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DepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Theseauthorscontributedequallytothiswork.
SearchformorepapersbythisauthorXuedongYanDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Theseauthorscontributedequallytothiswork.
Currentaddress:KeyLaboratoryofCarcinogenesisandTranslationalResearch(MinistryofEducation),PekingUniversitySchoolofOncology,BeijingCancerHospitalandInstitute,Beijing,China.
SearchformorepapersbythisauthorXinYaoDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorYongliZhangDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorYingShanDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorNingMaoDepartmentofCellBiology,InstituteofBasicMedicalSciences,Beijing,China
SearchformorepapersbythisauthorYiliYangCancerandDevelopmentalBiologyLaboratory,NationalCancerInstitute,Frederick,MD,USA
SearchformorepapersbythisauthorLingyaPanCorrespondingAuthor
DepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
ProfessorLingyaPAN,M.D.,DepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,1ShuaiFuYuan,WangFuJingStreet,Beijing100730,China.Tel.:+86‐10‐65296218Fax:+86‐10‐65124875E‐mail:lingyapan@hotmail.com
SearchformorepapersbythisauthorDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Theseauthorscontributedequallytothiswork.
SearchformorepapersbythisauthorXuedongYanDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
Theseauthorscontributedequallytothiswork.
Currentaddress:KeyLaboratoryofCarcinogenesisandTranslationalResearch(MinistryofEducation),PekingUniversitySchoolofOncology,BeijingCancerHospitalandInstitute,Beijing,China.
SearchformorepapersbythisauthorXinYaoDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorYongliZhangDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorYingShanDepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
SearchformorepapersbythisauthorNingMaoDepartmentofCellBiology,InstituteofBasicMedicalSciences,Beijing,China
SearchformorepapersbythisauthorYiliYangCancerandDevelopmentalBiologyLaboratory,NationalCancerInstitute,Frederick,MD,USA
SearchformorepapersbythisauthorLingyaPanCorrespondingAuthor
DepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,China
ProfessorLingyaPAN,M.D.,DepartmentofObstetricsandGynecology,PekingUnionMedicalCollegeHospital,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,1ShuaiFuYuan,WangFuJingStreet,Beijing100730,China.Tel.:+86‐10‐65296218Fax:+86‐10‐65124875E‐mail:lingyapan@hotmail.com
SearchformorepapersbythisauthorCurrentaddress:KeyLaboratoryofCarcinogenesisandTranslationalResearch(MinistryofEducation),PekingUniversitySchoolofOncology,BeijingCancerHospitalandInstitute,Beijing,China.
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ShareonEmailFacebookTwitterLinkedInRedditWechatAbstractEarlydetectionofresistancetoplatinum‐basedtherapyiscriticalforimprovingthetreatmentofovariancancers.WehavepreviouslyfoundthatincreasedexpressionofannexinA3isamechanismforplatinumresistanceinovariancancercells.HerewedemonstratethatannexinA3canbedetectedintheculturemediumofovariancancercells,particularlythesecellsthatexpresshighlevelsofannexinA3.LevelsofannexinA3werethendeterminedinserafromovariancancerpatientsusinganenzyme‐linkedimmunosorbentassay.Comparedwiththosefromnormaldonors,serafromovariancancerpatientscontainsignificantlyhigherlevelsofannexinA3.Furthermore,serumlevelsofannexinA3weresignificantlyhigherinplatinum‐resistantpatientsthaninplatinum‐sensitivepatients.Togaininsightintothemechanismofsecretion,theovariancancercelllineswereexaminedusingbothtransmissionelectronmicroscopyandimmunoelectronmicroscopy.Comparedwithparentcells,therearesignificantlymorevesiclesinthecytoplasmofovariancancercellsthatexpresshighlevelsofannexinA3,andatleastsomevesiclesareannexinA3‐positive.Moreover,somevesiclesappeartobefusedwiththecellmembrane,suggestingthatannexinA3secretionmaybeassociatedwithexocytosisandthereleaseofexosomes.ThisissupportedbyourobservationthatovariancancercellsexpressinghigherlevelsofannexinA3releasedincreasednumbersofexosomes.Furthermore,annexinA3canbedetectedinexosomesreleasedfromcisplatin‐resistantcells(SKOV3/Cis)byimmunoblottingandimmunoelectronmicroscopy.
IntroductionOvariancanceristheleadingcauseofgynaecologicalcancermortality.Itstreatmentusuallyconsistsofoptimalcytoreductivesurgeryandplatinum‐basedcombinationchemotherapies.Althoughtheplatinumcompoundscisplatinandcarboplatinareamongthemosteffectiveanticancerdrugs,developmentofresistancetotheplatinum‐basedtherapieshasemergedasamajorobstacleinthetreatmentofovariancancers.Asaresult,the5‐yearsurvivalrateforadvancedovariancancer(stagesIIIandIV)isonlyabout20–30%[1].Ithasbecomeapparentthatearlydetectionofdrugresistanceandpromptadjustmentofthechemotherapyregimenarecriticallyimportantforimprovingtheoutcomeofovariancancertreatment.
Therehavebeenextensivestudiesonthemechanismsofplatinumresistanceinovariancancers.Anumberofgenes,includingGST‐pi,LRP,MDR1,XIAP,HER2/neu,hMLH2andhMSH1havebeenfoundassociatedwithplatinumresistanceinovariancancercells[2-6].However,itisstillnotwellunderstoodwhetherandhowthesegenescanconferspecificresistancetotheplatinumdrugs.ItalsoremainstobedeterminedwhetheranyoftheassociatedgeneproductscanbeusedasbioMarkersfortheearlydetectionofplatinumresistanceinovariancancerpatients.Throughproteomicanalysis,wehavefoundthattheexpressionofanumberofproteins,includingannexinA3,destrin,cofilin1,glutathione‐S‐transferaseomega1andcytosolicNADP+‐dependentisocitratedehydrogenase,aresignificantlyalteredinplatinum‐resistantovariancancercells[7].FurtherstudiesdemonstratedthattheexpressionofannexinA3isalsosignificantlyincreasedintumoursfromplatinum‐resistantovariancancerpatients[8].Moreover,enforcedexpressionofannexinA3specificallyconferredplatinumresistancetoovariancancercellsbothincultureandinananimalmodel,whereasdown‐regulationofannexinA3intheplatinum‐resistantovariancancercellsmadethemmoresensitivetocisplatin[8].TheseresultsindicatedthatincreasedannexinA3playsacriticalroleinplatinumresistanceandraisedthepossibilitythatitcouldbeabiomarkerforresistancetoplatinum‐basedtherapiesinovariancancerpatients.
AnnexinsareafamilyofCa2+andphospholipid‐bindingproteinsthatareubiquitouslyexpressedinvariouscells.The12humanandvertebrateannexinsarecataloguedintogroupAofthefamilyanddesignedA1–A13[9].Theyallcontainaconserved∼70aminoacidresidueannexinrepeatthatisresponsibleforCa2+andphospholipidsbinding.Anumberofstudieshaveshownthatannexinsareabletoinducethegenerationofaninwardvesicle,aformationoflipiddomainsandintermembranecontact,whichparticipatescellularprocessessuchasmembrane‐cytoskeletonorganization,membranetrafficking,endocytosis,exocytosisandcytokinesis[10].Theseactivitiesappeartoberesponsibleforthediversifiedfunctionsofannexins,whichincludemediatingtheanti‐inflammationactionofglucocorticoids,participatingintheregulationofbloodcoagulationandmodulatingionchanneltransportation[11].Inaddition,theabilityofannexinA5tobindphosphatidylserinealsomakesitavaluabletooltodetectapoptoticcells[12].However,therearerelativelyfewstudiesonthefunctionsofannexinA3,the36‐kDproteinexpressedinthecytoplasmofmanycells[13].
Inthisstudy,wedemonstratethatannexinA3issecretedfromovariancancercells.TheamountofsecretionisassociatedwiththelevelofintracellularannexinA3andplatinumresistance.Bothelectronmicroscopystudiesandbiochemicalanalysisindicatedthatthesecretionisassociatedwithexocytosisandthereleaseofexosomes.WealsofoundthatthelevelofannexinA3inserafromplatinum‐resistantovariancancerpatientsissignificantlyhigherthanthatofnormaldonorsaswellasthatofplatinum‐sensitiveovariancancerpatients,indicatingthatannexinA3mayserveasapotentialbiomarkerforovariancancerandplatinumresistance.
Drug‐sensitiveepithelialovariancancercelllines(SKOV3andA2780),theircisplatin‐resistantderivatives(SKOV3/CisandA2780/Cis)andthecelllinestransfectedwithcontrolplasmid(SKOV3/m,A2780/m,SKOV3/Cis/mandA2780/Cis/m),anannexinA3‐expressingvector(SKOV3/AnnandA2780/Ann)oranantisenseannexinA3‐expressingvector(SKOV3/Cis/RandA2780/Cis/R)weredescribedpreviously[8].ThevectorswereconstructedbyinsertingPCR‐generatedannexinA3cDNAintotheEcoRIsiteofpcDNA3.1.Theirorientationsweredeterminedbydirectsequencing.TheprimersusedforgeneratingannexinA3cDNAare5’‐GAATTCCATCATGGCATCTATCTGGGTT‐3’(sense)and5’‐GAATTCGTCATCTCCACCACAGA‐3’)(antisense).ThecellsweremaintainedinDMEM(high‐glucose)supplementedwith10%foetalbovineserum(FBS).
Immunoblottingwasperformedasdescribed[14].Briefly,cellswerelysedwithRIPAbufferandproteinconcentrationsweredeterminedusingaBCAkit(ThermoFisherScientific,WI,USA).FollowingSDS‐PAGE,proteinsweretransferredontoPVDFmembrane,probedwithspecificantibodies(anti‐annexinA31:1000,anti‐Hsp701:1000andanti‐β‐actin1:1000),andvisualizedwithhorseradishperoxidase‐conjugatedsecondaryantibodyandchemiluminescence(ThermoFisherScientific).Anti‐annexinA3antibodywaskindlyprovidedbyDr.JoelD.Ernst(UniversityofCalifornia,SanFrancisco,CA,USA).Anti‐Hsp70andanti‐β‐actinantibodieswerefromSigma‐Aldrich(H5147andA5316,respectively).
Cells(1.5×106)seededin100‐mmculturedishwereculturedwithcompleteDMEMovernightandthenwith10mlDMEMwithout10%FBSfor48hrs.The10mlofconditionalmediumwascollectedandconcentratedto200μlwithanAmiconUltra‐15centrifugalfilter(Millipore,MA,USA).Equalamountsofconcentratedconditionalmediumfromdifferentovariancancercelllineswereanalysedbyanti‐annexinA3immunoblotting.Anotheraliquoteofconditionalmediumwascollectedwithoutconcentration,andannexinA3expressionwasmeasuredusingahumanannexinA3ELISAkit(Cusabio,Wuhan,China).
FiftyovariancancerpatientswhowerediagnosedwithepithelialovariancancersbetweenJanuary2007andSeptember2007atPekingUnionMedicalCollegeHospital,wereenrolledinthisstudy,whichwasreviewedandapprovedbytheEthicsCommitteeofPekingUnionMedicalCollegeHospital.Allclinicaldiagnoseswereconfirmedbyapathologyexamination.Bloodsampleswerecollectedwithconsensusfromthesepatientsbeforetheyreceivedcytoreductivesurgeryand/orstandardplatinum‐basedchemotherapies.Allpatientsenrolledunderwentcytoreductivesurgeriesthatleftnotumourtissueswith2cmdiametersandthereforeweredefinedasoptimalcytoreductionaccordingtotheguidelineofNationalComprehensiveCancerNetworkandourhospital.Amongthem,14patientsreceivedtheirsecondcytoreductivesurgery11–24monthsafterthelastchemotherapy.Thesepatientswerefollowedregularlyformorethan1yearaftercompletionoftheirchemotherapiestodeterminewhethertheyweresensitiveorresistanttoplatinum‐basedchemotherapiesaccordingtothecommonlyusedclinicaldefinition[15].Bloodsampleswerealsocollectedfrom30normalfemaledonorsduringthesameperiod.Serafromthesebloodsampleswerealiquotedin1.5‐or0.5‐mlambervialsandstoredunder−25°Cforamaximumofaweekbeforelong‐termstorageat−80°C.
ThelevelofannexinA3inseraofovariancancerpatientswasdeterminedusingtheELISAkitforhumanannexinA3(Cusabio)accordingtothemanufacturer’sinstructions.Eachsamplewasassessedintriplicate.
Cellswerefixedandprocessedasdescribed[16].Briefly,aftertreatmentwith1%osmiumtetroxidefor1hrat4°C,samplesweredehydratedinascendinggradesofethanolandembeddedinspurresin.Followingovernightpolymerization,ultrathinsections(70–80nm)weremadeusinganultramicrotome.Thesectionswerelaidoncoppergrids,stainedwithuranylacetateandleadcitrate,andexaminedunderthetransmissionelectronmicroscope(JEOLTEM1010,JEOL,Japan).Exosomesongridswerefixedin1%glutaraldehydeandnegativelystainedwith1%uranylacetatebeforeexaminingwithTEM.
IEMwasperformedasdescribedwithafewmodifications[17,18].Briefly,cellswerefixedwith4%paraformaldehydeand1%glutaralin0.1Mphosphatebufferat4°Cfor4hrs.AfterembeddinginL.R.White,ultrathinsections(70–80nm)werecutandlaidonnickelformvar‐carbonfilmedgrids.Followingincubationwiththerabbitanti‐annexinA3antibody(1:100)and12‐nmcolloidgold‐labelleddonkeyanti‐rabbitantibody(1:50)sequentially,thegridswerestainedwithammoniummolybdate‐trehaloseandexaminedwiththetransmissionelectronmicroscope(JEOLTEM1010,JEOL).
Cells(1×107)wereculturedwithDMEMwithoutFBSfor24hrstoavoidpotentialcontaminationofexosomesfromserum‐derivedproducts[19].Forpurificationofexosomes,60mlofculturemediumwasclearedbycentrifugationat1000×gfor10min.andconcentratedto∼1.5mlusingaCentriconPlus‐20filtercapsule(Millipore).Itwasthentransferredontothetopof30%sucrose‐deuteriumoxide(D2O)andultracentrifugedat100,000×gfor40min.at4°C.Theexosomelayerwascollected,washedandresuspendedwithphosphatebuffersaline(PBS)forfurtherexperiments.Quantitiesofexosomeswereexpressedastotalamountofproteinintheexosomepreparationfromonemillioncells(μg/106cells).ForIEM,freshexosomeswereadsorbedtoglow‐discharged400‐meshcarbon‐coatedparlodioncoppergrids(Pella)for2min.,rinsedbrieflywithPBS,andincubatedsequentiallywithanti‐annexinA3andgold‐labelledsecondaryantibody.
DatawereanalysedusingtheSPSS12.0statisticalsoftwarepackage.ContinuousvariableswereexaminedwithaStudent’st‐test.AMann–WhitneyU‐testwasusedtoanalyseclinicaldataandELISAresults.DifferencesbetweengroupswereconsideredtobesignificantwhenP0.05.ThereportedPvaluesweretwotailed.AscatterplotofannexinA3expressioninserumwasdrawnusingGraphpadPrism5.0.1software.AsurvivalcurvewasusedtodescribetheassociationbetweenannexinA3andprogress‐freetime.
Althoughannexinsdonotcontainasignalsequenceforproteinsecretion[20],somefamilymembers,includingA1,A2,A3andA6,havebeenfoundoutsidecellsundermanycircumstances[21-23].Therefore,weaskedwhetherincreasedexpressionofannexinA3inovariancancercellscanleadtotheirsecretiontoculturemedium.ComparedwiththosefromparentSKOV3andA2780cells,concentratedsupernatantsfromplatinum‐resistantcellsSKOV3/CisandA2780/CiscontainedsignificantlyhigherlevelsofannexinA3(Fig.1).SupernatantsfromSKOV3andA2780cellstransfectedwithanannexinA3expressingplasmidalsohadelevatedlevelsofannexinA3(Fig.1AandB).Furthermore,down‐regulationofannexinA3inSKOV3/CisandA2780/CiswithantisenseannexinA3significantlydecreasedtheamountofannexinA3inthemedium(Fig.1AandB).TheseresultsindicatethatannexinA3canbesecretedintoculturemediumandthesecretionissignificantlyincreasedincellsthatexpresselevatedlevelsofcytoplasmicannexinA3.
AnnexinA3levelsintheconditionalculturemediumfromtheovariancancercellsweremeasuredbyELISA.(B)Proteinsfromtheovariancancercelllysatesandconcentratedculturemediawereanalysedbyanti‐annexinA3immunoblotting.EnforcedexpressionofannexinA3inSKOV3andA2780cellsresultedintheincreasedsecretionofannexinA3intheculturemedium.Down‐regulationofannexinA3inplatinum‐resistantovariancancercellsSKOV3/CisandA2780/Cis,whichexpresshighlevelsofannexinA3,reducedthesecretionofannexinA3.(C)AnnexinA3levelsinserafromthe30normalfemaledonorsandthe50ovariancancerpatientsweredeterminedbyELISA[particularhighlevelsofannexinA3(2.0461,3.4453,8.8125and18.3081ng/ml,respectively)cannotbeseeninthegraphbecausetheY‐axisrangeis2.0ng/ml].Thepatientsaredividedintoplatinum‐sensitive(n=20)andresistant(n=30)groupsbasedonclinicaldata.TherearesignificantdifferencesintheserumlevelsofannexinA3amongnormaldonors,thesensitivegroupandtheresistantgroup.DatawereanalysedwithaMann–WhitneyU‐Test.Pvalueswerealltwo‐sided.(D)Progress‐freetimesofthe50ovariancancerpatients.ThereisasignificantdifferencebetweenthehighannexinA3group(serumA31.13ng/ml)andthelowannexinA3group(serumA31.13ng/ml)(P=0.0090.05).
TheroleofannexinA3inplatinumresistanceanditssecretionbyovariancancercellsledustoaskwhetheritcanbedetectedintheseraofnormaldonorsandpatientswithovariancancers.UsingacommerciallyavailableELISAkit,wefirstexaminedannexinA3inserafrom30normalfemaledonors.AsshowninTable1,theaveragelevelofannexinA3intheseserais0.8590ng/ml,witharelativelysmallvariationamongdifferentindividuals(S.D.=0.0744ng/ml),whichisclosetothestandarddeviationofassessingthesamesamplesintriplicates.ThelevelsofannexinA3intheserafrom50ovariancancerpatientsenrolledinourhospitalwerealsoassessed.Comparedwiththatofnormaldonors,theaverageannexinA3levelinserafromovariancancersissignificantlyelevated(1.6898ng/ml)(Table1).However,therearequitelargevariationsamongindividualpatients(S.D.=2.6563ng/ml).
Thecharacteristicsofthe50patientsareshowninTable2.Twentyofthepatientsareclinicallydefinedasplatinum‐sensitiveastheyrespondedtoinitialplatinum‐basedtherapyandexperiencedaprogress‐freeintervalofmorethan6months[15],whereastheother30patientsareclassifiedasplatinumresistant.Therearenosignificantdifferencesinallthecharacteristicsbetweenthesensitiveandresistantgroups.However,theaveragelevelsofannexinA3intheresistantandsensitivegroupare2.1145±3.3833and1.0528±0.1178ng/ml,respectively,whicharestatisticallysignificant(P=0.0009).ThereisalsoasignificantdifferencebetweenthelevelsofannexinA3intheserafromplatinum‐sensitivepatientsandthatofnormaldonors(P=0.006).
TheannexinA3levelsofindividualpatientsareshowninFigure1CandTable1.Itisworthnotingthatfourcisplatin‐resistantpatientshadparticularlyhighlevelsofannexinA3(2.0461,3.4453,8.8125and18.3081ng/ml,respectively)(Table3).TheyarelargelyresponsiblefortheconsiderablevariationofannexinA3levelsinovariancancerpatients.Furthermore,allpatientswhoseserumannexinA3levelsexceeded1.34ng/mlwereresistanttoplatinum‐basedchemotherapy(11of30).Amongtheresistantpatients,19of30hadserumannexinA3levelhigherthan1.13ng/ml,whereasonly4of20platinum‐sensitivepatientshadserumannexinA3levelabovethislevel.Analysisofthese50patientsalsoshowedthatelevatedlevelsofannexinA3(over1.13ng/ml)areassociatedwithsignificantlydecreaseddisease‐freetime[P=0.009(0.05);Fig.1D].TheseresultsindicatethatserumannexinA3levelsmaybeaprognosticbiomarkerforresistancetoplatinum‐basedchemotherapyinovariancancerpatientswithreasonablesensitivity(63%)andspecificity(80%).
Ithasbeenshowninavarietyofcellsthatannexinscanaffectvesicletraffickingandexocytosisbypromotingmembranefusion[24].Therefore,weexaminedtheultrastructureoftheovariancancercellsusingTEM.Thecellnucleus,manyorganellesincytoplasm,andthecellmembranecouldbeclearlyidentifiedunderTEM(10,000×).Comparedwiththatofplatinum‐sensitiveSKOV3andA2780cells,thecytoplasmofplatinum‐resistantcellsSKOV3/CisandA2780/Ciscontainssignificantlyincreasednumbersofmembrane‐boundroundorellipticalvesicles,rangingfrom0.1to1μminsize.Someofthesevesiclescontainhigh‐densityparticles,suggestingthattheymayberelatedtophagocytosisorendosomes(Fig.2A).Underhighermagnification(50,000×),itisevidentthatsomeofthevesicleslooklikemultivesicularbodies(MVBs)withcontentsofvariousdensities.Somevesiclescanbefoundfusingwithcellmembrane(Fig.2B),indicatingthattheyparticipateinexocytosis.Interestingly,SKOV3/AnnandA2780/Ann,theovariancancercellsthatweretransfectedwithanannexinA3‐expressingvector,alsopossessincreasednumbersofvesicles(Fig.2C).Furthermore,SKOV3/Cis/RandA2780/Cis/R,theplatinum‐resistantcelllinesthathadbeentransfectedwithavectorexpressinganantisenseannexinA3andhadreducedlevelsofannexinA3,alsohadfewervesicles(Fig.2C,lower).TheseresultsdemonstratethatincreasedexpressionofannexinA3intheovariancancercellsresultsintheformationofincreasednumbersofMVBsinthecytoplasm.
HighlevelsofannexinA3areassociatedwithincreasednumbersofvesiclesinthecytoplasm.(A)RepresentativeTEM(10,000×)photographsofpairedovariancancercelllinesSKOV3andSKOV3/CisandA2780andA2780/Cis.Thearrowsindicatetheincreasednumbersofvesiclesinplatinum‐resistantSKOV3/CisandA2780/Ciscells.(B)Highermagnification(50,000×)photographsofSKOV3/Ciscells.ArrowsindicatetheMVB‐likevesicle(upper)andexocytosisofavesicle(lower).(C)RepresentativeTEM(10,000×)photographsofovariancancercelllines.ArrowsintheupperpanelindicatedincreasedvesiclesinannexinA3‐expressingA2780/AnnandSKOV3/Anncells.ThelowerpanelsarepicturesofA2780/Cis/RandSKOV3/Cis/Rcells.ThevesiclesobservedinA2780/CisandSKOV3/Cis(A)havelargelydisappeared.
TofurtherexploretheassociationofannexinA3secretionandthecytoplasmicvesicle,cellswereexaminedbyIEMusingananti‐annexinA3antibodyandacolloidalgold‐labelledsecondaryantibody.Toavoiditspotentialeffectonantibody‐recognizedepitopesofannexinA3,osmicacidwasnotusedinthestainingofcellsforelectronmicroscopy.Therefore,thepicturesfromthesestudiesarelighterandhavelesscontrastcomparedtoregularTEMpictures.Nevertheless,theincreasednumbersofthevesiclescouldbeidentifiedinSKOV3/Ciscellsunderthiscondition(Fig.3).AsshowninFigure3B,thehigh‐densitygoldparticlesthatareindicativeofannexinA3werepresentinthecytoplasmicregionofthecells.Particularly,annexinA3appearedtobehighlyexpressedbotharoundandinsidethemembraneofthevesicles.PicturestakenunderhighermagnificationshowedthatsomeannexinA3‐containingvesiclesappeartobefusingwithcellmembranes(Fig.3Cand3D),indicatingthatannexinA3mightbesecretedthroughexocytosis.
ImmunoelectronmicroscopystudiesofannexinA3inplatinum‐resistantovariancancercellSKOV3/Cis.CellpreparationandtreatmentaredescribedinMaterialsandMethods.Ultrathinsectionswereexaminedbytransmissionelectronmicroscopy(left,50,000×;right,100,000×).Thehigh‐density12‐nmcolloidalgoldparticles,whichareindicativeofannexinA3,couldbeobservedinthecytoplasmofthecells(smallarrows).Somegoldparticlesexistedinoraroundvesicles(mediumarrows).(A)Control(sectionsweretreatedasdescribedwithoutanti‐annexinA3antibody).(B)AnnexinA3expressionincytoplasm.(C,D)AnnexinA3expressionincytoplamicvesicles.ThevesicleinCappearsreadytoundergoexocytosis.
Thesimilarityofthevesiclesinplatinum‐resistantcellsandMVBspromptedustoaskwhetherthesecretedannexinA3isassociatedwithexosomes,whichare40–100nmvesiclesthatresidedinsideMVBsandarereleasedfromcellsuponthefusionofMVBswiththecellmembrane.ExosomesisolatedfromtheculturesupernatantofSKOV3/Ciscellswereintherangeof40–100nmindiameterandexhibitedthetypicalcup‐shapedmorphology(Fig.4A).IEMwithanti‐annexinA3antibodyandgold‐coupledsecondaryantibodydemonstratedthatannexinA3isassociatedwiththeseexosomes(Fig.4BandC).However,becauseofthesizeofthegoldparticle(12nm),itisdifficulttodeterminewhetherannexinA3isassociatedwiththeexosomemembrane.TheassociationofannexinA3withtheseexosomeswasfurtherexaminedbyanti‐annexinA3immunoblotting.AsshowninFigure4D,exosomesfromSKOV3cellscontainbothannexinA3andthecharacteristichsp70.TheseresultsindicatethatatleastsomeannexinA3issecretedthroughexocytosisinsidetheexosomes.Theamountofexosomesreleasedfromvariousovariancancercellswasfurtherassessedbymeasuringtotalproteininexosomepreparations.AsshowninFigure5A,enforcedexpressionofannexinA3significantlyincreasedtheamountofexosomereleased,whereasdown‐regulationofannexinA3inplatinum‐resistantcellsreducedexosomereleasesignificantly.Thesamephenomenonwasobservedwhenexosomepreparationswereanalysedbyimmunoblottingwithanti‐hsp70andannexinA3antibodies(Fig.5B).
AnnexinA3isassociatedwithexosomesfromovariancancercellsSKOV3/Cis.(A)ElectronmicroscopicpictureofexosomesfromSKOV3/Ciscells.Theexosomesare40–100nmindiameterandexhibitthetypicalcup‐shapedmorphology.(B,C)IEMphotographsofexosomesfromSKOV3/Ciscells.Thehigh‐densitydotsrepresent12‐nmgoldparticles,whichareindicativesofannexinA3.(D)Conditionalmedium(30ml)fromculturedSKOV3/Ciscellswascollected,anddividedintotwoparts.Afterremovingtheexosomesfromoneofthem(DMEM2),bothwereconcentratedandanalysedbyimmunoblttingwithanti‐annexinA3andanti‐Hsp70antibodies.Theexosomepreparationandtheuppersucrosewerealsoexaminedbytheimmunoblotting.
TheamountofexosomesreleasedbytheovariancancercellsdependsontheexpressionofannexinA3.(A)Exosomeswereisolatedfromconditionedmediumofculturedovariancancercells.Thetotalamountofproteinwasusedtoquantifyrecoveredexosomes.(B)Comparisonoftheamountsofexosomesreleasedfromvariousovariancancercellsbyimmunoblttingwithanti‐annexinA3andanti‐Hsp70antibodies.
WehavefoundinthisstudythatannexinA3canbesecretedfromovariancancercells.Up‐regulationofannexinA3inthesecellsleadstoasignificantincreaseinitssecretion,whereasdown‐regulationofannexinA3resultsinitsreductioninculturemedium.ThisisconsistentwiththeobservationthatannexinA3canbedetectedinurinefromprostatecancerpatients[22].ExaminationoftheseovariancancercellsusingTEMandIEMrevealedthatincreasedexpressionofannexinA3resultsintheformationofMVBs‐likevesicles.AtleastsomeofthevesiclescontainannexinA3andappeartofusewiththecellmembrane.ItwasalsofoundthatannexinA3isassociatedwithexosomesreleasedfromplatinum‐resistantovariancancercells.BecauseMVBsoftencontainexosomesthatcanbereleasedthroughexocytosis[25],theseresultsledustoproposethatannexinA3promotestheformationofMVBsthataresecretedwithexosomesuponfusionoftheMVBswiththecellmembrane.Itisworthnotingthatotherannexins,suchasA1,A2andA6havealsobeenfoundtobesecretedthroughnonclassicalpathways[23,26,27].Furthermore,ithasbeenshownthatannexinA2isassociatedwithMVBsinenterocytes[26],suggestingthatexocytosisofMVBsmightbeacommonmechanismforannexinsecretion.Thishasbeenfurthersupportedbythefindingthatannexinsarecomponentsofexosomesfromavarietyofcells[28].
WealsoexaminedthelevelsofannexinA3inserafrom30normalfemaledonorsand50ovariancancerpatientsbyELISA.Comparedtothatofnormaldonors,thereisasignificantincreaseofannexinA3inserafromovariancancerpatients.Furthermore,theconcentrationofannexinA3inserafromplatinum‐resistantpatientsissignificantlyhigherthanthatofplatinum‐sensitivepatients.OvariancancerpatientswithhigherlevelsofserumannexinA3alsohaveasignificantlyshorterprogress‐freetime.TheseresultsindicatethatannexinA3maybeabiomarkerforovariancancerandaprognosticmarkerforplatinumresistance.Consistentwithourobservation,increasedannexinA3immunostaininghasbeenfoundinapproximatelytwo‐thirdsofcolorectalcancerpatients[29],inlungadenocarcinomawithlymphnodemetastasis[30]andinprostatetumours[31].Inparticularly,platinumdrugsarealsocommonlyusedinthetreatmentofpatientswithlungcancer.ItcouldbeveryinformativetofurtherassesstheserumannexinA3levelsinthesepatientsbefore,during,andafterchemotherapies.Interestingly,anumberofpatientsenrolledinourstudyhadparticularlyhighlevelsofserumannexinA3.Itisconceivablethat,byassessingthelevelofannexinA3inasignificantlylargergroupofpatients,wemaybeabletoassociatethehighserumA3levelwithaparticularpathologicaltype,stageandsizeofovariancancer.TofurtherexaminetheprognosticroleofannexinA3,wearealsoplanningtocompareserumannexinA3levelsinovariancancerpatientsbefore,duringandaftercytoreductivesurgeryandchemotherapy.Itisalsoworthnotingthatsomeoftheplatinum‐resistantpatientsdonothaveincreasedserumA3levels.Thismayreflectthenotionthatdrugresistanceinovariancancercanresultfromabnormalpharmacokineticsandtumourmicroenvironments[32].BecausewehaveshownpreviouslythatannexinA3specificallyconfersresistancetotheplatinumcompounds[8],itisalsoconceivablethattheinsensitivitytoplatinum‐basedchemotherapyinsomepatientsisbecauseofresistancetootheragentssuchaspaclitaxel[33,34].
Tworecentstudieshavefoundthat,whencomparedwithothertypesofovariancancer,annexinA4isup‐regulatedinovarianclearcellcarcinomasandisassociatedwithplatinumresistance[35,36].Giventhestructuralandfunctionalsimilaritiesamongtheannexinfamilymembers,itisconceivablethatannexinsA3andA4mayactthroughsimilarmechanismsinovariancancercells.Wearealsointerestedinexaminingtheexpressionofadditionalannexinsinvarioustypesofovariancancers.IthasbeenreportedthatannexinA3inurinedetectedbyimmunoblottingisahighlyspecificmarkerforearlydetectionofprostatecancer[22].GiventheincreasedserumannexinA3level,weareinterestedinexaminingannexinA3inurinefromovariancancerpatientsinthefuture.Inadditiontothe36‐kDannexinA3thatweinvestigatedinthisstudy,a33‐kDalternativelysplicedformofannexinA3hasbeenfoundinprimarycellculturesfromrenalcellcarcinomaandnormalrenalcortextissue[37].Intriguingly,although36‐kDisoformwasdown‐regulatedinrenalcellcarcinoma,the33‐kDisoformwasup‐regulatedinthesetumourcells[37].Weareinterestedinfurtherexploringwhetherthesmallerisoformisalsoassociatedwithothertypesoftumours.BecausetheN‐terminalregionofannexinA3playsanimportantroleinmediatingprotein–proteininteraction,itwouldbeinterestingtodeterminewhetherenforcedexpressionofthe33‐kDisoformcanstillincreasetheformationofvesiclesinovariancancercells,whichwouldhelptounderstandthemechanismofannexinA3‐promotedvesicleformation.
ThisworkwassupportedbytheKeyFoundationofPUMCHospital(Grant200203).WethankDr.JoelD.Ernst(UniversityofCalifornia,SanFrancisco,USA)foranti‐annexinA3antibody,ProfessorWeiDai(ElectronMicroscopyCenter,PekingUnionMedicalCollege,Beijing,China)fortechnicalassistanceinelectronmicroscopy.WearealsogratefultoourcolleaguesintheDepartmentofCellBiology,InstituteofBasicMedicalSciences,fortheirsupportandtechnicalassistance,andtoDr.AlanO.Perantoniforhiseditingofthepaper.
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