracSdoeKli iIOALSDAL sracSdoeK fo tnemtaerT roflTCNII esahP I esahP inks gnviltneavuqeiil dereyalib fargilpAsresalidoeKl iil tenragmunmuamuirttymumydoenideslupgno l dna OC lanoitcarFidoeKl fo tnemtaerTn ii sresaL tenraGmunmuAmuirttymumydoeNli fonoitneverPdna tnemtaerTeht rof farg ilpA foydutS toli PAisdoeKl ilsdoeKdesicxE foecnerruceR desluPgnoLdnaedixoDnobraCi l anoitcarFgnisUydutSA deunitnoC cihportrepyhTCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl eht rof eciveDdesaB saGnogrAnahti WyparehtoyrC lanoiseartnlI sracScihportrepyHdnadoeK fo tnemtaerTli TCNIII esahPxirtaciC fonoitacil ppa lacipoT sracs cihportrepyHcihportrepyhidoekl ilsracS sdoeKdna sracScihportrepyH fo tnemtaerTeht n i IXRTACCI sracScihportrepyHdnadoeK fo tnemtaerTli TCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl rof yparehtoyrC lanoiseartnl I foesUeht fonoitauavEevitcepsorPl 0cOjeh TCNVI esahPresal PTKmneht foycaciffE idoekl cihportrepyheahcun siliadoekl xirtacic xirtacic racs lacigrus racS reifitnedi vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoCdeunitnoC elbaTeltit ydutSTCNlebacilppa toNresal eyddesluP cihportrepyh idoeKl desluPmnagnisU tnemtaerT racSnohtdWesluP fo tceffEi TCNlebacilppa toN thgil AVUderutcafunamnamreG idoeklsracs amredorelcSsnoitidnoC rali imSdnaamredorelcS fo tnemtaerT rof thgLAVUi resaLeyD ecivedgnitti me gnisorbif rehtosnoitidnocTCNlebacilppa toN ecivederusserPTCNVI esahPl egenocilis lacipoT cihportrepyh idoeKlilERUSSERP sdoeK raE fo tnemtaerTeht n i eciveDerusserPidoeKl fo tnemtaerTeht no l eGenocili iSgnyrdfleS tnerapsnarT fo tceffEsracssracS l ianmodbAcihportrepyHTCNlebacilppa toNypareht noitadaRiidoeKl yrtsigeRnoitadaRdoeKlii TCNIII esahPnosahtemateB fonoitacil ppa lacipoT sracs cihportrepyHsretehtaC suoneV lartneC retfA sracSTCNI esahPII esahPTCN I esahPII esahP dna sll dicacidisuf dnaetareavl isdoeklidocitrococuglnoitadarri iBVU amredorelcs idoeKl inkS fo tnemtaerTeht n i yparehT thgL BVUil B teovartlUi leraunnaamounargl sracs siliadoekl enca desilacolxirtaM l amreDderetlAhti w snoitidnoC lecFVS suogootua fo snoitcenjI axal situc racs inkSCSDAxirtacic idoekl idevireDesopdA suogootuAhtil waxaL situCdna sracS foyparehT sll eCmetS lamyhcneseMTCNlebacilppa toN gnitti meAVUderutcafunamnamreG amredorelcs idoeKlsnoitidnoC ralimetsys thgil lamounarg sracs enca leraunna imSdnaamredorelcS rof thgLAVUi ilteovartlu VU FVS sll ecnoitcarf raucsavl lamorts FVS etahpsohp lynatit muissatop PTK sll ecmets l amyhcnesemdeviredesopdai hserF fo tnemtaerTeht rof resaLPTKmn l evoNa foydutS to liP CSDA licaruoroulf UFsracS lacigruS 0c Scars Burns HealingA variety of methods can be used for the surgical removal of keloids depending on the size of the keloid anatomical location skin type and age of patient52 These include linear closure and flap coverage excision with grafting Wplasty and Zplasty53 To reduce risk of keloid recurrence the surgeon performing the excision should establish tensionfree wound closure As a general rule closure of the wound should be accomplished with minimal tension and sutures leaving everted wound borders Zplasties threelayered sutures subcutaneousfascial tensile reduction sutures or local ï¬ap surgery can be employed on a casebycase basis5455 The final outcome of the scar is often positively correlated with the experience of the operating surgeon and technique utilised as well as the patients active participation in their wound care52Siliconebased productsSince the 1980s siliconebased products have been used in the treatment of keloids and hypertrophic scars with silicone gel or silicone gel sheeting considered as firstline therapy for minor keloids and hypertrophic scars2635 There are various other forms of silicone including creams sprays gel cushion and liquid24 The precise mechanism of action of silicone products is not fully understood but it is proposed that they enhance hydration and create an occlusive environment53 which influences fibroblast regulation and decreases collagen synthesis56 Silicone gel sheeting has been shown to have minimal side effects including local irritation which can be resolved quickly24 Studies have shown that the beneficial effects of silicone gel sheets include pain reduction tenderness and pruritus and flattening the keloid57 The silicone gel sheeting is recommended to be worn from two weeks after primary wound treatment for h for months58Studies have demonstrated an improvement of up to in keloid scars after using silicone gel sheeting35 and a decrease in the incidence rates of keloids and hypertrophic scars after surgery59 In addition controlled studies have reported the clinical effectiveness of silicone gel and silicone gel sheeting in the prevention and treatment of keloids3558 However a recent metaanalysis review60 and Cochrane review61 found that even though studies published data in support of the efficacy of silicone gel sheeting in the treatment and prevention of keloid and hypertrophic scarring they provided weak evidence and were of poor quality60 Therefore given Figure Auricular keloid surgical excision used as monotherapy Auricular keloid before a and after b surgical excisonsurgery all yielded significant improvements compared to treatment with TAC monotherapy339Intralesional steroid injections can cause several adverse side effects such as telangiectasis atrophy steroid acne pigmentary changes necrosis ulcerations and systemic side effects3 There is also significant pain associated with intradermal corticosteroid injections that can be reduced when local anaesthetic lidocaine is administered to control the pain44 Furthermore the side effects have been reported to diminish when intralesional TAC is used in combination with 5FU45Surgical excisionSurgical excision is a traditional method of removing keloids Figure However excision creates a new wound and can result in a similar or larger keloid47 Therefore surgical excision is not recommended as a monotherapy as it results in high recurrence rates in the range of For better postoperative surgical outcomes surgical excision is often combined with other forms of treatment including radiotherapy intralesional corticosteroid injections IFN injection bleomycin cryotherapy pressure therapy and silicone gel or sheeting82649 Successful use of dermal substitutes and epidermal skin grafting with keloid excision has also been reported50 A recent case series study in which patients with anterior chest wall keloids were given a treatment protocol consisting of complete excision Zplasty postoperative adjuvant radiotherapy and postsurgical wound selfmanagement reported excellent outcomes with a recurrence rate of only The use of steroid tape and injections helped to resolve the recurrence of keloids51 0cOjeh the lack of substantial evidence welldesigned clinical trials and studies are required to gain a better understanding of the effectiveness of siliconebased products in preventing and treating keloidsPressure therapyPressure therapy has been used to treat and manage keloids and hypertrophic scars for decades3563 It has been routinely employed as firstline treatment in the treatment of hypertrophic scarring resulting from burns64 The underlying mechanisms of action of compression techniques remain unclear however several hypotheses exist some of which include increased pressure to the scar surface reduces perfusion and decreased oxygen to the location of injury reduces collagen synthesis It is also thought that pressure increases apoptosis reduces scar hydration stabilising mast cells and decreases angiogenesis165 The application of pressure can be achieved using a variety of materials such as adhesive plaster moulds pressure earrings and customfitted splints16 which have improved scar cosmesis and rates of keloid recurrence66 A continuous pressure of mmHg preferably at the lower range soon after wound reepithelialisation for h per day for months is recommended166869 The efficacy of pressure therapy depends mainly on the anatomical location of the scar with trunk and limb areas being more appropriate sites for pressure therapy In addition a pressure garment is predominantly used for auricular keloids where pressure clips are commonly utilised after surgery6670 As an adjuvant therapy this form of pressure garment has also been successfully used to prevent the recurrence of keloids6667 In contrast a metaanalysis review that analysed the effectiveness of pressure garment therapy for the prevention of abnormal scarring after burn injury was unable to demonstrate any beneficial effects of pressure garment therapy on prevention or treatment of abnormal scarring59 Notwithstanding success rates of pressure therapy are contingent upon patient compliance69 which can sometimes be low due to discomfort Overall pressure therapy is tolerated better and is devoid of the pain often associated with intralesional therapies and hence can be considered as a good adjuvant therapy for keloid scarsRadiotherapyIn the treatment of keloids using superficial Xray irradiation was first described71 Since then it has been used less frequently as a monotherapy and more widely as an effective adjunct treatment after surgical excision72 with success rates in the range of and recurrence rates of about Radioactive skin patches have also been used in combination with other treatment modalities for keloids7879 Radiotherapy is most commonly used h after surgical excision7480 and acts by suppressing angiogenesis and inhibiting ï¬broblast activity1 Decreased fibroblast proliferation induced cell senescence and apoptosis leading to reduction in collagen production and suppression of keloid development have also been reported81Different radiotherapy modalities have been used after surgical excision including electron beam radiotherapy brachytherapy superï¬cial and orthovoltage radiotherapy with varying degrees of success84 Mankowski et a0 al conducted a literature review of studies to compare the clinical outcome of different forms of radiation treatment used for the management of keloids77 The metaanalysis demonstrated that radiation used as monotherapy yielded higher rates of recurrence compared to combinational therapy with postsurgery excision Comparison between the different radiationbased treatments revealed that the lowest rate of recurrence was observed with brachytherapy followed jointly by Xray and electron beam The authors also reported that the rate of recurrence was dependent on anatomical site of the keloid with chest keloids having the highest recurrence rate73The adverse effects of radiotherapy often linked with dose of radiation used can be grouped into acute skin reactions and late complications Acute reactions arise as early as seven days after keloid treatment and include oedema necrosis ulceration desquamation erythema and pigmentary changes with the latter two being the most common Late complications which include changes in pigment atrophy telangiectasis and alopecia may present several weeks after radiotherapy Emollient and steroid ointment used after radiotherapy can help alleviate the side effects19 A recommended radiation dose Gy over several sessions can also minimise adverse effects1980 Radiotherapy carries a risk of malignancy8586 Therefore caution should be used in radiationvulnerable sites such as the head neck thyroid and breast and in patients aged years2 Protecting fragile ans and selecting the most appropriate sitedependent dose protocol can help minimise further complications of radiotherapy87 0c Scars Burns HealingCryotherapyCryotherapy is a lowtemperature treatment that causes vascular damage resulting in tissue necrosis88 It has been used to treat keloids as a monotherapy or in combination with other treatment methods such as intralesional steroid injections89 Various delivery methods used for cryotherapy include spray and contact probes or the intralesionalneedle cryoprobe method Compared to contact and spray methods intralesionalneedle cryoprobe was found to be the most effective method in treating keloid scars90 Positive outcomes were observed in a number of studies that used liquid nitrogen and cryotherapy to treat keloids with success rates in the range of External cryotherapy has been associated with several side effects including hypopigmentation blistering pain delayed healing and infection9093 Moreover larger keloids have been shown to need multiple cryotherapy sessions9093 To minimise side effects intralesional cryotherapy was introduced and there are now a number of nitrogenbased cryodevices that have been described for the treatment of keloid scars with two commercially available a liquid nitrogenbased device88 and an argon gasbased device94 The intralesional cryotherapy was designed to overcome the hypopigmentation seen mostly in darkskinned individuals with external cryotherapy It works by destroying the core of the keloid sparing the surface epithelial cells including melanocytes9596 As a result it enhances volume decrease while minimising the risk of hypopigmentation and other surface reactions90 A recent comprehensive review based on the preferred reporting items for systematic reviews and metaanalysis was performed to investigate the efficacy of intralesional cryotherapy on keloid scars90 The review of eight studies that met the inclusion criteria revealed that average scar volume decreased in the range of but complete eradication of the scar on average was lacking Recurrence of keloid scars was in the range of The authors also reported that patients complaints of pain and pruritus was considerably reduced however hypopigmentation was seen mostly in Fitzpatrick skin type patients after treatment90Laser therapyLaser therapy for keloid treatment was introduced in the 1980s97 Since then different systems have been used for the treatment of keloid and hypertrophic scars4898 These lasers target skin chromophores like haemoglobin and melanin based on the principle of selective photothermolysis99 Lasers can be classified as ablative and nonablative The most common ablative lasers include the 2940nm erbiumdoped yttrium aluminium garnet ErYAG laser and the 10600nm carbon dioxide CO2 laser These emit a laser beam that is absorbed by water in the skin leading to local tissue destruction and reduction of lesion volume3 Common examples of nonablative lasers include 585nm or 595nm PDLs 1064nm neodymiumdopedyttriumaluminiumgarnet NdYAG neodymiumdopedvanadate NdVan laser and nm Qswitched NdYAG laser with low fluence100 These lasers induce thermal injury to the scars microvasculature leading to thrombosis and ischaemia which result in collagen denaturation and collagen fibre realignment101532nm laser Laser therapy requires several treatments at intervals of weeks depending on scar type and type of laser used98104 with possible side effects including itching pigmentary changes blister formation and postoperative purpura98 The use of the nonfractional vascular nm PDL in the treatment of keloid and hypertrophic scars has been welldocumented105 and has response rates in the range of PDL monotherapy has been shown to be effective108110 as well as CO2 laser monotherapy38111112 In a clinical study where patients with moderate to severe keloids were treated with highenergy pulsed CO2 laser the treatment was efficacious and welltolerated with minimal side effects112 In other studies where CO2 laser ablation was compared with other forms of treatment CO2 laser was as efficient as the other forms38111 It must be noted however that these studies are small and randomised controlled studies are lacking98Laser therapy such as PDL CO2 and NdYAG have been associated with a high rate of recurrence at months111113 However optimal results can be achieved with combination treatment especially with intralesional TAC injections116 Kumar and coworkers conducted a cohort study on patients with keloids previously treated with an NdYAG laser and reported complete scar resolution and flattening in seven patients only when intralesional TAC was used after laser therapy41 Moreover combined therapy with PDL and TAC119 and PDL TAC and 5FU36 were shown to produce better clinical results In a recent study that evaluated and compared the efficacy of combination therapy of 0cOjeh scars statistically fractional CO2 laser and intralesional TAC injection or TAC injection alone in keloid and hypertrophic significant improvements were reported in overall scar quality with the combined treatment options comto TAC monotherapy120 Moreover pared combined CO2 laser and IFNα2b injections given to patients with auricular keloids resulted in no recurrence in of patients three years after treatment121 Laser therapy can also be combined with other laser treatment topical corticosteroids and cyanoacrylate glue98 and have shown promising results however larger controlled clinical studies are needed to further evaluate their efficacy and safetyRecently lasers are also being explored as tools for assisted drug delivery Kraeva et a0al proposed an alternative technique of corticosteroid administration of laserassisted drug delivery of topical TAC This was shown to be effective when used on a keloid on the posterior scalp of a patient after each CO2 laser session122 More efficient intraepidermal drug delivery options are also being investigated Singhal et a0al developed TACcontaining polymeric microps that were prepared using a cryomilling technique for freezing fracture After ablation with a fractional ErYAG laser these microps can be deposited in cutaneous micropores and provide highdose intraepidermal reservoir systems with minimal transdermal permeation leading to sustained and targeted local drug delivery123It must be noted that one of the biggest limitations in studies available at present is the lack of histological definition between keloid and hypertrophic scars so conclusions are not valid on efficacyImiquimod creamImiquimod cream is approved for the treatment of basal cell carcinoma actinic keratoses and genital warts21 As an immuneresponse modifier it stimulates the production of proinflammatory cytokines such as TNFα interleukins and IFNs by activated Tcells124 thereby changing the expression of genes associated with apoptosis125 and reducing collagen production16 Studies have reported conflicting findings regarding efficacy of imiquimod cream postoperatively following keloid excision likely due to keloid location Many studie | 2 |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | 2 |
ovarian cancer is a silent and largely asymptomatic cancer leading to late diagnosis and worseprognosis the latestage detection and low survival rates makes the study of the spacetime evolution of ovariancancer particularly relevant in addition research of this cancer in small areas like provinces or counties is still scarcemethods the study presented here covers all ovarian cancer deaths for women over years of age in the provincesof spain during the period spatiotemporal models have been fitted to smooth ovarian cancer mortalityrates in age groups [ [ [ and [ borrowing information from spatial and temporal neighboursmodel fitting and inference has been carried out using the integrated nested laplace approximation inla techniqueresults large differences in ovarian cancer mortality among the age groups have been found with higher mortalityrates in the older age groups striking differences are observed between northern and southern spain the globaltemporal trends by age group reveal that the evolution of ovarian cancer over the whole of spain has remainednearly constant since the early 2000s differences in ovarian cancer mortality exist among the spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are essential to discoverinequalities in ovarian cancer mortality women over years of age should be the focus of followup studies as themortality rates remain constant since highmortality provinces should also be monitored to look for specific riskfactorskeywords agespacetime models disease mapping inla ovarian cancer mortality smoothing the number and scientific impact of publications on ovarian cancer are continuously increasing not in vainovarian cancer is the eighth most common cancer inwomen and the 18th most frequent overall with nearly new cases worldwide in around of cases are concentrated in developed countries whereovarian cancer is the most lethal gynecological tumorcorrespondence lolaunavarraes1department of statistics computer science and mathematics publicuniversity of navarre campus de arrosadia pamplona spain2inamat public university of navarre campus de arrosadia pamplonaspainthe highest incidences are found in northern and easterneurope austria the czech republic germany irelandlatvia lithuania the nordic countries slovakia and theuk and the united states whereas in africa andasia this tumor is virtually nonexistent this pattern isattributed mostly to the low birth rates found in developedcountries in the past years the overall number of tumors hasundergone a constant increase in spain this is due notonly to the population growth but also to the increasedlife expectancy and the use of early detection techniquesin the estimated number of new cases of ovarian the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0ctrandafir bmc public health page of cancer was representing of all female cancersbeing the fifth cause of cancer deaths in women afterlung breast colon and uterine tumors [ ] the agestandardized incidence rate calculated using the directmethod and the world standard population is per women which may be considered high as forits temporal evolution there was a slight decline between and but since then mortality rate starts toincrease slowly but constantly in addition there is a greatdeal of variability among the spanish provinces for example during the period one finds a rate of per women in the canary islands and a rate of per women in cuenca ovarian cancer is a disease affecting mostly older postmenopausal women with more than of cases beingdiagnosed in women over according to medicalexperts it is a silent and mostly asymptomatic cancer acircumstance that leads to late diagnosis and worse prognosis furthermore in the cases where symptoms doappear these may be confused with digestive problemsbloating early satiety abdominal andor pelvic pain orbenign gynecological alterations such as endometriosis orpolycystic ovary syndrome to date no method for earlydetection is available this is reflected in the fact that upto of cases are detected in the advanced stages of thedisease the etiology of ovarian cancer is poorly understoodhowever several factors associated with an increased riskof ovarian cancer have been identified age number ofovulations early menarche infertility low parity theuse of hormone replacement therapy hrt obesity physical inactivity a family history of breast andovarian cancer including brca1 and brca2 gene mutations and past and current smoking associations between exposure to asbestos in the workplace orat home and ovarian cancer have also been found further research is needed to corroborate this finding inspain as most jobs with a high exposure to asbestos arepredominantly maledominated eg mining milling orshipyard work nevertheless a study of asbestos exposure among italian women found that the main factorwas secondhand contact due to occupationally exposedrelatives for example from soiled work clothes broughthome as of this writing however there is no documented registry for asbestos exposure in the workplaceor at home in spain some epidemiological studies havedetected an increased risk of ovarian cancer in womenwith less exposure to sunlight and consequently with lowlevels of vitamin d in particular the higher the latitudethe less overall accumulated sunlight and the higher theincidence of ovarian cancer some protective factors against ovarian cancer have also been identified suchas multiparity oral contraceptives and tubal ligation orhysterectomy the 5year agestandardised relative survival in spain is estimated at less than similar tothe european average of as mentioned aboveovarian tumors are the eighth cause of cancer deathsamong women worldwide with deceases registered in overall among malign tumors in about women died yearly in spain from ovarian cancer representing of all cancer deaths and of all deaths among women the mean age of decease from ovarian cancer inspain is years cabanes analyzedthe ageadjusted mortality trends of ovarian cancer inspain for the period ovarian cancer caused deaths in this period with rates in women over showing a tenfold increase versus those in youngerwomen in women under rates increased peryear until and afterwards started to drop at a rateof per year in the age group mortalityrates significantly increased annually up to andbecame stable thereafter in women and older mortality rates increased annually up to the year anddecreased per year afterthe disproportionate impact on older women togetherwith the aforementioned concerns regarding latestagedetection and low survival rates makes the study of thespacetime evolution of ovarian cancer particularly relevant in addition it is important to mention that agegroups are not equally affected by ovarian cancer mortality and then it is necessary not just to standardize by agebut to analyze the different age groups hence the maingoal in this paper is to study the temporal evolution of thegeographical patterns of ovarian cancer mortality rates infour age groups of women aged years or moremethodsdata sourcethe study presented here covers all ovarian cancer deathscode c56 of the 10th edition of the international classification of diseases for women over years of agein the provinces of spain excluding the autonomouscities of ceuta and melilla recorded throughout theperiod by the spanish statistical officestatistical analysisa bayesian hierarchical spatiotemporal model is used toestimate rates the model is briefly described in whatfollows for better interpretation of resultsspain is divided into s provinces indexed by i s and data are available for t27 time periods corresponding to years labeled as t tfor each age group let nit represent the population at riskfor region i and time period t then conditional on themortality rates rit the number of ovarian cancer deathsoit is assumed to follow a poisson distribution with mean 0ctrandafir bmc public health page of table descriptive statistics of observed cases and crude mortality rates per women disaggregated by age groups provinceand year min minimum q1 first quartile q3 third quartile max maximumobserved casescrude ratesage group[ [ [ [ [ [ [ [ minq1medianmeanq3maxμit nitrit that isoitrit ¼ poissonμit nitritlog μit log nit log ritwhere the lograte is modelled aslog rit α ξi γt δithere α denotes the logarithm of the overall rate ξi andγt are the main spatial and temporal effects respectivelyand δit corresponds to the spacetime interaction effectssince each of these components are supposed to be gaussian markov random fields gmrf the integratednested laplace approximation inla technique hasbeen used for model fitting and inference specifically theleroux car prior distribution has been considered for the spatial random effects and a firstorderrandom walk prior distribution for the temporal randomeffects in addition the four different types of interactionintroduced by knorrheld have been considered forthe spatiotemporal random effects these interactionsallow the spacetime effects to be completely independenttype i interaction structured in time but not in spacetype ii interaction structured in space but not in timetype iii interaction or completely structured in spaceand time type iv interactionall the computations have been done using the interactive web application sstcdapp which can befound at httpsemisstcdappunavarraeslogin thisapplication provides a user interface for the analysis ofspatiotemporal areal count data allowing to fit a widevariety of spacetime models using the inla estimationtechnique in addition the application provides differentmodel selection criteria in this paper the model with the[[[[nemow rept shaed etar edurcyearfig temporal trends by age groups temporal trend of the ovarian cancer mortality crude rates by agegroups 0ctrandafir bmc public health page of lowest value of the deviance information criterion dic has been selected for further details about modelspecification prior distribution of the hyperparametersidentifiability constraints and additional model selectioncriteria see for example adin and the referencesthereinresultsa total of ovarian cancer deaths were registered inthe population of spanish women over years of age during the period since ovarian cancer is mainlyrelated to the onset of menopause the age groups we areconsidering in this paper are [ [ [ and [ a brief summary of observed cases and mortality rates per women by age groups provinceand year is shown in table clear differences areobserved in the mean and median mortality rates amongthe youngest and oldest age groups with values ranging from cases per women up to casesper women approximately respectively figure displays the global temporal trend of crude rates by agegroup here the different behaviour of the age groups iseven more evident a pronounced slope from the lastdecade of the twentieth century to the beginning of thetwentyfirst century is observed in the older age groupsmodel was fitted to smooth spatiotemporal rates ineach age group the interaction considered in the modelwas chosen on the basis of the dic values for each subgroup of ageclass the dic pointed toward a type ivinteraction for age groups [ [ and [ whereas a type ii interaction was selected for the agegroup [ to make the different terms in all themodels comparable a decomposition of the estimated logrates was computed by defining posterior spatial ξi t and spatiotemporal δtemporal γ it patterns see adini γ so that log rit α ξit note thatexpα represents the overall mortality rate for the wholeof spain during the period in order to facilitate interpretation of the results a map of spain showingits provinces is given in fig t δla coruñalugopontevedraorenseasturiasleoncantabriavizcayaguipuzcoaalavanavarrapalenciaburgosriojahuescaleridageronazamoravalladolidsoriazaragozabarcelonasegoviatarragonasalamancaguadalajaraavilamadridcacerestoledocuencabadajozciudad realalbaceteteruelcastellonvalenciaalicantebalearescordobajaenmurciahuelvasevillagranadaalmeriamalagacadizsanta cruz de tenerifepalmaslasfig administrative division of spain map with the administrative division of spain showing provinces source map was generated by the authorsusing the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of figure shows the map with the posterior meanieestimates of provincespecific mortality ratesexpα ξexceedance probabilitiesofrate being greaterthanthe overall spanish rate have also been computedsee fig i posteriorthis provincespecificthe estimated spatial pattern draws attention toasturias as a high ovarian cancer mortality rate provincefor all age groups in the age group [ the highest spatial rates are found in the northwestern provincesasturias lugo and la coruña but also in vizcaya andhuesca over cases per women in age group[ the regions with the highest estimated rates areasturias the balearic islands and valencia with an estimated rate of over cases per women in thethird age group [ the highest rates are located inthe centralnorthern areas with salamanca and asturiasleading the ranking and in the canary islands tenerifeprovince all of them with more than cases per women the oldest age group [ exhibits high rateareas in asturias barcelona gerona and guadalajarawith a rate of over cases per women all ofthese highrate provinces have a rate significantly higherthan the overall spanish rate in their respective age groupssee fig in general northern spain has greater ovarian cancermortality rates compared to the southern regions thelowest rates are found in guipúzcoa for age groups [and [ and in almerÃa for age groups [ and[ the northwestern province of la coruña showsage group [age group [ to to to to to to to to age group [age group [ to to to to to to to to fig provincespecific mortality rates estimates by agegroups posterior mean estimates of provincespecific mortality rates expα ξsource maps were generated by the authors using the library tmap from the r statistical software version no licenses are required touse or publishi 0ctrandafir bmc public health page of age group [age group [[][[[[[][[[[[][[[[age group [age group [[][[[[fig provincespecific posterior exceedance probabilities by agegroups posterior exceedance probabilities of each province in comparison withthe spanish overall rate pexpα ξstatistical software version no licenses are required to use or publish expαo source maps were generated by the authors using the library tmap from the ria surprising behaviour with high rates in the age group[ but one of the lowest rates in the age group [the estimated global temporal pattern expα γ t isvisualized in fig rates seem to have decreased duringthe last few years from to in age group [ but have remained nearly constant in the other threeage groups which experienced a sharp increase in ratesfrom up to the beginning of the twentyfirst centuryapproximatelyfigures and display maps showing the spatiotemporal evolution of ovarian cancer mortality rates foreach spanish province for the period dividedinto intervals of years for age groups [ [ [ and [ respectively specifically these mapsrepresent the posterior mean of rit expα ξi γ t δit the corresponding maps of probabilities showing theprobability that a particular rate in a given province andyear is greater than the spanish rate during that periodare not shown here to conserve space however we haveclassified a province as having a rate significantly greaterthan the spanish rate if this probability is greater than for age group [ we find that the mortality ratefor the period was highest in the northeasternregion with the province of huesca having a significantrate that lasted until in the latter years of the periodstudied only asturias showed a significant rate withinthis age group the percentage of the rates variabilityexplained by the spatiotemporal term was implying that the specific temporal evolution of each provinceis rather high in this age group 0ctrandafir bmc public health page of age group [age group [age group [age group [fig temporal trends estimates by agegroups for the whole of spain posterior mean estimates of yearspecific mortality rates expα γ and credible intervals dotted lines provide the estimated rate in each age group in the whole period in spain expαt to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publishin age group [ rates exhibit a greater variability among provinces between cases per women and about cases per women thehighest rates occurred in the late 1990s and early 2000smainly in the northern half of spain and in the balearicislands asturias lugo salamanca valladolid huescateruel gerona and the balearic islands show significantlyhigh rates at the end of the periodfor women between and years of age the lowest rates are found at the beginning of the study periodwith significantly low rates the highest significant ratesare located mainly in the northern and western parts ofspain and in the canary and balearic islands in the period the provinces with the lowest rates at the endof the period are gerona and madridwomen over years of age show significantly low ratesbetween again asturias shows the highestmortality rate at the end of the period some provinceslocated in southern spain mainly along the coastlineshow significantly low ratesthe temporal evolution of the rates for some selectedprovinces are plotted in fig the colors used in thebands are associated to the posterior exceedance probabilities of each province at year t in comparison withthe temporal pattern for the whole of spain in that yearnamely prit α γ t o in the case of barcelonathe probability that the mortality rate lies above the spanish rate reaches a maximum between for agegroups [ and [ and between forage groups [ and [ madrid behaves similar tospain in all age groups in asturias the probability is quitehigh for all age groups with a significant rising trend forage group [ from onwards la coruña showssignificantly low rates during the whole study period forthe age group [ whereas this provinces behaviourfor women over years of age is similar to the spanishratediscussionthe ovaries are one of the cancer sites where knownrisk factors are not enough to explain all the cases andthus spatiotemporal analyses provide additional important information allowing for the examination of thespatial temporal and spatiotemporal mortality patternsas the age groups are not equally affected by ovarian cancer mortality it is necessary not just to standardize by age but also to analyze the different agegroupsresults show large differences in mortality among theage groups with higher mortality rates in the older agegroups indeed the last age groups women of more than years of age double the rate of women between and more than cases in the last age groups vs casesper women on average in age group [ thiscan be explained by poor survival rates of gynaecologicalcancers in the elderly which in turn is influenced by late 0ctrandafir bmc public health page of barcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñafig temporal evolution of ovarian cancer mortality rates estimates for some selected provinces barcelona madrid asturias and la coruñatemporal evolutions of the posterior mean estimates of mortality rates rit for some selected provinces and twosided credible intervals for agegroups [ first row [ second row [ third row and [ fourth row the colors used in the bands are associated to theposterior exceedance probabilities of each province in time t in comparison with the temporal pattern of spain that is prit expα γ orepresented with a red line in the graphstdiagnoses and the failure of treatments due to comorbidity[ ]the global temporal trends by age group reveal thatthe evolution of ovarian cancer over the whole of spainhas remained nearly constant since the early 2000s particularly for women aged years or more after a sharpincrease during the period approximatelythe stabilization of the rates may be due to an increasedconcern among women regarding their personal health inparticular among older women that led them to be testedmore frequently access to better information via massmedia and the internet and the increasing effectiveness of 0ctrandafir bmc public health page of cancer treatments in the nineties access to informationvia the internet was more limited and health care was possibly less advanced which could explain in part the sharpincrease in mortality rates in the first half of the periodmortality in women between and years of age showsa slight decrease since until nearly althoughthis decrease does not seem to be significant with respectto the average mortality in this age group it seems thatin fact during the last two years of the study period ratesare starting to rise slightly but once again this trend is notsignificant as yetthe global geographical patterns show in general thatthe north has higher rates than the south a situation similar to that observed overall in europe the variabilityobserved in all age groups between northern and southernspain remains unknown although in general women inthe south are prone to marry earlier and have more children on average interestingly asturias the provincewith the highest rates in all age groups is one of thespanish provinces with the lowest average number of children the differences observed between the north and thesouth could also be explained taking into account the relationship between exposure to sunlight provitamin dand ovarian cancer although spain is in general a sunnycountry there is a great variability between the northand south in terms of average daily hours of sunlight forexample bilbao in the north receives about hoursof sunlight per year while sevilla in the south receives thus the observed increasing trend with latitude might be at least partly explained by a cumulativeexposure to sunlightheterogeneity among the provinces regarding ovariancancer mortality rates can be elucidated at least in partby a heterogeneous distribution of other risk factorsthere are differences among provinces in the age at whichwomen have their first period menarche the averageage of first childbirth and the total number of children[] the average fecundity rate in spain has beenmarkedly declining in particular the areas registering thelowest fertility rates were the basque country asturiasnavarre and aragón [ ] we should also point outthat the age of first birthing is closely related to socioeconomic development and is steadily increasing navarreand the basque country are the regions of spain wherewomen delayed childbearing the longest additionally hormonal replacement therapy has proven to have animportant influence on the appearance of ovarian cancerin postmenopausal women [] however its use inspain has been very limited another risk factor is the presence of a family history ofthe disease hereditary ovarian cancer syndrome presenting a mutation in brca genes is important between of ovarian cancer cases are linked to bcra mutation women who are carriers of the bcra1 mutationhave a chance of suffering ovarian cancer before age in spain the accumulated risk of developing ovarian cancer before age has been estimated at ci in carriers of a mutation in bcra1 and ci in carriers of a mutation in brca2 the prevalence of brca1 and brca2 mutation in spainis heterogeneous and varies according to geographical origin moreover blay showed that the brca1 andbrca2 spectrum of mutations in asturias was largely different from other areas of spain this could also explainin part the high mortality rates found in this provincediez studying a large group of spanish patientsshowed that there is only a slight difference betweenthe percentages of deleterious mutations in brca1 andbrca2 genes and respectively however somevariation due to geographic origin is present with a higherproportion of brca1 in families from the northwesternpart of spain according to vega the differencesfound in galicia could be due to founder effectsovarian cancer is also linked to lifestyle habits tobaccoand alcohol consumption the smoking habit is a riskfactor for epithelial ovarian cancer with an odds ratio of ci [ ] differences in alcoholand tobacco consumption can be found among spanishprovinces all in all to better understand the etiology of the disease and to better determine the effect of risk factors onthe spanish female population it would have been helpful to have the medical and workplace histories of all thewomen participating in this study this is the main limitation of the current work on the other hand as thereis a lack of scientific studies analyzing the associationbetween ovarian cancer mortality rates and risk factorsin the domains analyzed here age groups provinces andyears spatiotemporal analyses by age groups are essential to discover inequalities in ovarian cancer mortality todetect provinces with high risks in each age group and tokeep track of how the rates are evolving with timesdifferences in ovarian cancer mortality exist amongthe spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are very useful to look forpotential risk factors associated to the observed geographical patterns and to allocate funds among spanish regionsfor future clinical and epidemiological practice we recommend to followup women over years as the mortality rates remain constant since highmortalityprovinces should also be monitored to look more closelyfor specific risk factors some risk factors for ovarian cancer like getting old or having a family history cannot bechanged however women may slightly decrease their riskby avoiding other risk factors for example maintaining 0ctrandafir bmc public health page of a healthy weight avoiding tobacco and alcohol consumption or not receiving hormone replacement therapy aftermenopauseabbreviationsaei spanish research agency brca breast cancer dic deviance informationcriterion ue european union inla integrated nested laplaceapproximations gmrf gaussian random markov field sstcdapp spatialand spatiotemporal count data application uk united kingdomacknowledgementswe acknowledge the spanish statistical institute for providing the dataauthors contributionsstudy conception and design mdu pct acquisition of the data mdu aaanalysis of the data mdu pct aa interpretation of the data mdu aa pctwriting the pct mdu critical revision of the all authorsapproved the final manuscript and the decision to submit the manuscriptfundingthis research has been supported by the spanish ministry of science andinnovation project mtm 201782553r aeifeder ue the content of thispaper is solely the responsibility of the authors and does not represent theofficial views of the spanish ministry of science and innovationavailability of data and materialdata have been provided by the spanish statistical institute at municipalitylevel under a contract and aggregated later up on reasonable request thecorresponding author will make the datasets available mortality data fromcancer and other causes from by sex and province is available in theinteractive epidemiological information system ariadna httpariadnacneisciiiesevindexhtml of the spanish national center for epidemiologyethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesbrüggmann d pulch k klingelhöfer d pearce c groneberg d ovariancancer density equalizing mapping of the global research architectureint j health geogr bray f ferlay j soerjomataram i siegel r torre l jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin national institute for health and care excellence nice ovarian cancerthe recognition and initial management of ovarian cancer httpswwwniceukguidancecg122resourcesovariancancerrecognitionandinitialmanagementpdf35109446543557 accessed jan dos santos silva i beral v socioeconomic differences in reproductivebehaviour iarc sci publ ferlay j soerjomataram i ervik m dikshit r eser s mathers c rebelom parkin dm forman d bray f globocan v10 cancer incidenceand mortality worldwide iarc cancer base no [internet] lyoninternational agency for research on cancer available from httpglobocaniarcfr accessed jan ferlay j steliarovafoucher e lortettieulent j rosso s coebergh jcomber hea cancer incidence and mortality patterns in europeestimates for countries in eur j cancer forman d bray f brewster dh gombe mbalawa c kohler b piñerosm steliarovafoucher e swaminathan r ferlay j cancer incidence infive continents vol x iarc scientific publication no available from httppublicationsiarcfr_publicationsmediadownload3743e886b2754a75a0f70e190e9b56e5346047319c17pdfaccessed jan ledermann j raja f fotopoulou c gonzalezmartin a colombo n cs european society for medical oncology esmo guidelines workinggroup newly diagnosed and relapsed epithelial ovarian carcinomaesmo clinical practice guidelines for diagnosis treatment and followupann oncol coleman m gatta g verdecchia a esteve j sant m storm h allemanic ciccolallo l santaquilani m berrino f eurocare3 summary cancersurvival in europe at the end of the 20th century ann oncol bouchardy c rapiti e blagojevic s vlastos a vlastos g older femalecancer patients importance causes and consequences of undertreatment j clin oncol meindl a ditsch n kast k rhiem k schmutzler r hereditary breast andovarian cancer new genes new treatments new concepts deutschesärzteblatt | 0 |
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes | 2 |
" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse 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psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev wada i igarashi a the social costs of child abuse in japan child youth servrev miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e1545fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studiesa primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin 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devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
"Range 41.684.2 41.684.2 41.681.7 44.282.9 41.684.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?1020% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%). We successfully determined protein levels in all 85 patients. Ten of the 81 eligible patients did not achieve assignment to treatment versus observation within the 84-day time interval from surgical resection. The time interval from surgery to assignment ranged from 86 days to 105 days in these 10 patients. For 3 patients the specimens were received after the 84-day limit had passed. For the other 7 patients the time interval from receipt to reporting ranged from 7 days to 25 days (median 18 days). For the 71 patients with a successful assignment within the 84-day time interval from surgical resection the time from receipt to reporting ranged from 3 days to 26 days (median 8 days). The reasons for reporting results in excess of 14 days were equipment failure and inadequate expression values in control specimens which required equipment recalibration and a repeat processing of the specimens. Overall the time from receipt of specimens to reporting ranged from 1 day to 27 days (median 11 days; mean 12 days) which is similar to that reported for patients with advanced NSCLC (range 1 day-47 days; median 11 days; mean 12 days).18 Survival and Toxicity Survival analyses were performed on the 61 patients who accepted assignment to treatment (44 patients) or surveillance (17 patients). Patients who rejected their treatment assignment withdrew consent and thus could not be followed for survival. Fourteen patients had DFS events; 2 had died (1 from disease recurrence and the other from cardiac disease without recurrence). The median follow-up among those patients still alive at the time of last follow-up was 27 months (range 3 months-44 months). Six patients had <?24 months of follow-up. The collective 2-year DFS and OS rates were 80% (95% confidence interval [95% CI] 67%-88%) (Fig. 2A) and 96% (95% CI 87%-99%) from the date of registration. The 2-year DFS rate was 83% (95% CI 68%-92%) for patients who received chemotherapy (Fig. 2B) and it was 71% (95% CI 43%-87%) for those observed (Fig. 2C). includes 2-year DFS estimates within each of the 3 gene expression categories in the chemotherapy arm. The median time from surgery to enrollment was 41 days (range 11 days-79 days). The time from surgery was added as a covariate to a Cox regression model and was not found to be significantly related to DFS (P?=?.22) or OS (P?=?.36). Disease-Free Survival Rates Patient Group No. DFS (95% CI) 1-Year 2-Year Accepted assigned treatment 61 88% (77%-94%) 80% (67%-88%) Received chemotherapy 44 95% (83%-99%) 83% (68%-92%) By protein level category (for those that received chemotherapy) ?Low ERCC1/low RRM1 20 95% (69%-99%) 84% (59%-95%) ?Low ERCC1/high RRM1 18 94% (65%-99%) 82% (55%-94%) ?High ERCC1/low RRM1 6 100% (100%-100%) 100% (100%-100%) Abbreviations: 95% CI 95% confidence interval; DFS disease-free survival; ERCC1 excision repair cross-complementing group 1; RRM1 ribonucleotide reductase M1. Kaplan-Meier survival estimates are shown. (A) Collective disease-free survival is shown for patients who accepted adjuvant chemotherapy or observation based on gene expression analysis. (B) Disease-free survival is shown for patients who received adjuvant chemotherapy. (C) Disease-free survival is shown for patients in the observation group. Conf Int indicates confidence interval. A total of 22 patients discontinued chemotherapy because of treatment-related toxicity (50%). None of the patients died because of treatment-related toxicity. Details are provided in . Number of Patients With Grade 3 and Grade 4 Adverse Events Among the 44 Patients Who Received Chemotherapya Level of Severity Adverse Event Grade 3 Grade 4 No. of patients with events 13 14 Type of events ?Neutropenia 11 6 ?Thrombocytopenia 4 4 ?Nausea 4 0 ?Vomiting 4 0 ?Anemia 2 0 ?Anorexia 2 0 ?Fatigue 2 0 ?Febrile neutropenia 1 1 ?Thromboembolism 1 1 ?Dehydration 1 0 ?Hearing impairment 1 0 ?Mucositis 1 0 ?Pleural effusion 1 0 ?Renal failure 1 0 ?Bradycardia (sinus) 1 0 ?Syncope 1 0 ?ALT elevation 1 0 ?Hypokalemia 1 0 ?Hyponatremia 0 2 Abbreviation: ALT alanine aminotransferase. a Adverse events were assessed according to the Common Terminology Criteria for Adverse Events (version 3.0). In Situ ERCC1 and RRM1 Protein Levels RRM1 levels ranged from 2.4 to 234.3 (median 39.7; mean 48.1) which were not significantly different from the expected values (median 40.5; range 8.3-96.2) (P?=?.87).16 ERCC1 protein levels ranged from 4.3 to 211.2 (median 41.9; mean 58.8) and these values were significantly different from the expected values (median 65.9; range 1.9-178.7) (P?=? 0.02). There was a significant correlation noted between ERCC1 and RRM1 levels (correlation coefficient 0.39; P?=?.0003) (Fig. 3) as previously reported.91618 Distribution of excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) levels in eligible patients is shown. The median protein levels of ERCC1 in adenocarcinomas squamous cell carcinomas and the other histologies were 34.257.1 and 121.5 respectively. The corresponding median levels of RRM1 were 38.142.6 and 48.9 respectively. Although the levels were higher in squamous cell carcinomas compared with adenocarcinomas the medians were not statistically significant (ERCC1: P?=?.16; RRM1: P?=?.72). DISCUSSION Disease stage is a predictor of benefit from adjuvant chemotherapy in patients with NSCLC. Patients with stage III disease derive the most benefit and those with stage I are reported to derive the least.1241923 Although not statistically significant for patients with stage I disease and a tumor diameter >?3 cm a numerical risk reduction of 7% has been reported and for those with tumors measuring ??3 cm a numerical risk increase of 40% has been reported.23 A significant treatment-related toxicity is febrile neutropenia which has been reported in 7% to 24% of patients.242022 Treatment-related deaths occur in 0.5% to 2% of patients.122022 The inclusion of molecular markers predictive of therapeutic efficacy into adjuvant decision algorithms would greatly improve the clinical benefit and reduce toxicity for patients with NSCLC. This approach is particularly attractive for patients with stage I disease in whom the parameters for weighing risks and benefits are to our knowledge the least well defined. Recent advances in molecular diagnostics have resulted in improved outcomes for patients whose tumors harbor mutations in oncogenic signal transduction molecules that can be inactivated by therapeutic agents. Similarly platinum agents target DNA and gemcitabine targets ribonucleotide reductase; both are unequivocally required not only for cellular proliferation but also for other essential cellular functions. Although to our knowledge specific oncogenic mutations have not been identified to date ERCC1 and RRM1 have emerged as promising predictors of efficacy for cisplatin and gemcitabine respectively. We conducted a phase 2 trial of treatment selection based on the levels of protein expression of ERCC1 and RRM1 for patients with completely resected stage I NSCLC and tumor diameters ??2 cm primarily to establish feasibility but also to evaluate preliminary efficacy as assessed by 2-year survival rates. We achieved our primary goal by demonstrating within a cooperative group environment that treatment assignment can be achieved for >?85% of patients within 84 days (12 weeks) the established timeframe for the initiation of adjuvant therapy from surgery in patients with NSCLC.1242022 At first glance our demonstration of feasibility should not be surprising. However it is important to note that surgical practice has not usually engaged a medical oncologist at the time of initial therapeutic planning but rather after complete recovery which substantially reduces the time available for molecular testing before the initiation of adjuvant treatment. We found no difference (P?=?.20) between academic and community sites in the time elapsed from surgery to the receipt of specimens in the reference laboratory (community sites: 57 patients; median 48 days [range 18 days-90 days]; academic sites: 24 patients; median 53 days [range 20 days-90 days]). The time elapsed from specimen receipt to reporting (median 12 days; range 1 day-27 days) was similar to our previous experience in an international trial of patients with advanced NSCLC (median 11 days; range 1 day-47 days).18 Based on these observations we conclude that the current process for routine specimen procurement handling and shipping to a reference laboratory requires substantial improvements to facilitate implementation of molecularly based therapeutic decision-making. For example a developing National Cancer Institute-sponsored project Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trial (ALCHEMIST) which will randomize patients with epidermal growth factor receptor-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC to targeted therapy or not will need to carefully consider these logistical issues. Prior results from adjuvant trials and a retrospective staging project in patients with stage I disease after complete surgical resection have reported 2-year DFS rates of 72% to 74%20 and rates of 68% to 75% for patients with stage IB disease.4 The corresponding 2-year OS rates were 80% to 88% for patients with stage I disease2024 65% to 90% for patients with stage IB disease242225 and 85% for those with stage IA disease.25 Thus our results of a 2-year DFS rate of 80% and OS rate of 96% appear favorable by comparison. However it is prudent to be cautious because we lost 20 of 81 patients from the survival analysis because of consent withdrawal and a direct comparison of outcomes data among trials cannot account for differences in study populations eligibility and staging criteria and provisions for data collection and analysis. The spectrum of protein levels for ERCC1 and RRM1 significant correlation of levels between both molecules and distribution of patients into the 4 gene expression categories in the current study is consistent with previous experience.91213161826 However the current analysis method for biomarker evaluation (ie antibody-based assessment of in situ protein levels) is not suitable for general clinical implementation for several reasons. First ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents and only 1 isoform appears to be involved in platinum-induced DNA damage repair.27 Second the monoclonal antibody 8F1 which is consistently used for ERCC1 protein expression analysis detects a second and unrelated protein that shares a common epitope with ERCC1.2830 This observation may account for the highly batch-dependent performance of this antibody1827 which may explain the significantly lower ERCC1 values in the current study compared with prior results.16 Third protein levels for RRM1 in particular and to a lesser degree for ERCC1 appear to be influenced by the specimen processing and handling procedures used at collection sites.26 Finally although the method for immunofluorescence-based quantitative detection of both molecules performs well if all specimens to be analyzed are processed simultaneously there is considerable interassay variability if specimens need to be processed individually over an extended period of time as required for real-time patient decision-making.18 However it is important to note that the biochemical biophysical and cell biological evidence for ERCC1 and RRM1 as predictive molecules for platinum and gemcitabine efficacy remains undisputed.51012273132 A small number of recent clinical trials have used ERCC1 prospectively for therapeutic decision-making. These include 2 randomized phase 3 trials in patients with advanced-stage NSCLC (1 published [NCT00499109]18 and the other terminated and unpublished [NCT00801736]) and 2 adjuvant trials 1 of which was a terminated and not yet published phase 2 trial [TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE) NCT00775385] and the other an ongoing phase 3 trial [International TAilored Chemotherapy Adjuvant trial (ITACA); EudraCT 2008-001764-36]. Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however the authors raised the possibility of a false-negative result because of an inexplicably divergent survival in an internal control group.18 The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms27 and specificity problems with the 8F1 antibody.2830 The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin-based control treatment with OS as the primary endpoint and a planned accrual of 700 patients. Results from these trials will help to further delineate the feasibility and technical issues mentioned above. The results of the current study demonstrated the feasibility of our biomarker-based decision algorithm in a multiinstitutional cooperative group environment for patients with surgically resected NSCLC. We identified that the current practice of evaluation and treatment for these patients may present an obstacle to rapid molecular-based decision-making. Although encouraging efficacy data emerged from this trial bioassays that specifically measure platinum-induced DNA damage repair must be developed before further clinical trials are launched that seek to tailor the use of these agents. FUNDING SUPPORT Supported by National Cancer Institute grants CA014028 CA016385 CA020319 CA022453 CA027057 CA032102 CA 035090 CA035119 CA035178 CA035261 CA035431 CA 038926 CA042777 CA045377 CA045560 CA045807 CA0 46113 CA046368 CA046441 CA063844 CA063848 CA0 67575 CA067663 CA073590 CA074647 CA076429 CA10 5409 and CA129343. CONFLICT OF INTEREST DISCLOSURES Dr. Bepler has a patent pending for the use of RRM1 and ERCC1 as biomarkers of treatment benefit for therapeutic decision-making in patients with cancer. 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16980606 14 Bepler G Sharma S Cantor A RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non-small-cell lung cancer J Clin Oncol 2004 22 1878 1885 15143080 15 Simon GR Sharma S Cantor A Smith P Bepler G ERCC1 expression is a predictor of survival in resected patients with non-small cell lung cancer Chest 2005 127 978 983 15764785 16 Zheng Z Chen T Li X Haura E Sharma A Bepler G The DNA synthesis and repair genes RRM1 and ERCC1 in lung cancer N Engl J Med 2007 356 800 808 17314339 17 Camp RL Chung GG Rimm DL Automated subcellular localization and quantification of protein expression in tissue microarrays Nat Med 2002 8 1323 1327 12389040 18 Bepler G Williams C Schell MJ Randomized international phase III trial of ERCC1 and RRM1 expression-based chemotherapy versus gemcitabine/carboplatin in advanced non-small-cell lung cancer J Clin Oncol 2013 31 2404 2412 23690416 19 Chemotherapy in non-small cell lung cancer: a meta-analysis using updated 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Preliminary results in the surgical setting Lung Cancer 2003 41 54 25 Chansky K Sculier JP Crowley JJ Giroux D Van Meerbeeck J Goldstraw P International Staging Committee and Participating Institutions The International Association for the Study of Lung Cancer Staging Project: prognostic factors and pathologic TNM stage in surgically managed non-small cell lung cancer J Thorac Oncol 2009 4 792 801 19458556 26 Bepler G Olaussen KA Vataire AL ERCC1 and RRM1 in the International Adjuvant Lung Trial by automated quantitative in situ analysis Am J Pathol 2011 179 69 78 21224045 27 Friboulet L Olaussen KA Pignon JP ERCC1 isoform expression and DNA repair in non-small-cell lung cancer N Engl J Med 2013 369 1101 1110 23514287 28 Bhagwat NR Roginskaya VY Acquafondata MB Dhir R Wood RD Niedernhofer LJ Immunodetection of DNA repair endonuclease ERCC1-XPF in human tissue Cancer Res 2009 69 6831 6838 19723666 29 Ma D Baruch D Shu Y Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology BMC Biotechnol 2012 12 88 23171216 30 Vaezi A Bepler G Bhagwat N CCT? is a novel antigen detected by the anti-ERCC1 antibody 8F1 with biomarker value in lung and head and neck squamous cell carcinomas Cancer 31 Reed E Platinum-DNA adduct nucleotide excision repair and platinum based anti-cancer chemotherapy Cancer Treat Rev 1998 24 331 344 9861196 32 Chen Z Zhou J Zhang Y Bepler G Modulation of the ribonucleotide reductase M1-gemcitabine interaction in vivo by N-ethylmaleimide Biochem Biophys Res Commun 2011 413 383 388 21893046 World J Surg Oncol World J Surg Oncol World Journal of Surgical Oncology 1477-7819 BioMed Central 24755441 3999735 1477-7819-12-108 10.1186/1477-7819-12-108 Case Report Surgical treatment of a solitary pulmonary metastasis from eyelid sebaceous carcinoma: report of a case Kaseda Kaoru 1 [email protected] Ohtsuka Takashi 1 [email protected] Hayashi Yuichiro 2 [email protected] Emoto Katsura 2 [email protected] Asakura Keisuke 1 [email protected] Kamiyama Ikuo 1 [email protected] Goto Taichiro 1 [email protected] Kohno Mitsutomo 1 [email protected] 1Department of Surgery Section of General Thoracic Surgery Keio University 35 Shinanomachi Shinjuku-ku Tokyo 160-8582 Japan 2Department of Pathology School of Medicine Keio University Tokyo Japan 2014 23 4 2014 12 108 108 19 2 2014 7 4 2014 Copyright © 2014 Kaseda et al.; licensee BioMed Central Ltd. 2014 Kaseda et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article unless otherwise stated. Background Ocular sebaceous carcinoma is an uncommon aggressive ocular neoplasm with potential for regional and distant metastasis. Case presentation A 77-year-old woman was found to have a solitary pulmonary lesion 6 years after the initial treatment of sebaceous carcinoma of the eyelid. Video-assisted lung wedge resection of an undetermined pulmonary nodule was carried out successfully. Microscopically the tumor showed foamy cytoplasm and atypical nuclei consistent with metastasis of eyelid sebaceous carcinoma. Conclusion This is the first case report of resected solitary pulmonary metastasis of eyelid sebaceous carcinoma. Pulmonary resection is a good option for the treatment and diagnosis of metastatic eyelid sebaceous carcinoma. Sebaceous carcinoma Lung metastasis Solitary metastasis Background Sebaceous carcinoma of the eyelid is a relatively rare malignant tumor and accounts for less than 1% of all eyelid tumors [1]. As well as being a rare tumor sebaceous carcinoma can mimic other benign inflammatory and malignant processes thus errors or delays in diagnosis are not unusual [2-5]. Although local management strategies for this tumor have previously been described [6-10] very few reports have focused on the patterns of metastasis of this tumor and the treatment strategies for such metastases [78]. " | 1 |
"adipogenesis is the process through which mesenchymalstem cells mscs commit to the adipose lineage and diï¬erentiate into adipocytes during this process preadipocytescease to proliferate begin to accumulate lipid droplets anddevelop morphologic and biochemical characteristics ofmature adipocytes such as hormoneresponsive lipogenesisand lipolytic programs currently there are mainly twomodels of benign adipocyte diï¬erentiation in vitro one isï¬broid pluripotent stem cells which can diï¬erentiate intonot only adipocytes but also muscle cartilage and othercells there are two kinds of ï¬broid pluripotent stem cellsbone marrow and adipose mesenchymal stem cells anothergroup is ï¬broblastic preadipocytes which have a single direction of diï¬erentiation namely lipid diï¬erentiation including3t3l1 and 3t3f422a cells cancer cells with tumorinitiation ability designated as cancer stem cells cscshave the characteristics of tumorigenesis and the expressionof speciï¬c stem cell markers as well as the longterm selfrenewal proliferation capacity and adipose diï¬erentiationpotential in addition to cscs cancer cells undergoing epithelialmesenchymaltransformation emt havebeen reported to be induced to diï¬erentiate into adipocytes[] lung cancer ncih446 cells can be induced to differentiate into neurons adipocytes and bone cells in vitro the adipogenesis diï¬erentiation treatment is promisingin the p53 gene deletion type of ï¬broblastderived cancer cancer cells with homologous recombination defectssuch as ovarian and breast cancer cells with breast cancersusceptibility genes brca mutations can be inducedto diï¬erentiate by poly adpribose polymerase parp 0cstem cells internationalinhibitors the nuclear receptor peroxisome proliferatoractivated receptor Î pparÎ agonist antidiabetic thiazolidinedione drug can induce growth arrest and adipogenicdiï¬erentiation in human mouse and dog osteosarcoma cells thyroid cancer cells expressing the pparÎ fusion proteinppfp can be induced to diï¬erentiate into adipocytes bypioglitazone adipogenesis can be induced in welldiï¬erentiated liposarcoma wdlps and dediï¬erentiatedliposarcoma ddlps cells by dexamethasone indomethacininsulin and 3isobutyl1methyl xanthine ibmx in this review we highlight some of the crucial transcription factors that induce adipogenesis both in mscs and inincluding the wellstudied pparÎ and ccaatcscsenhancerbinding proteins cebps as well as othercell factors that have been recently shown to have an important role in adipocyte diï¬erentiation we focus on understanding the complex regulatory mechanism of adipocytediï¬erentiation that can contribute to the clinical treatmentof human diseases including those caused by obesity andadipocytes dysfunction especially for the malignant tumorwhich can be transdiï¬erentiated into mature adipocytes adipocyte differentiationcell proliferation and diï¬erentiation are two opposingprocesses and there is a transition between these two processes in the early stages of adipocyte diï¬erentiation theinteraction of cell cycle regulators and diï¬erentiation factors produces a cascade of events which ultimately resultsin the expression of adipocyte phenotype adipogenesishas diï¬erent stages each stage has a speciï¬c gene expression pattern in general adipocyte diï¬erentiation ofpluripotent stem cells is divided into two phases the ï¬rstphase known as determination involves the commitment ofpluripotent stem cells to preadipocytes the preadipocytescannot be distinguished morphologically from their precursor cells but also have lost the potential to diï¬erentiate intoother cell types in the second phase which is known asterminal diï¬erentiation the preadipocytes gradually acquirethe characteristics of mature adipocytes and acquire physiological functions including lipid transport and synthesisinsulin sensitivity and the secretion of adipocytespeciï¬cproteins the diï¬erentiation of precursor adipocytes is also dividedinto four stages proliferation mitotic cloning early diï¬erentiation and terminal diï¬erentiation after the precursors are inoculated into the cell culture plates the cellsgrow exponentially until they converge after reaching contact inhibition the growth rate slows and gradually stagnatesand the proliferation of precursor adipocytes stops which isvery necessary for initiating the diï¬erentiation of precursoradipocytes adipocyte precursors exhibit transient mitosiscalled clonal expansion a process that relies on the actionof induced diï¬erentiation factors some preadipocyte cellsmouse cell lines 3t3l1 3t3f442a undergo one or tworounds of cell division prior to diï¬erentiation whereasother cell lines mouse c3h10t12 diï¬erentiating into adipocyte do not undergo mitosis clonal expansion whether mitotic clonal expansion is required for adiposediï¬erentiation remains controversial however it is certainthat some of the checkpoint proteins for mitosis regulateaspects of adipogenesis [ ] when cells enter the terminaldiï¬erentiation stage the de novo synthesis of fatty acidsincreases significantly the transcription factors and adipocyterelated genes work cooperatively to maintain precursor adipocyte diï¬erentiation into mature adipocytes containing largelipid droplets regulatory pathways inpreadipocytes commitmentadipocyte diï¬erentiation is a complex process in which geneexpression is ï¬nely regulated the most basic regulatory network of adipose diï¬erentiation has not been updated inrecent years but some factors and signaling pathways thatdo aï¬ect adipose diï¬erentiation have been continuouslyreported adipocyte diï¬erentiation is the result of the geneexpression that determines the phenotype of adipocyteswhich is a complex and delicate regulatory process figure wnt signal pathway in adipogenesis wnt signaling isimportant for adipocytes proliferation and diï¬erentiationboth in vitro and in vivo the wnt family of secretedglycoproteins functions through paracrine and autocrinemechanisms to uence cell fate and development wntprotein binding to frizzled receptors initiates signalingthrough catenindependent and independent pathways wnt signaling inhibits adipocyte diï¬erentiation in vitroby blocking the expression of pparÎ and cebpα constitutive wnt10b expression inhibits adipogenesis wnt10b isexpressed in preadipocytes and stromal vascular cells butnot in adipocytes in vivo transgenic expression of wnt10bin adipocytes results in a reduction in white adipose tissuemass and absent brown adipose tissue development wnt10a and wnt6 have also been identiï¬ed as determinantsof brown adipocyte development [ ] wnt5b is transiently induced during adipogenesis and promotes diï¬erentiation indicating that preadipocytes integrate inputs fromseveral competing wnt signals the hedgehog hh signaling pathway mechanismthree vertebrate hh ligands including sonic hedgehogshhindian hedgehog ihh and desert hedgehogdhh have been identiï¬ed and initiated a signaling cascademediated by patched ptch1 and ptch2 receptors [ ]hh signaling had an inhibitory eï¬ect on adipogenesis inmurine cells such as c3h10t12 ks483 calvaria mscslines and mouse adiposederived stromal cells thesecells were visualized by decreased cytoplasmic fat accumulation and the expression of adipocyte marker genes after hhsignaling was inhibited although it is generally agreedthat hh expression has an inhibitory eï¬ect on preadipocytediï¬erentiation the mechanisms linking hh signaling andadipogenesis remain poorly deï¬ned erkmapkppar signal pathway extracellularregulated protein kinase erk is required in the proliferativephase of diï¬erentiation erk activity blockade in 3t3l1 0cstem cells internationaldex insulin demxwnt 10band othersshhpbc smotgfð½p smad3 smad3testosteroneð½catentinarirspi3kaktcrebpkapcrebfoxo1a2tcflef gata23cebpð½mapkg3k3ð½p2cebpð½cebpαppará½»bmpssmad1srebpadipocytegenesfigure regulation pathways in preadipocytes commitment bmp and wnt families are mediators of mscs commitment to producepreadipocytes exposure of growtharrested preadipocytes to diï¬erentiation inducers igf1 glucocorticoid and camp triggers dnareplication leading to adipocyte gene expression due to a transcription factor cascade the dotted line indicates an uncertain molecularregulatory mechanismcells and embryonic stem cells can inhibit adipogenesis inthe terminal diï¬erentiation phase erk1 activity leads topparÎ phosphorylation which inhibits adipocyte diï¬erentiation this implies that erk1 activity must be reduced afteradipocyte proliferation so that diï¬erentiation can proceedthis reduction is mediated in part by mitogenactivatedprotein kinase mapk phosphatase1 mkp1 [ ]these extracellular and intracellular regulation factors causeadipocytespeciï¬c gene expression and eventually lead toadipocyte formation adipocyte differentiationregulatory proteins pparÎ and adipocyte diï¬erentiation pparÎ is a member of the nuclearreceptor superfamily and is both necessaryand suï¬cient for adipogenesis forced expression ofpparÎ is suï¬cient to induce adipocyte diï¬erentiation broblasts indeedthe proadipogenic cebps andkrüppellike factors klfs have all been shown to induceat least one of the two pparÎ promoters in contrast antiadipogenic transcription factor gata functioned in part byrepressing pparÎ expression pparÎ itself has twoisomers the relative roles of pparÎ1 and pparÎ2 in adipogenesis remain an open question pparÎ2 is mainlyexpressed in adipose tissue while pparÎ1 is expressed inmany other tissues although both can promote adipocytediï¬erentiation pparÎ2 could do so eï¬ectively at very lowligand concentration compared with pparÎ1 the twoprotein isoforms are generated by alternative splicing andpromoter usage and both are induced during adipogenesispparÎ1 can also be expressed in cell types other than adipocytes ren et al used engineered zincï¬nger proteins tothe expression ofthe endogenous pparÎ1 andinhibitpparÎ2 promoters in 3t3l1 cells ectopic expression ofpparÎ2 promotes adipogenesis whereas that of pparÎ1does not zhang et al reported that pparÎ2 deï¬ciencyimpairs the development of adipose tissue and insulin sensitivity there are transcriptional cascades between adipocytesgenes including pparÎ and cebpα which are the coreadipocyte diï¬erentiation regulators in the early stage of adipocyte diï¬erentiation the expression of cebp and cebpδincrease which upregulates cebpα expressionfurtheractivate pparÎ pparÎ activating cebpα in turn resultsin a positive feedback pparÎ binding with retinoic acid xreceptor rxr forms diï¬erent heterodimers the variousdimmers can combine with the pparÎ response elementppre and initiate the transcription of downstream genesfor diï¬erentiation into adipocytes cebps participate in adipogenesis and several cebpfamily members are expressed in adipocytesincludingcebpα cebp cebpÎ cebpδ and cebphomologous protein chop the temporal expression of thesefactors during adipocyte diï¬erentiation triggers a cascadewhereby early induction of cebp and cebpδ leads tocebpα expression this notion is further supported by thesequential binding of these transcription factors to severaladipocyte promoters duringadipocyte diï¬erentiationcebp is crucial for adipogenesis in immortalized preadipocyte lines cebp and cebpδ promote adipogenesis atleast in part by inducing cebpα and pparÎ cebpαinduces many adipocyte genes directly and plays an important role in adipose tissue development once cebpα isexpressed its expression is maintained through autoactivation despite the importance of cebps in adipogenesis 0cstem cells internationalthese transcription factors clearly cannot function eï¬cientlyin the absence of pparÎ cebp cannot induce cebpαexpression in the absence of pparÎ which is required torelease histone deacetylase1 hdac1 from the cebpαpromoter furthermore ectopic cebpα expressioncannot induce adipogenesis in pparÎï¬broblasts however cebpα also plays an important role in diï¬erentiated adipocytes overexpression of exogenous pparÎ incebpαdeï¬cient cells showed that although cebpα isnot required for lipid accumulation and the expression ofmany adipocyte genes it is necessary for the acquisition ofinsulin sensitivity [ ] figure human ï¬broblasts withthe ability to diï¬erentiate into adipocytes also do not undergomitotic cloning ampliï¬cation however pparÎ exogenousligands need to be added to promote adipocyte diï¬erentiation therefore it can be inferred that mitotic cloning expansion can produce endogenous ligands of pparÎ bmp and transforming growth factor tgf inadipocyte diï¬erentiation a variety of extracellular factorsaï¬ect the preadipocyte commitment of stem cells includingbone morphogenetic protein bmp transforminggrowth factor tgf insulininsulinlike growthfactor igf1 tumor necrosis factor α and interleukin matrix metalloproteinase ï¬broblast growthfactor fgf and fgf2 bmp and tgf have variedeï¬ects on the diï¬erentiation fate of mesenchymal cells the tgf superfamily members bmps and myostatinregulate the diï¬erentiation of many cell types includingadipocytes tgf inhibitor can promote adipose diï¬erentiation of cancer cells with a mesenchymal phenotypein vitro and transgenic overexpression of tgf impairsadipocyte development inhibition of adipogenesis couldbe obtained through blocking of endogenous tgf with adominantnegative tgf receptor or drosophila mothersagainst decapentaplegic protein smad inhibitionsmad3 binds to cebps and inhibits their transcriptionalactivity including their ability to transactivate the pparÎ2promoter [ ] exposure of multipotent mesenchymalcells to bmp4 commits these cells to the adipocyte lineageallowing them to undergo adipose conversion theeï¬ects of bmp2 are more complex and depend on the presence of other signaling molecules bmp2 alone has little eï¬ecton adipogenesis and it interacts with other factors such astgf and insulin to stimulate adipogenesis of embryonicstem cells bmp2 stimulates adipogenesis of multipotentc3h10t12 cells at low concentrations and can contribute tochondrocyte and osteoblast development at higher concentrations klfs in adipocyte diï¬erentiation during adipocyte differentiation some klf family members are overexpressedsuch as klf4 klf5 klf9 and klf15 while klf16 expression is reduced [ ] klf15 is the ï¬rst klf family members which were identiï¬ed to be involved in adipocytediï¬erentiation its expression increased significantly on thesixth day of 3t3l1 adipocyte diï¬erentiation and peakedon the second day of adipocyte induction in mscs andmouse embryonic ï¬broblasts inhibition of klf15 by sirnaor mutation led to a decrease in pparÎ cebpα fatty acidbinding protein fabp4 and glucose transporter glut4 however overexpression of klf15 in nih3t3cells was found to be associated with lipid accumulation aswell as increases in pparÎ and fabp4 mice with complete absence of klf5 showed embryonal lethality and micewith singlechromosome klf5 knockout showed a significant reduction in white fat in adulthood suggesting thatklf5 plays an important role in adipocyte diï¬erentiationklf5 can be activated by cebp or cebpδ which isinvolved in early adipocyte diï¬erentiation klf5 can beactivated by cebp or cebpδ which is involved in earlyadipocyte diï¬erentiation direct binding of klf5 to thepparÎ2 promoter in combination with cebps inducespparÎ2 expression transfection of klf5 dominantnegative mutants in 3t3l1 cells reduced lipid droplet accumulation and inhibited pparÎ and cebpα expressionwhereas overexpression of wild klf5 significantly promotedadipocyte diï¬erentiation even without exogenous hormonestimulation similar to klf5 klf9 knockdown can inhibitthe expression of a series of adipocyte diï¬erentiation genessuch as pparÎ cebpα and fabp4 hence inhibitingadipocyte diï¬erentiation however klf9 overexpressiondid not upregulate the expression of pparÎ and cebpα in addition klf4 can transactivate cebp by bindingto the region of kb upstream of the cebp promoter and promote lipid diï¬erentiation klf6 can forma complex with histone deacetylase3 hdac3 inhibitingpreadipocyte factor1 pref1 expression and promotinglipid diï¬erentiation klf2 is highly expressed in adiposeprogenitors and its expression decreases during the processof lipid diï¬erentiation overexpressed klf2 can bind to thecaccc region of pparÎ2 proximal promoter and inhibitlipid diï¬erentiation as well as the expression of pparÎcebpα and sterolregulated elementbinding proteinssrebp by inhibiting the promoter activity rnasequence analysisshowed that klfl6 expression wasdecreased on the ï¬rst day of adipocyte diï¬erentiation of3t3l1 cells adipocyte diï¬erentiation was promoted byklf16 knockdown but was inhibited by klf16 overexpression via inhibition of pparÎ promoter activity in addition klf3 and klf7 were also found to play a negativeregulatory role in adipocyte diï¬erentiation [ ] signal transducers and activators of transcriptionstats and adipocyte diï¬erentiation the activated statprotein enters the nucleus as a dimer and binds to the targetgene to regulate gene transcription in the adipocyte diï¬erentiation of mouse 3t3l1 cells the expression of stat1 andstat5 was significantly increased while that of stat3and stat6 was not significantly changed in the adipocyte diï¬erentiation of human subcutaneous adipose precursor cells stat1 expression was significantly decreased while the expression of stat3 and stat5 wasincreased and stat6 expression was unchanged therole of stat1 in adipocyte diï¬erentiation is not clearbecause its expression trend in humans and mice diï¬ersduring the adipocyte diï¬erentiation process early adipocytediï¬erentiation of 3t3l1 cells was inhibited by stat1 0cstem cells internationalklf5srebp1cklf15klf2chopcebpá½»krox20ligandcebpð½cebpð¿gata23ppará½»cebpð¼proadipogenicantiadipogenicgenes of terminaladipocytedifferentiationfigure a cascade of transcription factors that regulate adipogenesis pparÎ is one of the key transcription factors in adipogenesis and thecore of the transcriptional cascade that regulates adipogenesis pparÎ expression is regulated by several proadipogenic blue andantiadipogenic red factors cebpα is regulated through a series of inhibitory proteinprotein interactions some transcription factorfamilies include several members that participate in adipogenesis such as the klfs black lines indicate eï¬ects on gene expression violetlines represent eï¬ects on protein activityagonist interferon Î loss of stat1 in 3t3l1 cells can rescue the inhibition of adipocyte diï¬erentiation caused byprostaglandin factor 2α other studies have found thatstat1 is required for adipose diï¬erentiation and stat1overexpression in c3h10t12 cells can prevent the inhibition of lipid diï¬erentiation caused by bcell lymphoma6knockdown there was no abnormal adipose tissuein stat1 knockout mice stat3 not only aï¬ectsthe proliferation of 3t3l1 cells but also coregulates theiradipocyte diï¬erentiation with high mobility group protein the fabp4 promoter was used to speciï¬callyknock out stat3 in the adipose tissue of mice and theresults showed that mice weight significantly increasedand the adipocyte quantity increased compared with thewildtype mice stat5a and stat5b have diï¬erenteï¬ects on adipocyte diï¬erentiation abnormal adipose tissuewas found in the mice with stat5a or stat5b knockout ordouble knockout and the amount of adipose tissue was onlyoneï¬fth of the original adipose tissue in mice withoutknockdown histone modiï¬cation in adipocyte diï¬erentiation histone deacetylase sirtuin sirt plays an important rolein biological processes such as stress tolerance energymetabolism and cell diï¬erentiation during the adipocyte diï¬erentiation of c3h1012 cells sirt1 expressiondecreased overexpression of sirt1 activated thewnt signal which caused the deacetylation of cateninthe accumulation of catenin in the nucleus could inhibitadipocyte diï¬erentiation sirt1 knockdown resulted inincreased acetylation of the histones h3k9 and h4k16 inthe secreted frizzledrelated protein sfrp and sfrp2 promoters thereby promoting transcription of these genes andpromoting lipid diï¬erentiation forkhead box proteino foxo is a member of the transcription factor foxofamily it can recruit cyclic amp response elementbindingprotein cbphistone acetyltransferase p300 to initiate anacetylation the acetylated foxo1 can be phosphorylatedby phosphorylated protein kinase b pkbakt the phosphorylation of foxo1 by akt inhibits the transcriptionalactivation of foxo1 the acetylation of foxo1 lost the ability of dnabinding aï¬nity and promoted its shuttling fromnuclei to cytoplasm sirt1 and sirt2 can deacetylateand active foxo1 activated foxo1 nonphosphorylatednuclear foxo1 in the nucleus binds to the promoters of target genes encoding p21 p27 and pparÎ and initiates subsequent transcriptions sirt2 inhibits the acetylation andphosphorylation of foxo1 thereby induces the accumulation of activated foxo1 in the nucleus activated foxo1could inhibit adipogenesis via pparÎ [] lysinespeciï¬c histone demethylase lsd1 expression increasedduring the adipocyte diï¬erentiation of 3t3l1 cells lsd1could reduce the dimethylation levels of histone h3k9 andh3k4 in the cebpα promoter region thereby promotingadipocyte diï¬erentiation set domaincontaining setd8 catalyzed the monomethylation of h4k20 andpromoted pparÎ expression the activation of pparÎ transcriptional activity leads to the induction of monomethylatedh4k20 and modiï¬cation of pparÎ and its targets therebypromoting adipogenesis enhancer of zeste homolog ezh2 is a methyltransferase and can bind methyl groupsto histone h3k27 which is also necessary for lipid diï¬erentiation the absence of ezh2 in brown fat precursors results inreduced levels of the wnt promoter histone h3k27me3which is also saved by the ectopic ezh2 expression or theuse of a wntcatenin signal inhibitor in addition histone demethylases such as lysinespeciï¬c histone demethylase lsdkdm kdm6 and histone lysine demethylasephf2 are also involved in adipose diï¬erentiation andkdm2b inhibits transcription factor activator protein 2αpromoter via h3k4me3 and h3k36me2 role of microrna and long noncodingrna in adipogenesismicrorna mir can bind and cut target genes or inhibittarget gene translation endogenous sirna can be producedby the action of dicer enzyme and bind to a speciï¬c proteinto change its cellular location many kinds of mirsare involved in regulating adipocyte diï¬erentiation the 0cstem cells internationalexpression of mir143 increased during the diï¬erentiationof adipose progenitor cells overexpression of mir143promoted gene expression involved in adipose diï¬erentiationand triglyceride accumulation inhibition of mir143 prevented the adipose diï¬erentiation of human fat progenitorcells [ ] additionally mir8 promotes adipocyte diï¬erentiation by inhibiting wnt signaling moreover mir mir103 mir21 mir519d mir210 mir30mir204211 and mir375 also play a certain role in promoting adipocyte diï¬erentiation while mir130 mir448and let7y inhibit lipid diï¬erentiation [ ] in additionto mirs long noncoding rna lncrna is a type of noncoding rna and is important during epigenetic regulationand can form a doublestranded rna complex with mrnacauses protein transcription lncu90926 inhibits adipocytediï¬erentiation by inhibiting the transactivation of pparÎ2 as a novel lncrna hoxaas3 expression increasedduring the adipose diï¬erentiation of mscs and hoxaas3 silencing reduced the marker gene of adipose diï¬erentiation and inhibited the adipose diï¬erentiation zhu et al reported that hoxaas3 interacted with ezh2 toregulate lineage commitment of mscs hoxa as3 canregulate the trimethylation level of h3k27 in the runx2promoter region by binding to ezh2 therefore hoxaas3 is considered to be an epigenetic switch regulating mscslineage speciï¬city adipocyte diï¬erentiationassociatedlncrna can act as a competitive endogenous rna of mir in the process of lipid diï¬erentiation thereby promotingthe expression of sirt1 the target gene of mir204 and thusinhibiting lipid diï¬erentiation the lncrna neat1can also regulate adipocyte diï¬erentiation under the uence of mirna140 other lncrna including lncrnablnc1 and plnc are also involved in regulating adipocytediï¬erentiation [ ] other biochemical response involved inadipocyte differentiation unfolded protein responses in adipocyte diï¬erentiationin the endoplasmic reticulum of eukaryotes unfolded protein response involves three proteinsinositolrequiringenzyme 1α doublestranded rnadependent proteinkinaselike er kinase and activating transcription factoratf 6α knockdown of atf6α aï¬ects the expressionof adipocytes genes and inhibits c3h10t12 adipocyte differentiation the inhibitory eï¬ect of berberine on adipocyte diï¬erentiation of 3t3l1 cells is also due to inducedchop and decorin expressions and this inhibitory eï¬ectis ameliorated by chop knockout in the adipocytediï¬erentiation process of 3t3l1 cells increases in pparÎand cebpα as markers of adipocyte diï¬erentiation wereaccompanied by an increase in the corresponding proteinexpressions of phosphorylated eukaryotic translation initiaeif 2α phosphorylated endoribonucleasetion factorire1α atf4 chop and other unfolded protein responsesendoplasmic reticulum stress inducer or hypoxic endoplasmic reticulum stress can inhibit adipocyte diï¬erentiationadditionally eif2α mutation results in continuous activation or overexpression of chop which also inhibits adipocyte diï¬erentiation after the initiation of adiposediï¬erentiation numerous diï¬erentiationassociated proteinsare synthesized exogenous endoplasmic reticulum stressinducers can lead to excessive endoplasmic reticulumresponse which in turn aï¬ects the synthesis of proteinsrelated to diï¬erentiation and inhibits adipocyte formationfigure role of oxidative stress in adipogenesis during thedirectional diï¬erentiation of mscs mitochondrial complexi and iii and nadph oxidase nox4 are the main sourcesof oxygen species ros production currently it is believedthat ros aï¬ects not only the cell cycle and apoptosis but alsodiï¬erentiation through uencing the signaling pathwaysincluding the wnt hh and foxo signaling cascade duringmscs diï¬erentiation the diï¬erentiation ability ofstem cells is determined by the arrangement of perinuclearmitochondria which speciï¬cally manifests as low atpcellcontents and a high rate of oxygen consumption the lackof these characteristics indicates stem cell diï¬erentiation adipocyte diï¬erentiation is a highly dependent rosactivation factor related to mitosis and cell maturation schroder et al found that exogenous h2o2 could stimulate adipocyte diï¬erentiation of mouse 3t3l1 cells andhuman adipocyte progenitor cells in the absence of insulinh2o2 regulates adipocyte diï¬erentiation of 3t3l1 cells ina dosedependent manner high doses of h2o2 and μm promote adipocyte diï¬erentiation [ ] tormos et al found that ros synthesis increased in humanmscs at the early stage of adipose diï¬erentiation and targeted antioxidants could inhibit lipid diï¬erentiation byknocking down rieske ironsulfur protein and ubiquinonebinding protein ros produced by mitochondrial complexiii was found to be necessary in initiating adipose diï¬erentiation however other studies have shown that theexpression levels of adiponectin and pparÎ were decreasedby using h2o2 mm in 3t3l1 cells free radical nitric oxide no also promotes lipid diï¬erentiationbecause treatment with no inducer hydroxylamine or nosynthase nos substrate arginine can significantly induceadipose diï¬erentiation of rat adipose progenitor cells nosinduced adipose diï¬erentiation mainly via enos rather thaninos ros can induce adipose diï¬erentiation primarily by inhibiting wnt foxo and hh signaling pathwaysthat inhibit lipid diï¬erentiation autophagy in adipocyte diï¬erentiation the increase inautophagosomes during lipid diï¬erentiation indicates thatautophagy may play an important role in lipid diï¬erentiation baerga et al conï¬rmed that the adipocyte diï¬erentiation eï¬ciency was significantly inhibited in mouse embryonic ï¬broblasts lacking autophagyrelated gene atg agene encoding an essential protein required for autophagy knockdown of atg5 in 3t3l1 cells promotesproteasomedependent degradation of pparÎ2therebyinhibiting adipocyte diï¬erentiation zhang reportedthat autophagyrelated gene 7atg7 is also crucial for adipose development atg7deï¬cient mice were slim and onlyhad of white fat compared to wildtype mice and the 0cstem cells internationalcebpð½ geneebf1 geneklf4egr2cebpð½cytosolcebpð¿ genecebpð¿klf5geneppará½» geneklf5nr2f2nfkb11433relasrebf1a2rxrappará½»ppará½»rxra heterodimerppará½»rxracorepressor complexfabp4ligands of ppará½»fam120bthrap3ep300ncoa2ncoa3helz2ncoa1crebbpebf1adipoq geneaidrfcebpð¼ geneznf638znf467cebpð¼ncor1hdac3ncor2 slc2a4 geneglut4 genelep genefabp4 genecdk4ccnd3plin1 genepck1 genefabp4cd36 geneppararxracoactivator complexppará½»fatty acidrxramediatorcoactivator complexangptlgeneppargc1amediator complex consensuslpl genenucleoplasmproteins bind to gene promoterstranscription of genes into proteinsacting on proteins compoundingtgfð½1wnt1wnt10btnf77233adipoqglut4slc2a4 tetramerlepfabp4lipid dropletplin1pck1papa pa4xpalmccd36paangptl4lplfigure regulation of adipocyte diï¬erentiation a regulatory loop exists between pparÎ and cebp activation transcription factor coeebf activates cebpα cebpα activates ebf1 and ebf1 activates pparÎ cebp and cebpδ act directly on the pparÎ gene bybinding its promoter and activating transcription cebpα cebp and cebpδ can activate the ebf1 gene and klf5 the ebf1 and klf5proteins in turn bind the promoter of pparÎ which becomes activated other hormones such as insulin can aï¬ect the expression ofpparÎ and other transcription factors such as srebp1c pparÎ can form a heterodimer with the rxrα in the absence of activatingligands the pparÎrxrα complex recruits transcription repressors such as nuclear receptor corepressor ncor ncor1 andhdac3 upon binding with activating ligands pparÎ causes a rearrangement of adjacent factors corepressors such as ncor2 are lostand coactivators such as transcription intermediary factor tif2 cbp and p300 are recruited which can result in the expression of cyclicampresponsive elementbinding protein creb followed by pparÎ pparÎ expression initiates the expression of downstream genesincluding angiopoietinrelated protein pgar perilipin fabp4 cebpα fatty acid transportrelated proteins carbohydrate metabolismrelated proteins and energy homeostasisrelated proteinslipid metabolism and hormoneinduced lipolysis in the adipocytes were altered autophagy related gene atg4b isactivated by cebp in the process of lipid diï¬erentiationand autophagy activation is necessary for the degradationof klf2 and klf3 two negative regulators of lipid diï¬erentiation these results showed that adipose diï¬erentiation andautophagy are mutually complementary in 3t3l1cells autophagy was inhibited by aspartate ammonia or 0cstem cells internationalmethyladenine at diï¬erent lipid induction periods and days and only autophagy inhibition at days hindered the formation of lipid droplets and the expression of lipid marker genes indicating that autophagy wasvery important in the early stage of lipid diï¬erentiation recent studies showed that lc3 is overexpressed in3t3l1 cells further demonstrating the important role ofautophagy in lipid diï¬erentiation role of alternative splicing in adipogenesis selectivesplicing is uenced by splicing regulators which regulateadipocyte diï¬erentiation by regulating the selective splicingof genes speciï¬c to this process lipin1 is an important regulator in the process of adipocyte diï¬erentiation and includestwo isomers lipin1α and lipin1 which have diï¬erenteï¬ects high expression of lipin1α promotes adipocyte differentiation while that of lipin1 promotes lipid droplet formation in sam68deï¬cient mice the ï¬fth intron ofserinethreonineprotein kinase mtor was retained resulting in unstable and rapid mtor degradation and inhibitionof adipocyte diï¬erentiation furthermore there arefour isomers of pref1 pref1a and pr | 0 |
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c | 0 |
netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimers and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth peoples hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term netosisshould only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3 levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimers disease alzheimers disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimers disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patients ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohns disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohns patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csas efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation httpsdoi101038s414180170012 gupta s and mj kaplan the role 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ting li sheng chen gu yueying and shuang ye neutrophil extracellular trap mitochondrial dna and its 0cautoantibody in systemic lupus erythematosus and a proofofconcept trial of metformin arthritis rheumatology kenny ef a herzig r kruger a muth s mondal prthompson v brinkmann hv bernuth and a zychlinsky diverse stimuli engage different neutrophil extracellular trappathways elife httpsdoi107554elife24437 hakkim a bg furnrohr k amann b laube ua abed vbrinkmann m herrmann re voll and a zychlinsky impairment of neutrophil extracellular trap degradation is associatedwith lupus nephritis proceedings of the national academy of sciences of the united states of america httpsdoi101073pnas0909927107 farrera c and b fadeel macrophage clearance of neutrophil extracellular traps is a silent process journal of immunology httpsdoi104049jimmunol1300436 maueroder c a mahajan s paulus s gosswein j hahn dkienhofer mh biermann et al menageatrois the ratio ofbicarbonate to co2 and the ph regulate the 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Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | 2 |
"annual meeting of the european associationfor the study of diabetes september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs casa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearsons regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimers diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether 1stresponder cells thatlead the 1st phase of ca2 uptake are the same as hub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as 1st responders and the of cellsresponding slower as last responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated hub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islets periphery at ± of the isletsradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from hub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on its role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio | 0 |
peripheral serum metabolomic profiles inform central cognitive impairmentJingye Wang1 Runmin Wei12 Guoxiang Xie1 Matthias Arnold Alexandra KueiderPaisley Gregory Louie Siamak Mahmoudian Dehkordi3 colette Blach5 Rebecca Baillie Xianlin Han7 Philip L De Jager David A Bennett9 Rima KaddurahDaouk Wei Jia The incidence of Alzheimers disease AD increases with age and is becoming a significant cause of worldwide morbidity and mortality However the metabolic perturbation behind the onset of AD remains unclear In this study we performed metabolite profiling in both brain n and matching serum samples n to identify differentially expressed metabolites and metabolic pathways associated with neuropathology and cognitive performance and to identify individuals at high risk of developing cognitive impairment The abundances of metabolites glycolithocholate GLCA petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and the deoxycholatecholate DCACA ratio along with the dysregulation scores of metabolic pathways primary bile acid biosynthesis fatty acid biosynthesis and biosynthesis of unsaturated fatty acids showed significant differences across both brain and serum diagnostic groups Pvalue Significant associations were observed between the levels of differential metabolitespathways and cognitive performance neurofibrillary tangles and neuritic plaque burden Metabolites abundances and personalized metabolic pathways scores were used to derive machine learning models respectively that could be used to differentiate cognitively impaired persons from those without cognitive impairment median area under the receiver operating characteristic curve AUC for the metabolite level model median AUC for the pathway level model Utilizing these two models on the entire baseline control group we identified those who experienced cognitive decline in the later years AUC sensitivity specificity for the metabolite level model AUC sensitivity specificity for the pathway level model and demonstrated their preAD onset prediction potentials Our study provides a proofofconcept that it is possible to discriminate antecedent cognitive impairment in older adults before the onset of overt clinical symptoms using metabolomics Our findings if validated in future studies could enable the earlier detection and intervention of cognitive impairment that may halt its progressionAlzheimers disease AD one of the top leading causes of death in the United States is an increasing challenge for health care systems and will result in increased economic burden as increasing numbers of new cases are diagnosed annually12 Currently there is no therapy to prevent or slow AD progression which may be due to the inability to detect AD before its progression into evident cognitive decline Identification of early 1University of Hawaii Cancer Center Ilalo Street Honolulu HI USA 2Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI USA 3Department of Psychiatry and Behavioral Sciences Duke University Durham NC USA 4Institute of Computational Biology Helmholtz Zentrum M¼nchen German Research Center for Environmental Health Neuherberg Germany 5Duke Molecular Physiology Institute Duke University Durham NC USA 6Rosa Co LLC San Carlos CA USA 7University of Texas Health Science Center at San Antonio San Antonio TX USA 8Center for Translational Computational Neuroimmunology Columbia University College of Physicians and Surgeons Department of Neurology New York NY USA 9Rush Alzheimers Disease Center Rush University Medical Center Chicago IL USA 10Institute of Brain Sciences Duke University Durham NC USA 11Department of Medicine Duke University Durham NC USA email kaddu001mcdukeedu wjiacchawaiieduScientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cbiomarkers associated with preclinical symptoms would allow early intervention or preventive strategies to be developed3 Research has identified multiple neurochemical perturbations in AD including amyloid precursor protein metabolism phosphorylation of tau protein and a wide range of metabolic perturbations4 Unfortunately current biomarkers for early disease including cerebrospinal fluid CSF betaamyloid and tau levels5 structural and functional magnetic resonance imaging6 the recent use of brain amyloid imaging7 or inflammaging8 and CSF markers to track brain atrophy and deposition of cortical betaamyloid and neurofibrillary tangles are limited because they are either invasive timeconsuming or expensiveRecent studies have focused on obtaining biomarkers to identify features that differentiate persons a0with cognitive impairment from persons without cognitive impairment Molecular markers sensitive to the underlying pathogenic factors would be highly relevant to early disease detection and facilitation of disease monitoring and treatment responses Metabolomics is an unbiased approach to study smallmolecule metabolites that offers hope for the discovery of more biomarkers for AD This profiling technology has already been used to identify differential metabolites that can distinguish mild cognitive impairment MCI subjects who will develop AD from stable MCI9 Mounting evidence suggests that AD is closely accompanied with the abnormal bile acid BA metabolism10 free fatty acid FFA metabolism14 lipid metabolism1718 and neurotransmitter metabolism19 BAs have become increasingly recognized as important metabolic signaling molecules that modulate lipid glucose and energy metabolism20 More importantly BAs in brain act as neuroactive steroids21 Different classes of BAs can either inhibit or potentiate GABAα a0and inhibit NMDA receptors while also exerting neuroprotective effects Recent crosssectional studies have shown differences in blood BAs in AD compared with noncognitively impaired individuals2425 Additionally researchers found an accumulation of FFAs in the hippocampus and cortex of AD mice compared to control mice2627 Another animal study examined the role of elevated FFA in the pathogenesis of AD and established a potential mechanism of FFA causing hyperphosphorylation of tau through astrogliamediated oxidative stress28 Alterations of FFAs have also been detected in postmortem AD brains tissues14 and serum samples16 which may indicate an alternative fuel source before the onset of clinical symptoms29 These observations have given rise to the possibility that metabolic perturbations could presage the onset of cognitive impairment and therefore aid in the identification of individuals with higher risks by providing additional information to use with standard clinical markersIn this study we performed metabolomic profiling in participants from a large longitudinal cohort with the goal of identifying metabolic changes as well as key metabolic pathways that might serve as new predictors of future cognitive impairment in older adultsMaterials and methodsParticipants The Religious Orders Study ROS which began in is a longitudinal clinicalpathologic cohort study of risk factors of cognitive decline and incident dementia run from the Rush Alzheimers Disease Center that is comprised of individuals from religious communities eg Catholic brothers nuns and priests across the USA3031 The Rush Memory and Aging Project MAP which began in includes participants from northeastern Illinois USA with a broader range of socioeconomic status and life experiences31 Participants in both studies enroll without known dementia agree to annual clinical evaluation and an donation Both studies were approved by an Intuitional Review Board of Rush University Medical Center All subjects signed an informed consent an Anatomic Gift Act and a repository consent to allow their biospecimens and data to be used for ancillary studies All research was performed in accordance with relevant guidelinesregulations set forth by the Rush University Medical Center Both studies are conducted by the same team of examiners and share a large common core of data collection at the item level to allow for efficient merging of dataCognitive performance tests Cognitive performance was measured using a battery of cognitive performance tests of which could be summarized in five cognitive domains ie episodic memory working memory semantic memory perceptual orientationvisuospatial ability and perceptual speed Table a0S1 Domains are created by averaging the zscores based on mean and standard deviation from all baseline data for tests in each domain The global cognitive function score is calculated by averaging zscores for all tests to yield a global measure of cognitive function Additionally the MiniMental State Examination was also administered to characterize the cohortClinical diagnoses Medical conditions were documented via selfreport and clinical evaluation Clinical diagnoses each year were determined blinded to previously collected data A threestep process starts with an actuarial decision tree based on the history of cognitive decline and impairment ratings in five cognitive domains based on cutoffs for cognitive tests32 a0followed by clinical judgment by a neuropsychologist for cognitive impairment and determination of dementia and its causes by a clinician ie neurologist geriatrician second neuropsychologist geriatric nurse practitioner33 The diagnosis of AD follows the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimers Disease and Related Disorders Association NINCDSADRDA34 Participants were categorized as a AD b MCI if diagnosed cognitive impairment by the neuropsychologist but not diagnosed dementia by the clinician32 and c no cognitive impairment NCI if diagnosed without AD or MCI35 At the time of death brain autopsies and histopathological exams were performed by clinicians to confirm the diagnosis After an autopsy was completed a spectrum of neuropathologic diagnoses was obtained such as a pathologic diagnosis of AD as defined using the modified NIA Reagan criteria However many other pathologies were present in the brains of older individuals the mean age of death is a0years old in ROSMAP and they were catalogued for each participantScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cNCI converters were those participants who were cognitively normal at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followupNeuropathology Upon death a postmortem neuropathological evaluation was implemented and the procedures follow those outlined by the pathologic dataset recommended by the National Alzheimers Disease Coordinating Center Brains of deceased subjects were removed weighed cut into one cmthick coronal slabs and stored Each brain was examined for the neuropathological indices of common pathologies that contribute to cognitive impairment The location age and volume of all macroscopic infarcts were recorded and tissue was obtained for histological confirmation in addition to the identification of microscopic infarctions as previously described3637 AD pathology was identified using the modified Bielschowsky silver stain technique and by use of the Consortium to Establish a Registry for Alzheimers Disease CERAD criteria38 and NIAReagan criteria39 while the assessment of neurofibrillary tangles was based on Braak criteria40 as described previously41 The CERAD score a semiquantitative measure of neuritic plaque burden is made of levels no AD possible AD probable AD and definite AD As recommended CERAD scores were reclassified to a binary level score score Seven categories of Braak stages were based on the region and severity of neurofibrillary tangles pathologyMetabolites quantification Using targeted metabolomics protocols42 and profiling protocols43 established in previous studies BAs were quantified by ultraperformance liquid chromatography triple quadrupole mass spectrometry UPLCTQMS Waters XEVO TQS Milford USA and other metabolites were quantified by gas chromatography timeofflight mass spectrometry GCTOFMS Leco Corporation St Joseph USA Details are described in the Supporting InformationStatistical analysis Stratifying by clinical diagnosis continuous demographic variables were expressed as mean [standard deviation SD] and tested by Wilcoxon ranksum test while categorical demographic variables were expressed as n percentage and tested by Chisquare test Missing values in quantitative metabolites due to limits of quantification were regarded as leftcensored missing and imputed by GSimp4445 Individual BA concentrations were normalized to the total BAs concentration ie the proportion of total BAs Metabolites were reported as median quantile quantile and tested by univariate analysis Wilcoxon ranksum test Due to the limited sample size of the AD group participants in serum samples we combined MCI and AD participants into an aggregate group MCIAD for the following data analysis Logtransformed abundances were used in the following data analysis We additionally generated BA ratios based on the BA metabolic pathway topologyTo identify metabolites differentially expressed in participants with cognitive decline we used ordinal logistic regression to compare metabolites across three groups NCI MCI AD for brain samples and logistic regression across two groups NCI MCIAD for serum samples To control the positive false discovery rate Qvalues were calculated based on Pvalues Additionally for serum samples we adjusted for potential confounders eg fasting status supplements diabetic and lipid lowering medications using logistic regressions The relationships between logtransformed brain metabolites levels with neurofibrillary tangle burden and neuritic plaque burden were expressed as boxplots across Braak scores KruskalWallis test and CERAD scores Wilcoxon ranksum test respectively Using Spearmans rank correlation test we further evaluated the associations between the abundances of each identified metabolite and the global cognitive function score in both brain and serum samples Linear regression models with each individual metabolite used as the predictor and each cognitive test as the response variable adjusted for age gender years of education and presence of APOE ε4 were used to test the associations between metabolite and cognitive function Similar analyses with an additional adjustment of BMI were conducted for serum samples The Wilcoxon ranksum test was carried out to explore whether identified variables were differentially expressed between NCI converters vs NCI nonconverters and between NCI converters vs MCIAD in sera Then we built a random forest RF predictive model to differentiate NCI nonconverters vs MCIAD using glycolithocholate GLCA deoxycholatecholate DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and age as the predictorsTo differentiate MCIAD vs NCI nonconverters we randomly split the data into training set and testing set times Each time we trained an RF model on the training set to differentiate the MCIAD from NCI nonconverters and evaluated it on the testing set using the area under the receiver operating characteristic curve AUROC sensitivity SE and specificity SP A final model was built on the whole NCI nonconverters and MCIAD dataTo investigate the preclinical predictive potentials as well as to validate the classification performance of our model we utilized this model on the entire baseline NCI group to identify those NCI converters from NCI nonconverters The differences of RF scores between NCI nonconverters vs NCI converters and NCI nonconverters vs MCIAD groups were tested by the Wilcoxon ranksum test To determine whether RF scores could independently differentiate NCI converters from NCI nonconverters in the presence of potential confounders we used the logistic regression method with RF scores as the predictor adjusting for gender years of education APOE ε4 and BMI Additionally we fit linear mixed effects models to evaluate correlations between RF scores with global cognitive function and each of the five cognitive domains separately with a random effects term for education and BMI and fixed effects terms for RF score gender and APOE ε4For the personalized pathway level analyses we extracted metabolite information from the Human Metabolome Database HMDB46 and metabolic pathway information from the Kyoto Encyclopedia of Genes and Genomes KEGG database47 to map affiliated metabolites to metabolic pathways We used the pathifier Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cOverallNCIMCIADBrain samplesNAge mean SDMale n Education mean SDAPOE ε4carrier n Serum samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Serum NCI samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Overall Overall NCI MCIAD NCI nonconverters NCI converters Table Detailed demographic characteristics of study samples Chisquare test Pvalue comparing AD vs NCI Wilcoxon rank sum test Pvalue comparing MCIAD vs NCI Wilcoxon rank sum test Pvalue comparing NCI converters vs NCI nonconverters Chisquare test Pvalue comparing NCI converter vs NCI nonconverter NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease APOE ε4 apolipoprotein E epsilon allele SD standard deviation algorithm48 to transfer metabolic level information of each sample to pathway level by generating a pathway dysregulation score PDS For each pathway each sample was projected onto a directed principal curve49 which was yielded depending on leading principal components of the pathway to optimally pass through the cloud of samples PDS was the distance along the curve between the projection of each sample and that of NCI Thus PDS could capture the pathwaylevel extent of abnormality increments or decrements for each participant relative to those with NCI We performed similar data analysis on pathway level data to what we did on metabolomics level data We tried to identify differential pathways using ordinal logistic regression across NCI MCI and a0AD groups in brain samples and logistic regression for NCI and MCIAD in serum samples Next we explored the associations between identified pathways with neuropathology KruskalWallis test for Braak scores Wilcoxon ranksum test for CERAD scores and cognitive performance Spearmans rank correlation test for the global cognitive function linear regression with adjustments for each cognitive test Then we examined the predictive potential of identified pathways in serum samples using univariate analysis Wilcoxon ranksum test for NCI converters vs NCI nonconverters NCI converters vs MCIAD Finally we built RF models on training sets and tested them on testing sets according to times random splitting on the model construction data and applied the final model on the validation data using ROC SE SP as evaluation methods The overall workflow chart of the data and the analysis are shown in Fig a0S9Data were analyzed using R version with packages including pROC pathifier randomForest ggplot2 ggsignif and MASS The statistically significance was determined by a threshold of unadjusted Pvalues and Qvalues ResultsParticipants and characteristics For the joint analyses of the ROSMAP study we measured metabolomics of serum samples NCI MCI and AD at the blood draw and postmortem brain tissues from dorsolateral prefrontal cortex NCI MCI and AD at the time of death Among brain samples and serum samples a total of participants had both brain and blood metabolomics data NCI participants n were further categorized into NCI converters and NCI nonconverters NCI converters were those participants who were cognitively normal NCI at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followup Among NCI participants the time between the blood draw and conversion ranged from to a0years with a median of a0years Fig a0S10 Detailed demographic characteristics of the serum samples and postmortem brain samples are included in Table a0 Among participants with postmortem brain samples AD patients tended to have at least one APOE ε4 allele compared to the NCI group as expected The mean age of NCI and MCIAD group at the time of blood draw among serum samples was a0years SD and SD respectively Similarly the age and the percentage of APOE ε4 Scientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cFigure a0 Brain metabolome and serum metabolome composition and alterations a Left panel the brain metabolome composition Right panel a0log10 Pvalue across clinical groups of brain tissues NCI MCI AD b Left panel the serum metabolome composition Right panel a0log10 Pvalue across clinical groups of serum tissues NCI MCIADcarriers were higher in the NCI converters group than the NCI nonconverters group We did not observe other significant demographic characteristics differences across clinical groups Table a0Identifying metabolites differentially expressed in participants with cognitive impairment In this study metabolites and metabolites overlapping metabolites were detected in brain tissues and serum samples respectively Tables a0S7 S8 Amino acids BAs carbohydrates anic acids and fatty acids were the predominant types of annotated metabolites accounting for of all the metabolites in brain tissues and in serum samples Fig a01ab left panel A total of seven a0metabolites BA BA ratio anic acid known as a longchain fatty acid fatty acids showed significant differences across clinical groups in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a01ab right panel Tables a0S14 S15 After adjusting for confounders ie fasting status supplement use diabetic and lipid lowering medications most of serum metabolites remained statistically significant Table a0S18 In brain tissues increments of the levels of GLCA DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid followed the pattern NCI MCI AD We observed increments of GLCA DCACA ratio and decrements of petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid in sera of MCIAD compared to controls Table a0 The trend of increments of identified metabolites in brain samples increments of BAs and decrements of FFAs in serum samples were further validated within individuals with both brain and serum samples From NCI to MCI and AD groups increments of identified metabolites were observed in brain samples Table a0S11 The increasing trend of GLCA DCACA ratio and decreasing trend of FFAs among MCIAD group relative to NCI group were detected in sera Table a0S11The seven brain metabolites were all negatively correlated with global cognitive function where higher scores indicate better cognitive performance Ï a0 for GLCA Ï a0 for DCACA ratio Ï a0 for petroselinic acid Ï a0 for linoleic acid Ï a0 for myristic acid Ï a0 for palmitic acid and Ï a0 for palmitoleic acid using Spearmans rank correlation analysis Fig a02a Similarly after adjusting for age gender years of education and APOE ε4 all identified metabolites remained negatively correlated with tests in five Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cBrain samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]Serum samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]NCIMCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ]NCI [ ] [ ] [ ] [ ] [ ] [ ] [ ]MCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Table Levels of metabolites differentially expressed in participants a0by diagnostic group Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing AD vs NCI Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing MCIAD vs NCI NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease IQR interquartile rangeFigure a0 Associations between metabolites level and global cognitive function a Boxplots showing group differences and P values for identified metabolites across Braak groups for brain tissue abundances b Boxplots showing group differences and significances for identified metabolites across CERAD groups for brain tissue abundances Ï correlation coefficient of Spearmans rank correlation testScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0ccognitive domains and the MiniMental State Exam see Table a0S2 for significant correlation pairs The serum concentration of two BAs showed negative correlations with global cognitive function Ï a0 for GLCA Ï a0 for DCACA ratio conversely fatty acids demonstrated positive correlations Ï for petroselinic acid Ï for linoleic acid Ï for myristic acid Ï for palmitic acid and Ï for palmitoleic acid Fig a02b Linear regression revealed similar consistent results in serum samples with adjustment for age gender years of education APOE ε4 and BMI see Table a0S2 Results of associations between identified metabolitesratio and each cognitive performance domains are shown in Table a0S13 Correlations with global cognitive function were further validated in individuals with both brain and serum samples and the directions were consistent with our previous findings among the a0entire cohort Seven identified metabolites were all negatively correlated with global cognitive function in brain samples while two BAs showed negative correlations and five FFAs showed positive correlations in serum samples Fig a0S6 Additionally the serumbrain ratio of identified FFAs were positively correlated with global cognitive function ie lower levels of identified FFAs in serum and higher levels of identified FFAs in brain were associated with worse cognition Fig a0S7Identified metabolites predicted antecedent cognitive impairment before the manifestation of clinical symptoms The concentrations of GLCA and DCACA were significantly lower in the NCI nonconverters group than in the NCI converters group By contrast the abundances of petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid were higher in the NCI nonconverters group than in the NCI converters group Fig a03a There were no significant differences in these metabolites between participants in NCI converters group vs MCIAD group Fig a03a Using the seven metabolites and age we built RF models on the training set according to 100times randomly splitting approach to differentiate MCIAD patients from NCI nonconverters group The median of times AUC on testing set was CIs with SE CIs and SP CIs using Youdens index to maximize the sum of SE and SP Fig a03b RF models showed decent classification performances in differentiating MCIAD group from NCI nonconvertersNext we were interested in studying the models early diagnostic capability for predicting NCI converters before clinical diagnosis The model was thus applied on the entire NCI group at baseline to differentiate NCI converters from NCI nonconverters We achieved an AUC of CIs SE SP at the cutoff value of Fig a03c with significant differences in RF scores between NCI converters vs NCI nonconverters between NCI nonconverters vs MCIAD group using the Wilcoxon ranksum test Pvalue Fig a03d After additional adjustment for gender years of education APOE ε4 and BMI fasting status and medications supplements diabetes lipid lowing the RF scores remained significant as an independent predictor with a coefficient of Pvalue Table a0S3 Additionally the RF scores showed significant negative correlations with global cognitive function and the five cognitive domains with the same adjustment in mixed effects models Table a0S12Personalized metabolic pathwaybased study for the association and prediction of cognitive impairment Considering altered metabolite levels were significantly associated with cognitive impairment and showed early predictive value of clinical symptoms onset we then employed the pathifier algorithm to summarize metabolite information to pathways level for further examinations All PDS scores ranged from to where larger scores represent the higher extent of the abnormality in the corresponding metabolic pathway out of metabolites detected in brain tissues and out of metabolites detected in sera were successfully mapped to the KEGG metabolic pathways This method identified metabolic pathways in brain tissues and metabolic pathways in serum samples overlapping pathways Figs a0S1ab left panel Table a0S9 Table a0S10 three of which ie primary BAs biosynthesis FFAs biosynthesis and biosynthesis of unsaturated FFAs were significantly shifted in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a0S1ab right panel Table a0S16 Table a0S17 We noted increased PDS for all three identified pathways from NCI to MCIAD that suggested dysregulation of these metabolic pathways in MCIAD patients compare to NCI Detailed PDS of these pathways stratified by diagnostic groups are described in Table a0S4 Results also indicated that higher PDS were significantly associated with lower global cognitive function ie worse cognitive performance in both brain Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S4 and serum samples Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S5 respectively In Table a0S5 we show the significant negative associations between each cognitive test and PDS of three pathways after adjusting for age gender years of education and APOE ε4 additional adjustment for BMI in serum samples Two fatty acid pathways showed significantly different PDS between the NCI nonconverters group and the NCI converters group Pvalue and Pvalue respectively A gradually increasing trend was noted for the BAs pathway across groups ie NCI nonconverters NCI converters and MCIAD Fig a04aWe then constructed a discriminant RF model in training data and tested on testing data based on three identified metabolic pathways along with age to differentiate MCIAD from NCI nonconverters in model construction data using 100times randomly splitting approach The median AUC on the a0testing set was CIs with SE CIs and SP CIs Fig a04b Applying the RF model to the whole NCI data at baseline could successfully discriminate NCI converters from NCI nonconverters with an AUC of CIs SE SP cutoff value Fig a04c Similarly predictive RF scores were significantly different between NCI converters vs NCI nonconverters and NCI nonconverters vs MCIAD group Pvalue Fig a04d After adjusting for gender Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cFigure a0 The identified panel of metabolites and its predictive performance a Boxplots showing group differences and P values for identified metabolites across NCI nonconverters NCI converters and MCIAD for serum abundances b ROC curves of metabolite models trained on the training data and tested on the testing data according to 100times randomly trainingtesting splitting c The ROC curve of the final metabolite model on the validation data d RF scores of the final metabolite model across NCI nonconverters NCI converters and MCIAD Pvalue Pvalue Pvalue Wilcoxon rank sum test The optimal cutoff was determined by the Youden index AD Alzheimers disease AUC area under | 2 |
as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is conï¬rmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors mnx1 was ï¬rstly foundto be expressed in lymphoid and pancreatic tissues and deï¬nedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia furthermore recent studiesconï¬rmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these ï¬ndingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression proï¬ling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at ¦c in a humidiï¬ed incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the aï¬liated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were conï¬rmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university aï¬liated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraï¬nembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was conï¬rmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the ï¬nalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturers protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcrampliï¬ed and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the cell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at ¦c and ï¬xed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with à clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of à hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using ï¬uorescencemicroscopy and proliferation cell ratios were counted fromï¬ve random ï¬elds in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were ï¬xedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artiï¬cial scratch on a conï¬uent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were ï¬lled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were ï¬xed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from ï¬ve random ï¬elds onevery ï¬lter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension à cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the cell index valuecell cycle analysiscells were digested with trypsinedta and ï¬xed with ethanol for h at ¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at ¦c a facscalibur ï¬ow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0g1 s and g2m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientiï¬c and scraped into a centrifuge tube the cells were centrifugedfor min at g at ¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at à g ¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at ¦c the chip elution buï¬ercontaining proteinase k was used for dna puriï¬cation thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr ampliï¬cation with primersspeciï¬c to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region bp wasampliï¬ed and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice weeks old were purchased from nanjingmedical university school of medicines accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group à exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the speciï¬cation of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length à width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression proï¬ling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage iii vs iiiiv p figure 1f t stage t1 vst2t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two speciï¬c sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression eï¬ciency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 conï¬rmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3cg of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in transcriptionand metabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been conï¬rmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no signiï¬cance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines cf two speciï¬c sirna si1 and si2 of mnx1 were designed and the transfection efï¬ciencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot gi the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefï¬ciencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespeciï¬c dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by ï¬ow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no signiï¬canceaacaataaat and bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efï¬ciency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments bd the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no signiï¬cancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efï¬ciency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacriï¬ced and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacriï¬ced andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no signiï¬cancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection eï¬ciency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6bf and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown eï¬ciency wasconï¬rmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7ce ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7hkfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identiï¬ed mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourï¬ndings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentiï¬ed to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebraï¬sh and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also conï¬rmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identiï¬ed as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identiï¬ed as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i | 0 |
Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po | 2 |
"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human AB the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [ ] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea nausea and vomiting and abdominal pain were common at presentation in COVID19 patients [ ] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto | 2 |
Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelialmesenchymal transition EMT and apoptosis in BC are not fully understood We ï¬nd that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4 EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8 Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspeciï¬c transmembrane protein foundin a variety of peripheral cells11 is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored at °C for western blotting and PCR analysis The othergroup of specimens was ï¬xed in formalin and parafï¬nizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and veriï¬ed for their authenticities by STR proï¬ling Epirubicin was purchased fromPï¬zer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The ï¬nal protein staining score was the percentage score multiplied by the intensity score ï¬nalprotein staining scores were divided into three categoriesas follows negative low expression and high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturers instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments à cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were ï¬xed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantiï¬edby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturers protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magniï¬cation à b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplanMeier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magniï¬cation à f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell ï¬uorescence was measured with a FACScan ï¬owcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefï¬ciency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of à stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacriï¬ced by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups à stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacriï¬ced by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were ï¬xed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were ï¬xed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunoï¬uorescenceImmunoï¬uorescence staining was performed as previously described19 In brief after corresponding treatments the cells ï¬xed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfï¬cial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by KaplanMeier analysis and thelogrank test was used to compare survival differencesbetween groups Pearsons correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically signiï¬cantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe ï¬rst measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfï¬cial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassigniï¬cantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and KaplanMeier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was signiï¬cantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot signiï¬cant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown signiï¬cantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further conï¬rm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efï¬ciency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was conï¬rmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1BE Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown signiï¬cantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunoï¬uorescence showed the same results as thoseOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magniï¬cation à f Correlation analyses of proteinexpression levels between DNER and catenin g KaplanMeier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further conï¬rm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were signiï¬cantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunoï¬uorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally KaplanMeier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfï¬cial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a speciï¬c Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were signiï¬cantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These ï¬ndingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown signiï¬cantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to conï¬rm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efï¬ciency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efï¬ciency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantiï¬cation of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see ï¬gure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightï¬eldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t | 2 |
" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11284f219 in lung cell lines and tissues was measured using reverse transcriptionquantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc nonsmall cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinomaassociated 0cli rp11284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfrtyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11284f219mir3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11284f219mir3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to Ëc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc clonetics corporation according to the manufacturer's instructions ncih460 and ncih1299 cells were cultured in rpmi medium cat no atcc and a549 cells in f12k medium cat no atcc supplemented with fbs cat no atcc and 1x penicillinstreptomycin thermo fisher scientific inc all cells were culture at Ëc with co2rna extraction and reverse transcriptionquantitative pcr rtqpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the firststrand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rtqpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward 'aac gac ccc ttc att gac c' and reverse 'tcc acg aca tac tca gca cc' rp11284f219 forward 'agg att ggc act cac ttc gg' and reverse 'tct ctc acc acg tct ggt ct' and ccar1 forward 'ctg atg gct agc cct agt atg ga' and reverse 'tgc ctt tca tgc cca cta aaa ' the temperature protocol used to perform rt was Ëc for h followed by Ëc for min thermal conditions of pcr reactions were initial denaturation at Ëc for min followed by cycles for sec at Ëc and sec at Ëc the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir3p mimics and mir3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck assay and edu labeling of proliferating cells a cck was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at Ëc and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckmancoulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at Ëc for h a total of 1x105 cells in µl serumfree medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides sinc sirp11284f219 mirnc mir3p mimics mir3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence ''uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at Ëc in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut 'untranslated region 'utr and ccar1 wt or mut 'utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were cotransfected with mirnc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigmaaldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sdspage transferred to a pvdf membrane and blocked with nonfat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at Ëc followed by incubation with goat antimouse or goat antirabbit igghorseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of Ëc with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature Ëc humidity and fed with sterile food and water free access to evaluate the effect of rp11284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated ncih1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ¥ independent experiments a twotailed unpaired student's ttest or oneway anova with tukey's posthoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 expression is upregulated in lc tissues and cell lines a expression of rp11284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas2b n3 lc lung carcinoma rtqpcr reverse transcriptionquantitative pcrresultsexpression of rp11284f219 is upregulated in lung carcinoma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11284f219 exerts antioncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck and edu assays fig 2bd the results suggested that knocking down rp11284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of sirp11284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rtqpcr assay was performed to detect the expression levels of invasionrelated genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11284f219 directly interacts with mir3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir3p was partially complementary with the 'utr of rp11284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 'utr with or without mir3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11284f219wt transfected cells but not the rp11284f219mut transfected cells indicating a direct interaction between the two noncoding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11284f219 leading to a significant increase in mir3p expression fig 3b thus these findings suggested an inhibitory effect of rp11284f219 on the expression of mir3p in lung carcinoma cellsthe expression of mir3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir3prp11284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir3p inhibitor which specifically downregulates the expression of mir3p was transfected into ncih1299 and ncih460 cells fig 4a the results from the cck and edu assays demonstrated that treatment with sirp11284f219 0concology reports figure rp11284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11284f219 knockdown was achieved via rp11284f219 sirna and the knockdown efficiency was verified using reverse transcriptionquantitative pcr n3 cell counting kit assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the sinc group n5 d an edu assay was performed to measure the proliferation of ncih1299 and ncih460 cells after transfection with sinc and sirp11284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11284f219 knockdown on apoptotic rates in ncih1299 and ncih460 cells n3 f transwell assay was performed to determine the effects of rp11284f219 knockdown on ncih1299 and ncih460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mirnc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure rp11284f219 directly interacts with mir3p a binding site between rp11284f219 and mir3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mirnc n3 p005 vs mirnc b expression of mir3p in ncih1299 and ncih460 cells transfected with sirp11284f219 was analyzed using rtqpcr p001 vs sinc n3 mir3p expression in c lc tissues and d ncih460 ncih1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rtqpcr n3 p005 p001 vs adjacent tissue or beas2b cells nc negative control sirna small interfering rna wt wildtype mut mutant mir microrna lc lung carcinoma mir3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by sirp11284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mirnc group fig s3 in addition transfection with mir3p inhibitor also diminished the proapoptosis effect of sirp11284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir3prp11284f219 regulates ccar1 via targeting mir3p to further evaluate how rp11284f219 exerts an oncogenic role via mir3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir3p and the ccar1 'utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1wt treated cells but not the ccar1mut treated cells suggesting a direct binding of mir3p to the 'utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir3p in lung carcinoma cells and tissuesrp11284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir3p a expression of mir3p in ncih1299 and ncih460 cells transfected with mirnc or mir3p inhibitor was detected using rtqpcr analysis n3 p005 vs mirnc cell counting kit assay was performed in b ncih1299 and c ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n5 d edu assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 e flow cytometry analysis was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor n3 f transwell assay was performed in ncih1299 and ncih460 cells stably transfected with sirp11284f219 in the presence of mirnc or mir3p inhibitor magnification x200 n3 p005 vs sinc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11284f219 promotes lung carcinoma proliferation and invasionfigure mir3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 'untranslated region and mir3p b luciferase reporter assay in ccar1wt or ccar1mut treated cells in the presence of mirnc or mir3p mimic n3 p005 vs mirnc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rtqpcr n13 p001 vs adjacent tissue expression of ccar1 in ncih1299 and ncih460 cells transfected with mirnc or mir3p mimics was detected using d rtqpcr and e western blotting n3 p005 vs mirnc mir microrna nc negative control wt wildtype mut mutant rtqpcr reverse transcriptionquantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the sirp11284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the sirp11284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11284f219mir3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir3p directly interacts with rp11284f219 by binding to its 'utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir in vitamin denhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzymemediated intratumoral drug metabolism mir is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11284f219 on the expression of mir3p in addition it was identified that the mir3p inhibitor can neutralize the antitumor effects of rp11284f219 knockdown indicating that rp11284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir3p this antitumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of ncih1299 cells transfected with sinc or sirp11284f219 n5 expression levels of d mir3p and e ccar1 in the tumor tissues from nude mice injected with ncih1299 cells transfected with sinc or sirp11284f219 for weeks were detected using reverse transcriptionquantitative pcr n5 p005 p001 p0001 vs sinc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11284f219 furthermore the regulatory axis of rp11284f219mir3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11284f219 and mir3p and the interaction between mir3p and ccar1 were demonstrated by the dualluciferase assay although this method has been used to validate rnarna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rnaprotein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicininduced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir3p the expr | 0 |
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