requires PKC-#_#x03B4; mediated p53 activation in malignant pleural me

16 Pages·2017·13.59 MB·English

RESEARCHARTICLE
Apoptosis by [Pt(O,O0-acac)(γ-acac)(DMS)]
requires PKC-δ mediated p53 activation in
malignant pleural mesothelioma
AntonellaMuscella1*,CarlaVetrugno1,LucaGiulioCossa2,GiovannaAntonaci2,
AmilcareBarca3,SandraAngelicaDePascali4,FrancescoPaoloFanizzi4,
SantoMarsigliante2
1 LaboratoryofCellPathology,DepartmentofBiologicalandEnvironmentalSciencesandTechnologies(Di.
a1111111111 S.Te.B.A.),UniversityofSalento,Lecce,Italy,2 LaboratoryofCellPhysiologyDi.S.Te.B.A.,Universityof
Salento,Lecce,Italy,3 LaboratoryofPhysiologyDi.S.Te.B.A.,UniversityofSalento,Lecce,Italy,
a1111111111
4 LaboratoryofInorganicChemistry,Di.S.Te.B.A.,UniversityofSalento,Lecce,Italy
a1111111111
a1111111111 *[email protected]
a1111111111
Abstract
Mesotheliomacancercellshaveepithelioidorsarcomatoidmorphology.Theworstprogno-
OPENACCESS
sisisassociatedwithsarcomatoidphenotypeandresistancetotherapyisaffectedbycells
Citation:MuscellaA,VetrugnoC,CossaLG,
heterogeneity.WerecentlyshowedthatinZL55mesotheliomacelllineofepithelioidorigin
AntonaciG,BarcaA,DePascaliSA,etal.(2017)
Apoptosisby[Pt(O,O0-acac)(γ-acac)(DMS)] [Pt(O,O0-acac)(γ-acac)(DMS)](Ptac2S)hasanantiproliferativeeffectinvitroandinvivo.
requiresPKC-δmediatedp53activationin AimofthisworkwastoextendthestudyontheeffectsofPtac2SonZL34cellline,represen-
malignantpleuralmesothelioma.PLoSONE12(7): tativeofsarcomatoidmesothelioma.ZL34cellswereusedtoassaytheantitumoractivityof
e0181114.https://doi.org/10.1371/journal.
Ptac2Sinamousexenograftmodelinvivo.Then,bothZL34andZL55cellswereusedin
pone.0181114
ordertoassesstheinvolvementofp53proteinin(a)theprocessesunderlyingthesensitivity
Editor:Yi-HsienHsieh,InstituteofBiochemistry
tochemotherapyand(b)theactivationofvarioustransductionproteinsinvolvedinapopto-
andBiotechnology,TAIWAN
sis/survivalprocesses.Ptac2SincreasesZL34celldeathinvivocomparedwithcisplatin
Received:March7,2017
and,invitro,Ptac2Swasmoreefficaciousthancisplatinininducingapoptosis.InPtac2S-
Accepted:June25,2017 treatedZL34andZL55cells,p53regulatedgeneproductsofapoptoticBAXandanti-apo-
Published:July12,2017 ptoticBcl-2proteinsviatranscriptionalactivation.Ptac2SactivatedPKC-δandPKC-ε;their
inhibitionbyPKC–siRNAdecreasedtheapoptoticdeathofcells.PKC-δwasresponsiblefor
Copyright:©2017Muscellaetal.Thisisanopen
accessarticledistributedunderthetermsofthe JNK1/2activationthathasaroleinp53activation.Inaddition,PKC-εactivationprovoked
CreativeCommonsAttributionLicense,which phosphorylationofp38MAPK,concurringtoapoptosis.InZL34cells,Ptac2Salsoactivated
permitsunrestricteduse,distribution,and
PKC-αthusprovokingERK1/2activation;inhibitionofPKC-α,orERK1/2,increasedPtac2S
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. cytotoxicity.ResultsconfirmthatPtac2Sisapromisingtherapeuticagentformalignant
mesothelioma,givingasubstantialstartingpointforitsfurthervalidation.
DataAvailabilityStatement:Allrelevantdataare
availableinthebodyofthemanuscript.
Funding:Theauthorsreceivednospecificfunding
forthiswork.
Competinginterests:Theauthorshavedeclared
Introduction
thatnocompetinginterestsexist.
Theincidenceofmalignantpleuralmesothelioma(MPM)isgrowingduetowideasbestos
Abbreviations:FBS,fetalbovineserum;MPM,
usageinvariousdevelopingcountries[1].ThemostefficaciousMPMtreatmentableto
malignantpleuralmesothelioma;MTT,3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenoltetrazolium lengthensufferers’lifeisthecombinationofpemetrexedorraltitrexed,multi-folateinhibitors
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
bromide;PK,pharmacokinetics;Ptac2S,[Pt(O,O0- andcisplatin;nevertheless,themediansurvivalis12months,withresponseratesofabout40%
acac)(γ-acac)(DMS)];SRB,sulforhodamineB. [2,3].Biologicagentstargetingoncogeneticpathways,suchashistonedeacetylases,phosphati-
dylinositol3-kinase/mammaliantargetofrapamycin,nuclearfactorkBandneoangiogenesis
havealsobeentested[4].However,noneofthesetreatmentsshowedtoimpactsignificantlyon
thisneoplasm;thus,thereisanurgentneedfornewdrugs.Histologically,MPMcanbeclassi-
fiedinthefollowingthreesubtypes:epithelioid(50%),sarcomatoid(16%),andmixedtypeor
biphasic(34%).Sarcomatoidmesotheliomasarecharacterizedbyaggressivebiologicalbehav-
iour,resistancetosystemictreatments,morefrequentdistantspreadandpoorprognosis.
Greatcarehasbeengivenondesigningnewplatinum-basedcompoundshavingfewertoxicity
andmorefavourabletherapeuticindicesthancisplatin.Inregardtothis,itwassynthesizedthe
Pt(II)-deriveddrug[Pt(O,O'-acac)(γ-acac)(DMS)](Ptac2S)havingnon-genomictargets[5].
Ptac2Sachievedincreasingheedaspotentialanticancerdrugasitshighandselectivecancer
cellcytotoxicityobservedinimmortalizedcelllinesandinbreastcancercellsinprimarycul-
ture[6–10]andinvivo[11–13].Notably,inapreclinicalmodelmadeofhypodermicinjection
ofbreastcancercells,Ptac2Sshowsupforananticanceractivityhigherthancisplatin;inWis-
tarratsitaswellshowedincreasedpharmacokinetics,biodistributionandtolerabilityincom-
parisontocisplatin.PharmacokineticsstudieswithPtac2Suncoveredlengthenedsystemic
bloodpersistenceofPtanddiminishednephrotoxicityandhepatotoxicity.Inprinciple,this
Pt-compoundwouldyieldawideruse,sincePtac2Salsoexertsspecificantimetastatic
responsesinvitro[13–14].Assaid,itseemsnotabletounderstandwhetherPtac2Shasalso
cytotoxiceffectsonMPM.Previously,weusedtheepithelioidZL55cellsandshowedthatcis-
platinprovokedapoptosistogetherwiththeactivationofPKC-αandERK1/2pro-survival
pathwaysbythesynthesisofROS[15].InthesameZL55cellswealsotestedtheeffectsof
Ptac2Sandobservedagreatercytotoxicitythancisplatin.Ptac2Swasabletoactivatedifferent
transductionpathwayswithstrongpro-apoptoticactivity(p38andPKC-δ),whilethePKC-α
pro-survivalpathwayactivatedbycisplatinwasnotobserved.Therefore,thehighercytotoxic-
ityofPtac2SinthesecellsmaybeduetothefactthatitdoesnotactivatePKC-α[12].Inthe
currentinvestigation,weassessthecytotoxicityofPtac2Salsoonmesotheliomacellsofsarco-
matoidoriginthataregenerallymoreaggressiveandlesssusceptibletochemotherapy.There-
fore,thisstudywasconductedusingtheZL34cellsbothinvitroandinvivowiththetechnique
ofthexenograftonnudemice.Furthermore,wealsolookedforthedifferencesbetween
responsestoPtac2SandcisplatinandthemolecularmechanismsthatdeterminetheZL34cell
death/survivalfate.
Materialsandmethods
Cellculture
ThehumanmesotheliomacelllinesZL34andZL55[15]weregrowninRPMI1640medium
(Sigma,St.Louis,MO,USA)supplementedwith10%fetalbovineserum(FBS),penicillin(100
U/ml)andstreptomycin(100mg/ml).Thecellsweremaintainedat37˚Cinthepresenceof
5%CO inair.Cellsweregrownto70–80%confluenceandthentreatedwithPt-compounds
2
atvariousconcentrationsandfordifferentincubationperiods.
Invivoxenograftexperiments
Athymicnudemice(6wks.old,female,20to30gbodyweight)werepurchasedfromHarlan
Laboratories(SanPietroalNatisoneUD,Italy)andmaintainedunderpathogen-freecondi-
tions.Theyweregivenfreeaccesstostandardfoodandwater,witha12hlight-darkcycleata
temperatureof22+/−2˚C.Approximately6x106ZL34cells(8mice)wereinjectedsubcutane-
ouslyintotheflank.Animalsweremonitoreddailyforgeneralhealthandbodyweightswere
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
measuredtwiceweekly.Tumoursizewasmeasuredwithslidecallipersandvolumeswerecal-
culatedas(LxW2)/2,whereLandWarethemajorandminordiameters,respectively.Once
tumourvolumesreached~50mm3,micewererandomlydividedintothreegroupsandtreated
byasingleintravenousofsalineasacontrol,or10mg/kgofPtac2Sor10mg/kgcisplatin.The
miceweresacrificedafter35daysoftreatmentandthetumourswereexcised.Asdescribed
previously[11],allanimalsreceivedcareincompliancewiththePrinciplesofLaboratoryAni-
malCareformulatedbytheNationalSocietyforMedicalResearchandtheGuidefortheCare
andUseofLaboratoryAnimalspreparedbytheInstituteofLaboratoryAnimalResources,
publishedbytheNationalInstitutesofHealth(NIHPublicationNo.86–23,revised1985),as
wellasinaccordancewiththeItalianlawsonanimalexperimentation(art.4and5ofD.L.
116/92).EthicalCommitteeonAnimalResearch(MinisterodellaSaluteD.M.109/2014-B)
approvedtheprotocols.Alleffortsweremadetominimizesufferingtoanimals;thus,the
experimentalproceduresusedintheworkdescribedinthisarticlewereincompliancewith
theguidelinesforreportingexperimentsinvolvinganimals[16].
Cytotoxicityassay
WeevaluatedtheIC inZL34cellswithSRBandMTTassays.TheSRB(sulforhodamineB)
50
assayandtheconversionofMTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazoliumbro-
mide)bymesotheliomacellswereusedasindicatorofcellnumberasdescribedpreviously[7].
Viablecellswerealsocountedbythetrypanblueexclusionassayandlightmicroscopy.The
datapresentedaremeans±standarddeviation(S.D.)fromeightreplicatewellspermicrotitre
plate.
Clonogenicsurvivalassay
ZL34cellswereseededin100mmPetridishesatlowdensity(~3X104perdish)andleftto
adherefor24hinastandardmedium.CrescentconcentrationsofPtac2Sorcisplatinwere
addedandclonogenicsurvivalassaywasperformedasdescribedpreviously[8].
Preparationofsubcellularfractionandwesternblots
Preparationofsubcellularfraction,westernblottinganalysisandimmunodetectionwereper-
formedaspreviouslyreported[17].Westernblottingandimmunodetectionanalyseswereper-
formedaspreviouslydescribed[18].
Reversetranscriptionandpolymerasechainreaction(RT-PCR)
TotalRNAwasextractedfromZL34andZL55cellsusinganSVTotalRNAisolationkitand
performedaccordingtothemanufacturer’sprotocols(Promega,Madison,WI,USA)asprevi-
ouslydescribed[8].Ameltcurveanalysiswasperformedfollowingeveryruntoensureasingle
amplifiedproductforeveryreaction.Foreachgene,relativeexpressionwasdeterminedusing
the2-ΔΔCTmethodsandnormalizedtoβ-actinexpression.
DesignandpreparationofsmallinterferingRNA(siRNA)
PKC-α,PKC-δandPKC-εsiRNAswerepreparedbyaninvitrotranscriptionmethod,accord-
ingtothemanufacturer’sprotocol(Promega,Madison,WI,USA)aspreviouslydescribed[8].
siRNAtransfection
Thecells(50–70%confluence)weretransfectedwithsiRNAduplexesusingtheprotocolsup-
pliedwiththeCodeBreakersiRNAtransfectionreagent(Promega,Madison,WI,USA)as
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
describedpreviously[15].Quantitativeanalysisofproteinexpression,asmeasuredbyintensity
ofimmunoreactivityinsiRNA-transfectedcells,revealedaveryhighreductioninPKC-α,
PKC-δandPKC-εexpression.
Statisticalanalysis
Theexperimentermeasuringthetumoursandthedataanalystwereunawareofthetreatments
giventotheanimals.Data,presentedasmeans±SD,werecollectedinblindedfashionand
analysedusingGRAPHPADPRISM5software(GraphPadSoftware,LaJolla,CA,USA).
UnpairedStudent’st-testorone-wayANOVA,andwhenthisreturnedP<0.05,posthoc
analysisusingBonferronitest,wereperformed;weusedtheBonferroni-Dunnposthoctestin
theANOVAafterasignificantomnibusF-test.P<0.05wasacceptedasalevelofstatistical
significance.
Materials. Ptac2Swaspreparedaspreviouslyreported[5,19].Cisplatinwaspurchased
fromSigma(Milan,Italy).RPMI1640medium,antibiotics,glutamineandfoetalbovine
serumwerepurchasedfromCelbio(Milan,Italy).Caspase-9and-7,BAX,PARP-1,phospho
p38MAPK,phosphoJNK1/2antibodieswereobtainedfromCellSignalling(Celbio,Milan,
Italy).PKCisoformsandphosphoERK1/2antibodies,goatanti-rabbitconjugatedwithperoxi-
dase,aswellascontrolantibodieswereobtainedfromSantaCruzBiotechnology(SantaCruz,
CA,USA).AllothersreagentswerefromSigma(Milan,Italy).
Results
AnticanceractivityofPtac2SinaMPMpreclinicalmodel
ZL34andZL55celllinesrepresentsarcomatoidandepithelioidMPM,respectively.Thein
vivoefficacyofPtac2SinpreclinicalmodelofepithelioidMPMwasalreadydetermined[12].
Here,sarcomatoidmodelwasassessedbythehypodermicinjectionofZL34cellsintheflank
ofBALB/cnudemice.Whencancersreachedthesizeof~50mm3,inordertoreduceweight
andtumoursizeodds,micewererandomizedinthreegroups.10mg/kgofPtac2Swasfound
beforetobeeffectivewithoutnotablesideeffectsinanimalstudieswithxenograftsofhuman
breastcancerouscells[11].Hence,afterthatanonlyintravenousofsaline(control)or10mg/
kgofPtac2Sor10mg/kgofcisplatinwasdispensed,tumourvolumeswereevaluatedbyver-
niercalliperevery3daysfor5weeks.Themeanvolumesofthetumoursineachgroupwere
assessed,andwedrewtherelatedtumourgrowthcurves.Ptac2Sdisplayedhigheranticancer
activitythancisplatintowardZL34tumoursexamined,inducingupto50%growthinhibition.
InmiceinoculatedwithZL34,during5weeksmeantumourvolumeaugmentedfrom46.6
±6.78to285.11±38.69mm3forthesalinegroup,to251.87±49.36mm3forthecisplatingroup
(10mg/kg;p>0.05)andto133.72±41.22mm3forthePtac2Sgroup(Fig1).Micedisplayeda
significantdecreaseoftumourmassforeachexperimentaltimeconsideredinthePtac2S
groupscomparedwithbothcontrolandcisplatin-treatedmice(p<0.05).Inaddition,during
observationtime,nohealthproblemswereobservedandtheoverallbehaviourwassimilarto
thatofthecontrolanimals.
CytotoxicityofPtac2S
InvitrocytotoxicitydatawereachievedbyMTTandvalidatedbySRBassaystoexcludeconse-
quencesofPtac2Sonenzymesofmitochondria.Furthermore,similarresultsareattained
whencellnumberisdefinedthroughtheircounting(datanotshown);thus,SRBassaywas
usedforalltheexperimentsshownherein.Adose-dependentdecrementofcellsurvivalwas
obtainedwhenMPMcellswereincubatedwithcisplatinorPtac2S(from1μMto200μM,
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
Fig1.GrowthinhibitoryeffectofPtac2Sandcisplatininaxenograftmodelofmesothelioma.Balb/cnude
micecarryingsarcomatoidorepithelioidmesotheliomadevelopedbyinjectionofZL34(around50mm3)received
intravenousPtac2S(10mg/kg)orcisplatin(10mg/kg).Tumourvolumewasmeasuredevery3daysforatotalof35
days.Resultsareshowedasmean±S.D.(animalspergroupn=8).*P<0.05,significantlydifferentfromsaline
control;#P<0.05,significantlydifferentbetweenPtac2Sandcisplatin.(Table)Afterkilling,tumourswerecollected
andmeasured.
https://doi.org/10.1371/journal.pone.0181114.g001
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
Fig2.SensitivityofMPMcellstoPtac2Sandcisplatin.(A)ZL34cellsweretreatedornotwithincreasing
concentrationsofPtac2Sorcisplatinfor24and48h,orcontinuouslyexposedto50μMcisplatinor5μM
Ptac2S.CellviabilitywasobtainedbySRBassayanddataaremeans±S.D.of6independentexperiments
with8replicatesineach,andarepresentedaspercentofcontrol.ForbothcisplatinandPtac2S,P<0.0001
byone-wayANOVA(n=6);valueswithsharedlettersarenotsignificantlydifferentaccordingtoBonferroni/
Dunnposthoctests.(B)ClonogenicsurvivalassayinZL34cellstreatedwiththeindicatedamountsofPtac2S
orcisplatinfor2h,andafter15daysofgrowth;onlycoloniesconsistingofmorethan50cellswerescored.
Thepercentageofnumbercoloniesrepresentsthemeans±S.D.ofsix-independentexperiments.For
cisplatinandPtac2S,P<0.001andP<0.0001byone-wayANOVA(n=6),respectively;valueswithshared
lettersarenotsignificantlydifferentaccordingtoBonferroni/Dunnposthoctests.(C)Cytosolicandnuclear
proteinswereobtainedfromZL34cellstreatedornotwith5μMPtac2Sor50μMcisplatin.Sampleswere
dissolvedinSDSbufferandseparatedonSDSgel.Immunoblottingwasperformedusingmonoclonal
antibodiesspecifictoPARP(fromnuclearfractions)andtocaspases-9,and-7(cytosolicfractions).
Sequentialincubationwithanti-β-actinconfirmedtheequalproteinloading.Figuresarerepresentativeof6
independentexperiments.Inset:TheIC valuestocisplatinandPtac2Scalculatedafter48h.
50
https://doi.org/10.1371/journal.pone.0181114.g002
Fig2A).InsarcomatoidcellscisplatinwassignificantlylesscytotoxicthanPtac2S(IC 4.64
50
±0.13and48.63±0.72μMn=6,forPtac2Sandcisplatin,respectively;p<0.001,seetableinFig
2).ClonogenicassayperformedonZL34showedthatPtac2Swasmorecytotoxicthancisplatin
(Fig2B).
Ptac2Scausescaspasesproteolysis
Fig2Cshowswesternblottingofcaspase-9and-7activationandproteolysisofPARPinZL34
andZL55cells.PARPwascleavedincellstreatedwith5μMPtac2Sorwith50μMcisplatin;
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
however,proteolysiswasfasterwithPtac2S.Subsequentincubationofblotswithanantibody
againstβ-actinvalidatedthatloadedproteinamountwasthesame.
Ptac2Sinducesp53activation
Sincedrugsmaystabilizep53,thatisnotmutatedinmanyMPMspecimens[20],weassessed
Ptac2Seffectsonp53anditsrelatedgenesBcl-2andBAX.Ptac2Streatmentincreasedp53pro-
teinlevels(Fig3A),increasedBAXanddecreasedBcl-2proteins(Fig3A)inbothZL34and
ZL55cells.ByRT-PCRwefoundthatPtac2Sup-regulatedp53andBAXmRNAexpression,
anddecreasedBcl-2mRNAinatime-dependentway(ANOVAp<0.01,Fig3B,3Cand3D).
Wenextusedaninhibitorofp53transcriptionaltargets,PFT-α[21].Fig4showsthat30μM
PFT-αinhibitedp53andBAXup-regulationandBcl-2down-regulationduetoPtac2Sinboth
celllines,indicatingthatapoptosisinducedbyPtac2Swasmediatedbyp53andBAX.
Ptac2S-inducesMAPKsphosphorylation
WedemonstratedpreviouslythatPtac2SactivatestheMAPKssignallingpathwaysinseveral
tumourcelllines[6,8,9,22],ZL55cellsincluded[12]andthatcisplatinactivatesERK1/2in
ZL55cells[15].WeshowedthatwhereasPtac2SactivatedallthreeMAPKsinZL34cells,in
ZL55cellsitactivatedp38MAPKandJNK1/2butnotERK1/2(Fig4Aandref.[12]).Pre-incu-
bationwithJNK1/2inhibitorSP600125,orwiththep38MAPKinhibitorSB203580,signifi-
cantlyreducedPtac2S-inducedcytotoxicityinbothcelllines(Fig4).Furthermore,SP600125
markedlyinhibitedPtac2S-inducedactivationofp53(Fig4),thussuggestingthatJNK1/2
mediatesp53induction.PD98059,aninhibitorofMEK1/2,theERK1/2upstreamkinase,sig-
nificantlyincreasedPtac2S-inducedcytotoxicity,inZL34cells(Fig4).Jointlytheseresults
indicatedthatJNK1/2andp38MAPKarepro-apoptoticpathwayswhereasERK1/2,inZL34
cells,behaveslikeananti-apoptoticsurvivalpathway.
RoleofPKCsinPtac2S-inducedapoptosisinMPMcells
AsthecellulareffectsofPtac2SgotogetherwiththeactivationofvariousPKCisoforms,we
herehavestudiedtheiractivation.Inthepreviousstudy[12]inZL55cells,weshowedthat
PKC-εandPKC-δtranslocatedfromthecytosoltothemembranes;similarlytowhathap-
penedwithcisplatin[15],thecellstreatedwithPtac2Salsoshowtheproteolyticactivationof
PKC-δ.Whilethefull-lengthPKC-δmovedtothemembraneandnuclei,itsfragmentwas
locatedtothemitochondria.Incontrasttocisplatin,thePKC-αwasnotactivated(datanot
shown).
Here,thecytosol-to-membranetranslocationofPKCswasfollowedbyimmunoblottingin
ZL34cellsincubatedwithPtac2S(0–20min).Ofthevariousisoformsexpressed,PKC-ε,PKC-
δandPKC-αwereactivatedbytranslocation:PKC-εtranslocatedtotheplasmamembrane
andPKC-δtoplasmamembraneandnucleus(Fig5A).TherolesofPKCswereevaluated
usingsiRNAtechniquetoinhibitPKC-ε,PKC-δorPKC-α.Afterseeingbywesternblotting
thatPKC-siRNAsdecreasedPKC-ε,PKC-δandPKC-αexpressions(Fig5B)itwasshownthat
PKC-δinhibitionincreasedsurvival(Fig5Cand5D)anddecreasedcaspase-9activationand
PARPcleavageinZL34cellstreatedwithPtac2S(Fig5C).Thus,inZL34cellstheroleofacti-
vatedPKC-δappearsthesameastheroleitplayswhentheZL55cellsareincubatedwithcis-
platin[15]orwithPtac2S[12].Inaddition,PKC-δ–siRNA(10nM)inhibitedPtac2S-induced
JNK1/2phosphorylation,p53andBAXinductionaswellasBcl-2down-regulation(Fig5C
and5D),inbothcelllines.PKC-ε–siRNA(10nM)inhibitedp38MAPKphosphorylation,cas-
pase-9activationandPARPcleavagebutalsoincreasedthesurvivalofPtac2S-treatedZL34
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Fig3.Ptac2Sinducesp53inMPMcells.(A)CytosolicproteinswereobtainedfromZL34orZL55cells,
treatedornotwith5μMPtac2S.Immunoblottingwasperformedusingmonoclonalantibodiesspecifictop53,
BaxandBcl2.Sequentialincubationwithanti-β-actinconfirmedtheequalproteinloading.Thesefiguresare
representativeofsixindependentexperiments.(B-D)Cells,pre-treatedornotwith30μMPFT-α,weretreated
ornotwith5μMPtac2SfordifferenttimesandthenRNAwasextracted.RNAwasreverse-transcribedand
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
analysedbyreal-timePCR,withspecificprimersforp53(B),BAX(C)andBcl-2(D)andforthehousekeeping
geneβ-actin.mRNAlevelswerepresentedasfoldchangevaluesrelativetocontrol.Datawereexpressedas
themean±S.D.sixdifferentexperiments.*P<0.05,significantlydifferentfromsalinecontrol;#P<0.05,
significantlydifferentbetweenPtac2SandPFT-α.
https://doi.org/10.1371/journal.pone.0181114.g003
cells(Fig5C).PKC-α-siRNA(10nM)inhibitedthephosphorylationofERK1/2(Fig5C)indi-
catingthatPKC-αactivationiscrucialforthesurvivalofPtac2S-treatedZL34cells.Thesame
resultswereobtainedwhen1μMGo6976(inhibitorofconventionalPKCs)wasused(datanot
shown).
Fig4.Ptac2SinducesMAPKsphosphorylationinMPMcells.(A)ZL34andZL55cellsweretreatedornot
with5μMPtac2Sforindicatedtime.Celllysateswereanalysedbywesternblottingwithanti-phosphorylated
p38MAPK,JNK1/2andERK1/2antibodies.(B)Cells,pre-treatedornotwiththep38MAPKinhibitor
SB203580(1and10μM),theJNKinhibitorSP600125(1and10μM)orMEKinhibitor(10and20μM),were
thenincubatedwithPtac2S.Celllysateswereanalysedbywesternblottingusingmonoclonalantibody
specifictop53.Sequentialincubationwithanti-β-actinconfirmedtheequalproteinloading.Thesefiguresare
representativeofsixindependentexperiments.Viablecellnumberwasdetermined24hlaterbySRBassay.
Thedataaremeans±S.D.offivedifferentexperimentsrunineightreplicatesandarepresentedaspercentof
control.P<0.0001byone-wayANOVA(n=5);valueswithsharedlettersarenotsignificantlydifferent
accordingtoBonferroni/Dunnposthoctests.
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ApoptosisbyPtac2SrequiresPKC-δmediatedp53activationinmalignantpleuralmesothelioma
Fig5.RoleofPKCsinPtac2S-inducedapoptosisinMPMcells.(A)ZL34cellsweretreatedwithoutorwith5μMPtac2Sfortheindicatedtimes.
ForPKCstranslocationstudies,cytosol(cyt),membrane(mem),nuclei(nuc)fractionswereanalysedbywesternblottingwithspecificantibodies.The
figuresarerepresentativeofsixindependentexperiments.(B)MPMcellsweretransfectedwithsiRNA–PKC-δorsiRNA–PKC-εorsiRNA–PKC-αand
thenwereincubatedwith5μMPtac2S;westernblottingoftotallysateswasthenperformedwithspecificanti-PKCsantibodiesinordertoshowthe
decrementofPKC-δ,PKC-εorPKC-αexpressions.Thefiguresarerepresentativeofsixindependentexperiments.(C)ZL34cellsweretransfected
withsiRNA–PKC-δorsiRNA–PKC-εorsiRNA–PKC-αwhilst(D)ZL55cellsweretransfectedwithsiRNA–PKC-δorsiRNA–PKC-εonlyandthen
incubatedwithPtac2S.Viablecellnumberwasdetermined24hlaterbySRBassay.Thedataaremeans±S.D.offivedifferentexperimentsrunin
eightreplicatesandarepresentedaspercentofcontrol.P<0.0001byone-wayANOVA(n=5);valueswithsharedlettersarenotsignificantlydifferent
accordingtoBonferroni/Dunnposthoctests.Cytosolicornuclear(forPARP-1)fractionswereanalysedbywesternblottingwithantibodiesagainst
PKC-δ,PKC-ε,PKC-αphosphorylatedp38MAPK,phosphorylatedJNK1/2,phosphorylatedERK1/2,p53,BAX,Bcl2,caspase-9(Casp-9)andPARP-
1;ß-actinwasusedasacontrolforproteinloading.Representativeimmunoblotsoffiveexperimentsaredepicted.Inset:crosstalkbetweenMAPKs,
p53andPKCspathways,keyfactorsaffectingcelldeathandsurvivalinPtac2S-treatedMPMcells.
https://doi.org/10.1371/journal.pone.0181114.g005
Discussion
MPMoriginatesfrommesotheliumcellsthatformaspecialisedmonolayerthatlineserous
cavitiesofpleura,pericardiumorperitoneum[23].MPMcellscanbeeitherepithelioidorsar-
comatoid[24]andseveralstudiesdemonstratedaresponsivenesstochemotherapeuticsdepen-
dentfromthephenotypes[25–26].TherapiesforMPMassociatespemetrexedtocisplatin
gettingsoa40%responserate,and3monthsand1yearaveragesurvivalandmediansurvival
times,respectively[3,27,28];thesamehappensassociatingcarboplatin,liposomizeddoxoru-
bicinandgemcitabine[29].Thereisanurgentneedforeffectivetherapysinceabout50%of
PLOSONE|https://doi.org/10.1371/journal.pone.0181114 July12,2017 10/16