Adiponectin reverses the proliferative effects of estradiol and IGF-1 in human epithelial ovarian cancer cells by downregulating the expression of their receptors

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HormonesandCancer(2018)9:166–

174https://doi.org/10.1007/s12672-018- 0331-z

ORIGINALPAPER

AdiponectinReversestheProliferativeEffectsofEstradiolandIGF- 1inHumanEpithelialOvarianCancerCellsbyDownregulating theExpressionofTheirReceptors

MartaHoffmann¹^&JustynaGogola¹^&AnnaPtak¹

Received:12February2018/Accepted:14March2018/Publishedonline:30March2018

#TheAuthor(s)2018

Abstract

TheexpressionofadiponectinreceptorsAdipoR1andAdipoR2hasbeenreportedinthehumanovaryandovariancancertissues.More over,adiponectinhasbeenreportedtoactasananti-

tumorfactorbyinhibitingcancercellproliferation.Thus,weinvestigatewhetheradiponectinanditsreceptorsinfluenceovariancanc erdevelopment.Inthepresentstudy,wefoundthatadiponectinwasnotexpressedinthegranulosacellline(COV434),andepithelialo variancancercelllines(OVCAR-3,SKOV-3,andCaov-

3).Additionally,wefoundthatAdipoR1andAdipoR2expressionislowerinepithelialovariancancercellsthaningranulosatumorcell s.Endogenous17β-

estradiolaswellasexogenousestrogens,suchasbisphenolAanditschlorinatedandbrominatedanalogsdonotaffectadiponectinrece ptorexpression.WefoundthatadiponectininhibitedthegrowthofOVCAR-3andSKOV-

3cells,andthatthiseffectwasindependentofapoptosis.Moreover,adiponectinreversesthestimulatoryeffectsof17β- estradiolandinsulin-

likegrowthfactor1oncellproliferationbydownregulatingtheexpressionoftheirreceptors,whereasprogesteroneincreasedthese nsitivityofcancercellstoadiponectinbyupregulatingAdipoR1andAdipoR2expression.Theseresultssuggestinteractionsbetween adiponectinandvariousovariansteroidhormoneandgrowthfactorpathwaysinovariancancercells.

Introduction

Ovariancancerremainstheleadingcauseofdeathamon gwomen,withanestimated150,000annualdeathsworldwide

Highlights

• Adiponectinreceptorexpressionislowerinepithelialovariancancercell sthaningranulosatumorcells.

• Adiponectinrepressestheproliferationofepithelialovariancancercells.

• Estrogens,suchasE2andBPAanditsanalogs,donotaffectadiponectinrec eptorexpression.

• Progesteronepotentiatestheeffectsofadiponectinbyupregulatingtheex pressionofitsreceptors.

• AdiponectinreversestheproliferativeeffectsofE2andIGF-1bydown- regulatingtheexpressionofitsreceptors.

*AnnaPtak

anna.ptak@uj.edu.pl

MartaHoffmannmarta.hoffmann@

doctoral.uj.edu.pl

JustynaGogolajustyna.gogola@d octoral.uj.edu.pl

1

DepartmentofPhysiologyandToxicologyofReproduction,ChairofAni malPhysiology,InstituteofZoologyandBiomedicalResearch,Jagiello nianUniversity,Krakow,Poland

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[1].Duetoitsnon-

specificsymptoms,mostcasesofovariancanceraredetected whenthediseasehasadvancedtoalatestagethatassociateswit hpoorsurvival.Thus,approachesthatwouldincreaseitsearly detectionareurgentlyneededtore-

ducemortality.Ovariancancercanbeclassifiedintothre etypesbasedonthecellofitsorigin,namely,epithelial,stro- mal,andgerm,witheachtypeconferringdifferenthistopath- ologicalfeaturesandclinicaloutcomes[2].Epithelialovarian canceristhemostcommonovarianmalignancy;itoriginatesin epithelialcellsfoundonthesurfaceoftheovaryandac- countsfor~80–

90%ofovarianmalignancies.Stromaltu-

mors,ontheotherhand,accountfor~7%ofovarianmalignancies,andthemostfrequentlydiagnosedstromaltumo rtypeisthegranulosacelltumor(GCT).

Thereisemergingevidencetoindicatethatobesityisthem ainindependentriskfactorforovariancancer[3–

5].Althoughthecorrelationbetweenovariancancerandobe- sityhasbeenlinkedtohormones,itisnotclearhowtheycantrig gercancerinobesewomen.Hormonesandgrowthfactorshav eimportantrolesinregulatingcellproliferation,differentiati on,andapoptosis.Forexample,17β-

estradiol(E2),progesterone(P4),andinsulin- likegrowthfactor1(IGF-

1)haveallbeenproposedtoinfluenceovariancancerdevelop ment[6,7].Adipokines,hormonessecretedfrom

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HORMCANC(2018)9:166–174 16 7 adiposetissuesthatmaypromoteobesity,mayalsoaffectcance

rdevelopment.

Adiponectin,anadipokinewithamolecularweightof30kD a,isfoundintheserum,whereitexistsinfourisoforms,namely,tri meric(90kDa),hexameric(180kDa),andhigh-molecular- weight(360and400kDa)isoforms[8].Ataserumconcentrationo f5–

30μg/ml,itisthemostabundantcirculatingpeptidehormone.In obeseadults,however,theserumadiponectinlevelisreduced[

9].Adiponectinhasbeenreportedtoactasananti- tumorfactorbyinhibitingcancercellprolifer-

ation[10,11].Otherstudiesreportaroleforadiponectininobesity -

associatedcancersuchasthoseofthebreast,cervix,andendometri um.However,theroleofadiponectininovariancancerhasbeenst udiedmuchless.Forexample,Jinetal.re-

portedthatadiponectinlevelsweresignificantlylowerinovar- iancancerpatientsthaninhealthyindividuals,butthereasonforth isisnotclear[12].Furthermore,thebiologicalactionsofadiponec tinaremediatedthroughinteractionswithitsreceptorsubtypes,A dipoR1andAdipoR2.Lietal.showedthatalowAdipoR1expressi onlevelincancerousovariantissuesservesasanindependentprog nosticindicatorofthedisease[13].InthehumangranulosaKGNc ellline,AdipoR1functionsincellsurvival,whereasAdipoR2reg ulatessteroidproduction[14].

Severalendogenous,aswellasexogenousfactors,includ- inginsulin,thiazolidinediones,metformin,andbisphenolA(B PA),canregulatetheproductionandsecretionofadiponect ininthe3T3-L1adipocytecellline[15–

18].Ontheotherhand,severallinesofevidenceindicatethaten do-

crinedisruptingchemicals,suchasBPA,caninduceobesity[19, 20].BPA,acommercialproductcommonlyusedinpoly- carbonateplasticsandepoxyresins[21],possessesestrogenica ctivityandpromotesovariancancercellproliferation[22,23]an dmigration[24].Epidemiologicalstudiesreportthathumans havedetectableserumlevelsofnotonlyBPA,butalsoitshalog enatedderivatives,tetrabromobisphenolA(TBBPA)andtetr achlorobisphenolA(TCBPA)[25–27].

Weaimedtoinvestigatewhetheradiponectinanditsrecep- tors,AdipoR1andAdipoR2,areexpressedinhumanepithe- lialovariancancercelllines.WealsoexaminedwhetherBPAan ditsanalogscanaffecttheexpressionofadiponectinanditsrecep torsinovariancancercells.Theeffectsofadiponectinoncellproli ferationandapoptosiswerealsoexamined.Finally,weinvestig atedwhetherE2,P4,andIGF-

1canregulateAdipoR1andAdipoR2expressionandmodulate theeffectsofadiponectinontheproliferationofovariancancerc ells.

MaterialsandMethods

CellCultureandChemicals

OVCAR-3,SKOV-3,andCaov-

3werepurchasedfromtheAmericanTypeCultureCollection(

Manassas,VA,USA).

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168 HORMCANC(2018)9:166–174

COV434cellswereobtainedfromtheEuropeanCollectionof AuthenticatedCellCultures(ECACC,Sigma-

Aldrich,St.Louis,MO,USA).Thehumanovarianserouscarci nomacelllinesOVCAR-3,SKOV-3,andCaov-

3wereculturedinRPMI1640,McCoy’s,andDulbecco’smodi fiedEagle’sme-

dium(DMEM),respectively.Mediaweresupplementedwith 10%fetalbovineserum(FBS)

(ThermoFisherScientificInc.,Carlsbad,CA,USA).Thehu manmetastaticgranulosacelllineCOV434wasculturedin DMEMsupplementedwith2mM^L-

glutamine(Biowest,Nuaillé,France)and10%FBS.Allcellc ulturesweremaintainedat37°Cinahumidifiedatmosphere containing5%CO2.

Humanadiponectin,17β-estradiol,progesterone,and insulin-likegrowthfactor1werepurchasedfromSigma- Aldrich.BPA(AccuStandard,NewHaven,CT,USA)a ndE2weredissolvedinabsoluteethanol.TBBPAandTCBP AwerepurchasedfromSantaCruzBiotechnology(SantaCruz ,CA,USA)anddissolvedinDMSO.Thefinalconcentrationof ethanolandDMSOinthemediumwas0.1%.Viabilityofcellsw asnotaffectedatthisconcentration.

Real-

TimeP CRA nalysis

COV434,OVCAR-3,SKOV-3,andCaov- 3cellswereseededin96-

wellplatesandcultureduntil70%confluentintheindi- catedmediafor24h.Theculturemediumwasthenremoved,an dthecellswererinsedwithPBSandstoredat−20°C.Toinvesti gatetheeffectsofE2,P4,IGF-

1,BPA,TBBPA,andTCBPAontheexpressionofAdipoR1an dAdipoR2,thecul-

turemediumwasreplacedwithfreshmedium24hbeforethecel lsweretreatedwithcompounds.Thecellswerethenex- posedtovehicle(medium,0.1%DMSO,or0.1%ethanol),E2(1 0nM),P4(100μM),IGF-1(100ng/ml),orBPA,TBBPA, orTCBPA(1,10,and100nM)for24h.Toinvestigatetheeffec tsofadiponectinontheexpressionofERα,ERβ,PR,andIGF 1R,theculturemediumwasreplacedwithfreshme-

dium24hbeforethecellsweretreatedwithadiponectinat25 μg/mlfor24h.

TotalRNAwasisolatedfromcontrolandtreatedcells,foll owedbycDNAsynthesisusingtheTaqManGeneExpressi onCells-to-

CTKit(AppliedBiosystems,FosterCity,CA,USA),accordi ngtothemanufacturer’sinstructions.Thelysissolutioncontai nedDNaseItodigestcontaminatinggenomicDNA.Theresult ingcDNAwasanalyzedbyreal-

timePCRusinga StepOnePlusReal-

TimePCRSystem(AppliedBiosystems)andTaqManGeneE xpressionAssaystogetherwithaTaqManGeneExpression MasterMixandROXReferenceDye(AppliedBiosystems).

Thethermalcy-

clingconditionswereasfollows:50°Cfor2minand95°Cfor10 min,followedby40cyclesof95°Cfor15sand60°Cfor60s.Dupli catecontrolcDNA-

freesampleswerepreparedforeachgene.TheTaqManGeneEx pressionAssaysusedforreal-

timePCRwereasfollows:adiponectin[ADIPOQ,assay

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nos.Hs00605917_m1andHs00977214_m1],AdipoR1[A DIPOR1,assayno.Hs01114951_m1],AdipoR2[ADIP OR2,assayno.Hs00226105_m1],ERα[ESR1,assayn o.Hs 0 01 7 4 860 _m 1 ] ,E Rβ[E SR 2,assayno.Hs001100353_m1], PR[PGR,assayno.Hs01556702_m1],

andIGF1R[IGF1R,assayno.Hs00609566_m1].Expressionl evelswerenormalizedtothatofGADPH(assayno.431088 4E),andtherelativeexpressionwasquantifiedusingthe2^−ΔΔCtm ethod[28].

WesternBlotAnalysis

COV434,OVCAR-3,andSKOV-3cellswereseededin24- wellplatesandcultureduntil70%confluentintheindicatedmed iafor24h.Thecellswerethenremovedfromplates,trans ferredtoice-

coldlysisbuffer,andstoredat−20°C.Theproteinconcentrati onsofthelysatesweredeterminedbytheBradfordmethod(Bio -RadProteinAssay,Bio-

RadLaboratories,Hercules,CA,USA).Anequalamountofpro -

tein(60μg)fromeachsamplewasassayed.Proteinsweresep aratedby10%SDS-

PAGE,andthentransferredtopolyvinylidenefluoride(PVD F)microporousmembranes(Millipore,Billerica,MA,USA)u singtheBio-RadMini-

Protean3System.Themembraneswereblockedfor1hin 0.02MTris-bufferedsalinecontaining5%bovineserumal- buminand0.1%Tween20,andthenincubatedovernightat4°C withantibodiesspecificforhumanAdipoR1(cat.no.sc- 46749,SantaCruzBiotechnology),humanAdipoR2(cat.no.sc -46751,SantaCruzBiotechnology),ERα(cat.no.sc-

542,SantaCruzBiotechnology),PR(cat.no.sc-

539,SantaCruzBiotechnology),IGF1R(cat.no.ab39675, Abcam,Cambridge,UK),andPARP(cat.no.#9542,CellSign alingTechnology).Themembraneswerethenwashedthreetim esinTris-

bufferedsalinecontaining0.1%Tween20(TBST)andincubate dfor1 h atroomtemperaturewitha species-

compatiblehorseradishperoxidase-conjugatedsecondaryan- tibody(cat.no.sc-2020,SantaCruzBiotechnology).β- Actin(cat.no.A5316,Sigma-

Aldrich)wasusedastheloadingcon-

trol.ImmunopositivebandswerevisualizedusingWeste rnBrightQuantumHRPSubstrate(cat.no.K-

12043D20,AdvanstaInc.,MenloPark,CA,USA).Quantificat ionofproteinbandswasperformedbydensitometryusingVisi onWorksLSAcquisitionandAnalysissoftware(UVP,LL CUpland,CA).

CellProliferation

CellproliferationwasmeasuredusingAlamarBlueCellViabi lityReagent(Invitrogen,Paisley,UK),accordingtothemanufa

cturer’sinstructions.OVCAR-3andSKOV-

3cellswereexposedtoadiponectin(0.025,0.25,2.5,or25μg/ml) for48h.Inco-treatmentexperiments,OVCAR-

3cellsweretreatedwithadiponectin(25μg/ml)andE2(10nM),P 4

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(100μM),orIGF-

1(100ng/ml)foranadditional48h.TheAlamarBluestocksolu tionwasasepticallyaddedtothewellsafter48hofcultureinana mountequaling10%ofthevolumeofculturemedium.Theredu ctionofresazurintoresorufinwasdeterminedafter4hofincuba tionbymeasuringthefluores-

cenceatanexcitationwavelengthof560nmandanemissionwa velengthof590nmusinganFLx800fluorescencemicro- platereader(BioTekInstruments,Winooski,VT,USA).Data wereanalyzedusingKCJUNIORSoftware(BioTekInstr uments).

Caspase- 3Activity

Twenty-fourhoursbeforeeachexperiment,theculturemedi- umwasreplacedwithserum-

freemedium.Treatmentsconsistedofvehicleoradiponecti n(0.025,0.25,2.5,or25μg/ml)for24h.Themediumwasthenr emoved,andtheplateswerestoredat−70°C.Cellswerely sedincaspaseassaybuffer(50mMHEPES,pH7.4,100mM NaCl,0.1%CHAPS,1mMEDTA,10%glycerol,and10mMD TT).Theproteinconcentrationsofthelysatesweredetermined bytheBradfordmethod(Bio-

RadProteinAssay).Anequalamountofthecytosolicextract(1 00μgofprotein)fromeachsamplewasanalyzed.Theassaywa scarriedoutbyadding100μMAc-DEVDAMC(Sigma- Aldrich)andincubatingtheplatesat37°C.Theamountoffluore scentproductwasmonitoredcon-

tinuouslyfor120minwithaspectrofluorometer(FLx800Bi oTekInstruments)atanexcitationwavelengthof355nmanda nemissionwavelengthof460nm.DatawereanalyzedusingK CJUNIORSoftware,andnormalizedtofluorescencelevelsinv ehicle-treatedcells.

StatisticalA nalysi s

Statisticaldataarepresentedasmeans±SDofthreeindepen- dentexperimentsperformedintriplicate.Statisticalanalysis wascarriedoutusingone-wayortwo-

wayANOVA,followedbyTukey’stest(GraphPadSoftware, LaJolla,CA,USA).Thelevelofsignificancewassetatp<0.05.

Resul ts

ExpressionofAdiponectinandItsReceptorsi nEpithelialOvarianCancerCellLines

Beforeinvestigatingtheeffectsofadiponectinonhumanep- ithelialovariancancercells,weexaminedtheexpressionofad iponectinanditsreceptorsinepithelialovariancancercelllines (OVCAR-3,SKOV-3,andCaov-

3)andcomparedthemRNAlevelswiththoseinthegranulosatu morcellline(COV434).Byreal-

timePCRanalyses,allcancercelllinesfailedtoexpressadipo nectin(datanotshown).Both

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ADIPOR1andADIPOR2receptorswereexpressedbyallcellli nes.Therelativequantity(RQ)ofeachtranscript(ADIPOR1an dADIPOR2)expressedbythegranulosatumorcellline(CO V434)wasarbitrarilysetat1.ThebasalADIPOR1mRNAleve lwaslowerintheepithelialovariancancercells(3.5-,2-,and1.3- foldinOVCAR-3,SKOV-3,andCaov-

3,respectively)thaninthegranulosatumorcellline(Fig.1a;p<

0.05).Similarly,thebasalADIPOR2transcriptlevelwasloweri nOVCAR-3,SKOV-3,andCaov-3cells(6-,4.3-,and2.2- foldinOVCAR-3,SKOV-3,andCaov-

3,respectively)thaninCOV434cells(Fig.1b;p <0.05).The basalAdipoR1andAdipoR2proteinlevelswereexaminedby WesternblotanalysisinCOV434,OVCAR-3,andSKOV- 3cells,andtheresultsmirroredthosefromreal-timePCRanal- yses(Fig.1c).

EffectsofEnvironmentalEstrogens(BPAandItsAnalo gs)ontheExpressionofAdiponectinReceptors ApreviousstudyreportedthatBPAregulatesadiponectinpr oductionandsecretionin3T3-L1adipocytes.Forthisrea- son,weassessedtheeffectsofBPA,TBBPA,andTCBPAonthe expressionofAdipoR1andAd ipoR2inSKOV-

3andOVCAR-3cellsbyreal-

timePCRanalysis.BPA,TBBPA,andTCBPAatalltestedcon centrations(1,10,and100nM)hadnoeffectsonADIPOR1and ADIPOR2expressioninSKOV-3(Fig.2a)andOVCAR- 3cells(Fig.2b).

EffectofAdiponectinonOVCAR-3andSKOV- 3CellProliferation

WeexaminedtheabilityofadiponectintoaffectOVCAR- 3andSKOV-

3cellproliferation.After48hoftreatment,weobservedadose- dependentinhibitionintheproliferationofbothOVCAR- 3andSKOV-

3cells.Comparedwiththatofcontrolcells,therewasastatistic allysignificantdecreasein

proliferationafterexposingOVCAR-

3cellstoadiponectinatconcentrationsof2.5and25μg/ml(85±1 .5%and76±3%relativetothecontrol,respectively)

(Fig.3a;p <0.01,p<0.001).Adiponectinatconcentrationsof 0.025and

0.25μg/mlhadnoeffectontheproliferationofOVCAR-3 cells.Similarly,adiponectindecreasedSKOV-3cellprolifer- ationdose-

dependently(87±1.7%,79±2.9%and75±2.5%at0.25,2.5,and 25μg/mlrelativetothecontrol,respectively)

(Fig.3b;p<0.05,p<0.001).

Epithelialovariancancercellsdifferfromgranulosatumorce llsintermsoftheirmorphologyandclinicalbehavior.Wefoundt hatADIPOR1andADIPOR2expressionwaslowestinOVCA R-

3cellscomparedwiththatintheothercells.Moreover,ourfin dingsuggestedthatadiponectininhibitOVCAR-3andSKOV- 3cellproliferationinasimilarmanner.Forthisreason,OVCAR- 3cellswerechosenasaninvitromodelofserousepithelialovari ancancerforfurtheranalysis.

EffectofAdiponectinonCaspase-3Activity andCleavedPARPProteinExpressioninOVCAR- 3Cells

Inaparallelexperimentthatemployedacaspase-

3activityassay,weevaluatedtheeffectofadiponectinoncas pase-

3activityandcleavedPARPproteinexpressioninOVCAR- 3cells.AdiponectinatalltestedconcentrationshadnoeffectonO VCAR-3caspase-

3activityaswellascleavedPARPproteinexpression(Fig.4).

EffectsofE2,IGF-

1,andP4onAdiponectinReducestheProliferationofC ancerCells

PreviousstudiesreportedE2,P4,andIGF-

1toaffectovariancancerdevelopment,whichpromptedustoe xaminetheef-fectsofadiponectinandE2,P4,orIGF-

1ontheproliferationofcancercells.Consistentwithpreviousre sults,wefoundthat

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Fig.1B a s a l mRNAandproteinexpressionofadiponectinreceptor s(AdipoR1andAdipoR2)invariouscancercelllines.AdipoR1mRNA(a), AdipoR2mRNA(b),andproteinexpression(c)inahumanovariangranulos atumorcellline(COV434cells)andepithelialovariancancer

celllines(Caov-3,SKOV-3,andOVCAR-

3).RQ,relativequantity.ThemRNAlevelsofADIPOR1andADIPOR2in COV434cellsweresetat

1.0.Statisticallysignificantdifferencesareindicatedwithdifferentletters(a< b

< c <d,p<0.05)

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170 HORMCANC(2018)9:166–174

Fig.2EffectsofBPA,TBBPA,andTCBPA(1,10,and100nM)onAdipo R1andAdipoR2mRNAexpressioninepithelialovariancancercelllines(

SKOV-3,a)and(OVCAR-

3,b).ADIPOR1andADIPOR2mRNAexpressionafterexposingcellstot heindicatedcompoundsfor24h.RQ,relativequantity;C,control.ThemRN AlevelsofADIPOR1andADIPOR2invehicle-treatedcellsweresetat1

E2andIGF-1stimulatedOVCAR-

3cellproliferation(115±3%and138±5%relativetothatoftheco ntrol,respectively)

(Fig.5;p<0.05).However,adiponectinreversedE2- andIGF-1-inducedOVCAR-

3cellproliferation;thecombinedtreatmentofadiponectina ndE2oradiponectinandIGF-

1decreasedcellproliferationtothebasaladiponectinlevelafter 48h,(75±7%and86±9%,respectively,vs80±5%),

(Fig.5;p<0.05).AlthoughP4hadnoeffectonOVCAR- 3cellproliferation,thecombinedtreatmentofadiponectinand P4decreasedcellproliferationtobelowthebasaladipone ctinlevel(67±5%vs80±5%),(Fig.5;p<0.05).

EffectsofE2,P4,andIGF-

1onAdiponectinReceptorExpression

ToinvestigatethemechanismbywhichadiponectinreducedE2 -andIGF-1-

inducedcellproliferation,wemeasuredthemRNAlevelsofA dipoR1andAdipoR2inOVCAR-3cellsafterE2,IGF- 1,andP4treatment.AsshowninFig.6,E2andIGF-

1hadnoeffectonthebasalAdipoR1andAdipoR2levels.Howe ver,thelevelsofAdipoR1andAdipoR2werehigherinOVCA R-

3cellsculturedinthepresenceofP4thanincontrolcells(35±6%f orAdipoR1and31±8%forAdipoR2comparedwiththerespecti vebasallevels)(Fig.6).

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HORMCANC(2018)9:166–174

Fig.3E ffectofadiponectin(0.025,0.25,2.5,and25μg/ml)onthepr oliferationofOVCAR-3(a)andSKOV-3(b)cellsafter48hoftreat- ment.Ccontrol.Theproliferationrateinvehicle-

treatedcellswassetat100%.*p<0.05,**p<0.01,***p<0.001compared withcontrolcells

EffectsofAdiponectinonERα,ERβ,PGR,andIGF1RR eceptorExpression

Next,weexaminedwhetheradiponectin(25μg/ml)cou ldaffectthemRNAexpressionofESR1,ESR2,IGF1R,and PGRinOVCAR-

3cells.WeobservednodifferenceinESR2expressionbetw eenadiponectin-

treatedandcontrolcells(datanotshown).Bycontrast,theleve lsofIGF1R,ESR1,andPGRweresignificantlylowerinadi ponectin-

treatedcellsthanincontrolcells(0.83±0.04,0.74±0.06,a

nd0.43±0.1vs1RQ,r e s pectively).TheE Rα,P R , a ndIGF1 RproteinlevelswereexaminedbyWesternblotanalysis,

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Fig.4E ffectofadiponectin(0.02 5,0.25,2.5,and25μg/ml) oncaspase-

3activity(a)andcleavedPARPprote inexpression

(b) inOVCAR-

3cellsafter24hoftreatment.Ccon trol

andtheresultsmirroredthosefromreal- timePCRanalyses(Fig.7).

Discussion

TheexpressionofAdipoR1andAdipoR2hasbeenreportedinah umangranulosatumorKGNcellline[14].However,themajorit y(~90%)ofmalignantovariantumorsareofepithelialcellorigin, andonly~7%areclassifiedasovariansexcordtumors,withthe mostcommontypebeingthegranulosatu-

mor[2].Theexpressionofadiponectinanditsreceptorsinepit helialovariancancercellshasnotyetbeenreported.Inthepres entstudy,wefoundthatadiponectinwasnotexpressedintheg ranulosacellline,aswellasepithelialovar-

iancancercelllines.Thesefindingssuggestthatadiponectincan actonovariancancercellsinvivoonlyasanendocrinefactor.Ad ditionally,ourresultsindicatedthatbothAdipoR1andAdipoR 2areexpressedinvariousepithelialovarian

Fig.5Effectsofadiponectin(25μg/ml)andE2(10nM),P4(100μM),orIGF -1(100ng/ml)ontheproliferationofOVCAR-

3cellsafter48hoftreatment.Ccontrol.Statisticallysignificantdifferencesa reindicatedwithdifferentletters(a<b<c<d<e<f,p<0.05)

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cancercelllines,andthattheirexpressioninthesecelllineswas lowerthaninthegranulosatumorcellline(COV434).Ourob servationsareconsistentwiththoseofLietal.

[13]andTiwarietal.

[29],whoreportedAdipoR1expressionincan-

cerousepithelialovariantissues.Moreover,Lietal.[13]illus- tratedthatepithelialovariancancerpatientswithAdipoR1- positiveexpressionsurvivedlongerthanthosewithAdip oR1-

negativeexpression.Toourknowledge,thereisnoinformatio nonAdipoR2expressioninhumanovariancancertissues.This studyisthefirsttoreportalowerexpressionofAdipoR1andAd ipoR2inepithelialovariancancercellscom-

paredwithgranulosatumorcells.Thisobservationmaypartly explainwhygranulosatumorsaregenerallyconsideredt ohaveabetterprognosisthanepithelialovariantumors.

Second,weinvestigatedwhetherBPAanditsanalogscoul dregulatetheexpressionofAdipoR1andAdipoR2inepithe lialovariancancercells.Wefoundthatlownanomolar

Fig.6EffectsofE2(10nM),P4(100μM),orIGF-

1(100ng/ml)onADIPOR1andADIPOR2expressioninOVCAR- 3cells.ADIPOR1andADIPOR2mRNAexpressionafterexposingcells tocompoundsfor24h.RQrelativequantity,Ccontrol.ThemRNAlevels ofADIPOR1andADIPOR2weresetat1.**p<0.01,***p<0.001comp aredwithcontrolcells

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172 HORMCANC(2018)9:166–174

Fig.7E ffectsofadiponectin(25μg/ml)ontheestrogenreceptor(ESR1,a), progesteronereceptor(PGR,b),andinsulin-

likegrowthfactortype1receptor(IGF1R,c)mRNAat24handproteinat4 8hexpressioninOVCAR-

3cells.RQrelativequantity,Ccontrol.Thecontrolvaluewasarbitrarilyseta t1.*p<0.05,**p<0.01,***p<0.001comparedwithcontrolcells

concentrationsofBPA,aswellasTBBPAandTCBPA,hadnoe ffectsontheexpressionofbothreceptorsinepithelialovaria ncancercelllines(OVCAR-3andSKOV-

3).Previously,Hugoetal.

[30]observedthatBPAat0.1and1nMinhibitedadiponectin secretionfromhumanbreastadi-

posetissueexplantsandabdominalsubcutaneousexplants.O ntheotherhand,Kidanietal.[18]indictedthatBPAandBPA- relatedchemicals,suchasbisphenolF,bisphenolE,andbisphen olB,decreasedadiponectinsecretionin3T3-L1adi-

pocytes.However,thereisnoinformationontheeffectsof

BPA,TBBPA,orTCBPAonAdipoR1andAdipoR2expres- sioninhumanovariancells.

Becausetheeffectsofadiponectinonepithelialovarianc ancercellsareunknown,weexaminedwhetheradiponectinatco ncentrationsreportedintheserumandlowercanaffectOVCAR -3andSKOV-

3cellproliferation.Wefoundthatadiponectininhibitedthegro wthofbothcelllines.Similarresultshavebeenobservedinvari ousbreastcancercelllines,includingMCF-7[10],MDA-MB- 231[11,31],andT47D

[11].Theanti-

proliferativeeffectsofadiponectininbreastcancercellsar esometimes[31],butnotalways[11,32],as-

sociatedwithapoptosis.Consideringthis,weanalyzedthee ffectoftheadiponectinonovariancancercellapoptosis.

WeobservedthatadiponectinhadnoeffectonOVCAR- 3apoptosis.Thesedataindicatedthatadiponectindecreas edepithelialovariancancercellproliferation,andthatthiseffect wasindependentofapoptosis.

Next,weinvestigatedwhetherovarianhormonesanda growthfactor(E2,P4,andIGF-

1)couldaffectadiponectinactivityinepithelialovariancancer cells.Wedemonstratedtheantagonisticeffectofadiponectino nE2-andIGF-1-

inducedepithelialovariancancercellproliferation.Cons istentwithpreviousdata,weobservedmitogenicactivityaftertr eatmentofepithelialovariancancercellswithE2[22,33]andIG F-

1[34].Inthepresentstudy,proliferationwasreducedtotheba saladiponectinlevelincellsexposedtoadiponectinandE2ora diponectinandIGF-

1.TheseresultsareconsistentwiththoseofDieudonneetal.

[10],whoreport-

edthatadiponectinsuppressedtheproliferationofE2- treatedbreastcancerMCF-

7cells.However,thereisnoinformationontheeffectsofadipone ctinonIGF-1-inducedproliferationinovariancancercells.

Togaininsightintothemechanismunderlyingtheantago- nisticeffectsofadiponectinonE2-andIGF1-

inducedcellproliferation,weexaminedAdipoR1andAdipoR 2expressionafterE2 andIGF -

1treatme ntand ERα,ERβ,an d IGF1Rexpressionafteradip onectintreatment.Similartoexogenousestrogens,suchasBPA, andendogenousestrogens,wefoundthatE2hadnoeffectonAdi poR1andAdipoR2expression.WealsofoundthatIGF- 1didnotaffectAdipoR1andAdipoR2expression.However,a diponectinreducedtheex-

pressionofERαandIGF1R,butnotthatofERβ,inepithelialovar iancancercells.IncontrasttoERα,ERβactivatesanti-

proliferativepathwaysinovariancancercells[35].Thereisalso aninversecorrelationbetweenERβexpressionandovar- iantumorprogression[36,37].Resultsfromthisstudydem- onstratedthatadiponectinchangestheERα/βratiobyde- creasingERαexpressioninepithelialovariancancercells.Si

(14)

HORMCANC(2018)9:166–174 3 milartoourobservations,Jardeetal.

[38]notedthatadiponectindownregulatedERαgeneexpre ssionintheMCF-7breastcancercellline.However,anti- proliferativeadiponectineffectshavebeenreportedforER- positive[10]andER-

negative[31]breastcancercelllines,suggestingthat

(15)

theeffectsofadiponectinarenotduetointeractionswiththeestr ogenpathway.Indeed,ourresultsindicatedthatadiponecti nnotonlyinteractswiththeestrogenpathway,butalsowiththeI GF-1pathway.

WealsofoundthatP4hadnoeffectontheproliferationofOVC AR-3cells.Murdochetal.[39]arrivedatasimilarcon-

clusionafterperformingexperimentsonP4-treatedOVCAR- 3andSKOV-3cells.Interestingly,weobservedthatco- treatmentwithadiponectinandP4potentiatedtheeffectsofadi ponectinbyreducingcellproliferationtobelowthebasaladipo nectinlevel.WealsofoundthatP4upregulatedAdipoR1and AdipoR2expression,whereasadiponectindownregulatedP GRexpression.Thus,thepotentiationoftheeffectsofadipone ctinbyP4enabledustoinvestigatetheresponsivenessofdiffere ntcancercelllinestothehormone.Ontheotherhand,thedecreas edPGRexpressionalteredtheinsensitivityofvariousepithelia lovariancancercellstotheanti-

proliferativepropertiesofP4[40].Ourresultsmaybepartlyex plainedbyinteractionsbetweenP4andleptinpath-

ways.Itiswelldocumentedthatleptin,anadipocyte-

secretedhormone,hasoppositeeffectsontheproliferationofca ncercells[41].Forexample,Jardéetal.

[38]demonstratedthatleptinupregulatedPRmRNAexpressi oninMCF-

7cells.Moreover,P4inhibitedleptinreceptorgeneexpressioni nthehumanendometrium[42].Ourresults,togetherwiththose ofpreviousstudies,supportthecontentionthattheeffectsofadip onectinoncancercellsareoppositetothoseofleptin.

Takentogether,thesefindingsindicatethatadiponectinre- pressestheproliferationofepithelialovariancancercellsandre versesthestimulatoryeffectsofE2andIGF-1oncellpro- liferationbydownregulatingtheexpressionoftheirreceptors.In addition,P4increasedthesensitivityofcancercellstoadipon ectinbyupregulatingtheexpressionofitreceptors.These resultsalsosuggestinteractionsbetweenadiponectinandvario usovariansteroidhormoneandgrowthfactorpath-

waysinovariancancercells.

FundingThisstudywasfundedbytheNationalScienceCentre(NCN)Pol and(grantnumber2013/09/B/NZ7/00405)andJagiellonianUniversi ty(grantnumberDS/MND/WBiNOZ/IZ/17/2016).

Compliancew ithE thicalStandards

Thisarticledoesnotcontainanystudieswithhumanparticipantsoran- imalsperformedbyanyoftheauthors.

Conflictof InterestThea uthorsd eclaret hatt heyh aven o c onflicto f interest .

OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCo m mo nsAt tribu t ion4. 0In t ernat iona lLicen se(ht tp://creativecommo ns.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andr eproductioninanymedium,providedyougiveappro-

priatecredittotheoriginalauthor(s)andthesource,providealinktotheCreat iveCommonslicense,andindicateifchangesweremade.

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