p38
MAPK
and
ERK1/2
dictate
cell
death/survival
response
to
different
pro-oxidant
stimuli
via
p53
and
Nrf2
in
neuroblastoma
cells
SH-SY5Y
Giuseppe
Filomeni
a,b,
Sara
Piccirillo
b,
Giuseppe
Rotilio
a,b,
Maria
R.
Ciriolo
a,b,*
aDepartmentofBiology,UniversityofRome‘‘TorVergata’’,viadellaRicercaScientifica,00133Rome,Italy
b
ResearchCentreIRCCSSanRaffaelePisana,ViadeiBonacolsi,00163,Rome,Italy
1. Introduction
Sinceitsfirstidentificationin1985byCerutti[1],theintimate relationshipbetweenpro-oxidantconditionsandcancer develop-ment has been widely investigated. Today, non-physiological alterations of the intracellular redoxstate are well established
hallmarks oftumor biology,due to their implication inseveral transformingevents,suchasself-sufficiencyingrowthsignals[2], apoptosis evading [3], sustained angiogenesis [4,5], autophagy
[6,7] and tissue invasion [8,9]. This has been exploited by chemotherapeuticstrategiesbasedontheuseofROS-generating molecules (e.g., 2-metoxyestradiol and anthracyclins) to selec-tively kill transformed cells [10–12]. In order to optimize therapeuticdesignandreducepossibledevelopmentofresistance phenomena, research is attempting to deeply investigate the mechanisms underlying such an intrinsic alteration of redox homeostasisincancercells.Anintensemetabolismandmultiple mutationsinproteinscontrollingredoxhomeostasisareimportant causativedeterminantsofthehighlevelsofROS[13],which,in turn,induceaselectivepressuretosurviveundertheseextreme conditions.Inthisscenario,themodulationofglutathione(GSH), whichrepresentsoneofthemajorindicatorsoftheintracellular redox environment, as well as the control on transcriptional activityofredoxsensitivefactorsisemergingaspossiblestrategies
[12].Amongthetranscriptionfactorsabletosenseoxidativestress, thenuclearerythroidfactor2(NE-F2)-relatedfactor2(Nrf2)and p53arewell-characterizedexamples[14].Besidesthecanonical
A R T I C L E I N F O
Articlehistory:
Received23November2011
Accepted1February2012
Availableonline9February2012
Keywords: p53 Nrf2 MAPK Glutathione Oxidativestress Diamide Glutathioneperoxidase A B S T R A C T
Redoxchangesareoftenreportedascausativeofneoplastictransformationandchemoresistance,butare also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derivedtumorhistotypesareresistanttoROS-basedtreatmentsbymeansoftheredox activationofNrf2,buthighlysensitivetodisulfidestressorstriggeringapoptosisviatheredoxinduction ofTrx1/p38MAPK/p53signalingpathway.
Here,weprovideevidencethatneuroblastomaSH-SY5Yhasacompleteoppositebehavior,being sensitivetoH2O2,butresistanttotheglutathione(GSH)-oxidizingmoleculediamide.Consistentwith
theseobservations,theapoptoticpathwayactivateduponH2O2treatmentreliesuponTrx1oxidation,
and is mediated by the p38MAPK/p53 signaling axis. Pre-treatment with different antioxidants,
pharmacologicalinhibitorofp38MAPK,orsmallinterferingRNAagainstp53rescuecellviability.Onthe
contrary,cellsurvivaltodiamidereliesuponredoxactivationofNrf2,inawayindependentonKeap1 oxidation,butresponsivetoERK1/2activation.ChemicalinhibitionofGSHneo-synthesisorERK1/2 phosphorylation,aswellasoverexpressionofthedominant-negativeformofNrf2sensitizescellsto diamidetoxicity.Inthesearchingforthemoleculardeterminant(s)unifyingthesephenomena,wefound thatSH-SY5YcellsshowhighGSHlevels,butexhibitverylowGPxactivity.Thisfeatureallowsto efficientlybuffer disulfide stress, but leaves them beingvulnerable to H2O2-mediated insult. The
increaseofGPxactivitybymeansofseleniumsupplementationorGPx1ectopicexpressioncompletely reversesdeathphenotype,indicatingthattheresponseoftumorcellstodiverseoxidativestimulideeply involvestheentireGSHredoxsystem.
ß2012ElsevierInc.Allrightsreserved.
Abbreviations:AMS,4-acetamido-4-maleimidylstilbene-2,2-disulfonicacid; ARE,
antioxidantresponseelements;BSO,buthionine sulfoximine;DMSO,dimethyl
sulfoxide;DMTU,dimethylthiourea;DNNrf2,dominantnegativeNrf2protein;
DNP,2,4-dinitrophenylhydrazine;ERK1/2,extracellularsignalrelatedkinase1and
2;GI-GPx,gastrointestinalisoformofglutathioneperoxidase;GPx, glutathione
peroxidase;GSH,reducedglutathione;GSHest,reducedglutathioneethylester;
GSSG,glutathionedisulfide;HO-1,hemeoxygenase-1;JNK,c-Jun-NH2-terminal
kinase;Keap1,kelch-like ECH-associated protein 1; MAPK, mitogenactivated
proteinkinase;NAC,N-cetylcysteine;Nrf2,nuclearerythroidfactor2
(NE-F2)-relatedfactor2;p62/SQSTM1,proteinof62kDasequestrosoma1;PARP,
polyADP-ribosepolymerase;ROS,reactiveoxygenspecies;Trx1,thioredoxin1.
*Correspondingauthorat: DepartmentofBiology, UniversityofRome‘‘Tor
Vergata’’,ViadellaRicercaScientifica,00133Rome,Italy.Tel.:+390672594369;
fax:+390672594311.
E-mailaddress:ciriolo@bio.uniroma2.it(M.R.Ciriolo).
ContentslistsavailableatSciVerseScienceDirect
Biochemical
Pharmacology
j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm
0006-2952/$–seefrontmatterß2012ElsevierInc.Allrightsreserved.
regulationofNrf2mediatedbyKelch-likeECH-AssociatedProtein 1(Keap1),recentpiecesofevidenceindicatethatNrf2andp53are functionalcompetitorsforthesameDNApromoterregions[15], therebyidentifyinganewsophisticatedlevel ofcontrolof Nrf2 trans-activation.Inparticular,ithasbeenindicatedthatp53binds totheantioxidantresponseelements(ARE),inhibiting,insucha way,Nrf2transcriptionalactivityofthebatteryofgenesinvolved intheantioxidantdefense.Thehighincidenceofp53mutationsin humantumors togetherwithitspivotalroleinprotectingfrom DNAdamage[16,17]andintheinductionofapoptosis[18]makes thecrosstalkbetweenthesetranscriptionfactorsaveryintriguing subjectoftumorbiologythatdeservestobeinvestigatedindepth. Indeed,theinvolvementofNrf2inresistancedevelopmentduring cancertreatmenthasbeenextensivelyreported[19,20]andarole forsomedysfunctionsintheNrf2/Keap1interactionhasbeenalso suggestedtoallowcancerprogression[21].Inthisregard,itisalso worthtomentionthat a novelredox-independent activationof Nrf2hasbeenshowntoproceedthroughtheinteractionofKeap1 withp62/SQSTM1[22,23]. Theformation of Keap1/p62 hetero-dimerdoesnotinvolveanyredoxalterationofKeap1cysteines. However,itallowsNrf2tobereleasedandtoaccumulateintothe nucleus,bymeansofadirectcompetitionbetweenKeap1/p62and Keap1/Nrf2complexesforKeap1binding.Thetumorigenicroleof Nrf2hasbeenalsoveryrecentlyreportedbyDeNicolaetal.[24],who demonstratedthattheoverexpressionoftheoncogenicmutantsof Ras,RafandMycresultsintheup-regulationofNrf2 andinthe activationoftheantioxidant/detoxifyingsystemsdownstreamofit. Thegreatimpactthatthisfindingmighthaveincancerresearch reliesuponthe evidencethatNrf2canrepresentthe converging point of different oncogenic pathways, in a way completely independentonthemutageniceventscausingcelltransformation. Inthiscontext,wehavepreviouslydemonstratedthatoxidative stressofdifferentnature(ROSversusdisulfidestress)canevocate opposite responses (cell death or survival), in the gastric adenocarcinomacelllineAGS,bymeansoftheredoxactivation ofspecificsignalingpathways[14].Inparticular,weidentifiedin the redox-dependent dissociation of Keap1/Nrf2 complex the eventresponsiblefortheresistancetoROS-mediatedinsults,and in the redox-activation of trx1/p38MAPK/p53 signaling axis the
pathwaygoverningtheinductionoftheapoptosisupontreatment withdisulfidestressors(e.g.,diamide).Here,wetrytoextendthe knowledge of cell response to oxidative stress and provide evidencethat GSH concentration and glutathione peroxidase 1 (GPx1)activityarethemaindeterminantsoftumorcellresponse todisulfidemoleculesand H2O2,respectively. In particular,we
demonstratethat,incontrasttoAGScells,neuroblastomaSH-SY5Y havehighintracellularlevelsofGSH,whichallowthemtodirectly dampendisulfidestressors-mediatedinsultviaERK/Nrf2signaling pathway. Nevertheless, the lack of a significant GPx activity rendersthemsensitivetoH2O2treatmentandinducesapoptosis
inap53-dependentmanner. 2. Materialsandmethods 2.1. Materials
Diamide, dimethyl sulfoxide (DMSO), dimethylthiourea
(DMTU),H2O2,sodiumselenite(Na2SeO3),propidiumiodide,goat
anti-mouse and anti-rabbit IgG (H+L)-horseradish peroxidase conjugate, buthionine sulfoximine (BSO) and N-acetylcysteine (NAC)werefromSigma(St.Louis,MO);GSHandGSSGwerefrom RocheAppliedScience(Monza,Italy);Oxyblotdetectionkitwas fromIntergen(Purchase,NY);nitrocellulosemembranewasfrom Bio-RadLaboratories(Hercules,CA);AlexaFluor488-conjugated secondary antibody was from Molecular Probes (Eugene, OR); ChemiGlowchemoluminescencesubstratewasfromAlpha
Inno-techCorporation(SanLeandro,CA).Allotherchemicalswerefrom Merck(Darmstadt,Germany).
2.2. Cellculturesandtransfection
HumanneuroblastomacellsSH-SY5Yandgastric adenocarci-nomaAGSwerepurchasedfromtheEuropeanCollectionofCell Culture and grown at 378C in an atmosphere of 5% CO2 in
Dulbecco’smodifiedEagle’s-F12medium(DMEM-F12)mediumor F12,respectively,supplementedwith10%fetalcalfserum,2mML
-glutamine,and0.1%penicillin/streptomycin(Lonza,Milan,Italy). 24hafterplating,SH-SY5Ycellsweretransfectedby electropora-tionusingaGenePulserxcellsystem(Bio-Rad).Downregulationof
p53 was obtained by means of transfection with a small
interferenceRNA(siRNA)againstp53[25].Proteinoverexpression wasobtainedbymeansoftransfectionwith:(i)pmaxFPTM
-Green-CvectorcontainingthedominantnegativeNrf2protein(DN-Nrf2)
[25];(ii)pcDNA3vectorcontainingthewild-typeformofmouse glutathioneperoxidase1(GPx1)kindlyprovidedbyProf.Regina Brigelius-Flohe´, Department of Biochemistry of Micronutrients,
German Institute of Human Nutrition Potsdam-Rehbruecke,
Nuthetal, Germany. Cells transfected with siRNA duplex not sharinghomologywithanyotherhumanmRNAs(siScr),orempty pmaxFPTM-Green-CandpcDNA3vectorswereusedascontrol.
2.3. Treatments
Diamide solution (10mM)was prepared in DMSO, whereas H2O2wasdissolvedinwatertoreachtheconcentrationof100
m
M.All solutions were prepared just before the experiments and treatmentsweredonefor1hinserum-containingmedium.Then themediumwasremoved,cellswerewashedandletgrowingin freshmedium.Ascontrol,equalvolumesofDMSOorwaterwere addedtountreated cells.BSOwasused attheconcentration of 1mM,added12hbeforetreatmentsandmaintainedthroughout theexperiments.DMTU,GSHestandNACweresupplementedto
culture medium at the concentration of 20, 10 and 5mM,
respectively, added 1h before treatments and maintained throughout the experiments.Na2SeO3 was used toinduce GPx
expression.Inparticularitwasdissolvedinwaterandaddedtocell mediumatthefinalconcentrationof25or50nMandmaintained fortwoweeks.CellpermeableERK1/2,JNKandp38MAPKinhibitors,
namelyU0126,SP600125andSB203580, (Calbiochem-Novabio-chem,LaJolla,CA)wereaddedatconcentrationof0.26,10and 15
m
M,for1hbeforetheadditionofdiamideorH2O2,maintainedduringtreatment,andre-addedduringtherecoveryphase. 2.4. Analysisofcellviabilityandapoptosis
After 24h of recovery, adherent and detached cells were combinedandstainedwith50
m
g/mlpropidiumiodidepriorto analysisby aFACScalibur instrument(BD Biosciences,San Jose`, CA).Apoptoticcellswereevaluatedbycalculatingpeakareasof hypodiploidnuclei(sub-G1)[26].Alternatively,cellswerecounted afterTrypanbluestainingbyopticmicroscope.2.5. Westernblotanalyses
Totalandnuclearextractswereobtainedaspreviouslyreported
[25],electrophoresedbySDS-PAGEandblottedontonitrocellulose orPVDFmembrane(Bio-Rad). Polyclonalantiphospho-p38MAPK
(Thr180/Tyr182), anti phospho-ERK1/2-p42/44 (Cell Signaling Technology, Beverly, MA), anti-Nrf2, anti-Bax, anti-JNK, anti-p38MAPK, anti-histone H2B, anti ERK1/2, anti Trx1 (Santa Cruz
Biotechnology,Santa Cruz, CA);monoclonal anti-p53 and anti-tubulin(Sigma),anti-hemeoxygenase-1(HO-1)(BDBiosciences),
antipolyADP-ribosepolymerase(PARP),anti-phospho-JNK(Santa CruzBiotechnology), anti-caspase9and caspase3(Cell Signaling Technology)wereusedasprimaryantibodies.Thespecificprotein complex,formedupon incubationwithspecificsecondary anti-bodies,wasidentifiedusingaFluorchemImagingsystem(Alpha Innotech,M-Medical,Milano,Italy)afterincubationwith Chemi-Glowchemiluminescencesubstrate(AlphaInnotech).
2.6. RedoxWesternblot
Trx1 redox statewas analyzed as previously described[14]. Briefly,cellsweretreatedwithcoldTCAatfinalconcentrationof10% for30minat48C.Proteinswerethenprecipitatedat12,000gfor 10minandpelletsincubated for30minincoldacetoneat48C. Further,pelletsweredissolvedin20mMTris/HCl,pH8.0containing 15mMAMS(4-acetamido-4-maleimidylstilbene-2,2-disulfonic ac-id,MolecularProbes)andincubatedatroomtemperaturefor3hto allowmodifyingfreethiols.Trx1redoxformswerethenseparated on 4–12% bis–TrisMES gel in non-reducing loading buffer and blottedontonitrocelluloseorPVDF.Polyclonalanti-Trx1(SantaCruz Biotechnology)wasusedasprimaryantibody.
2.7. Measurementofglutathione,proteincarbonylsandGPxactivity IntracellularGSHandGSSGwereassayedbyHPLCaspreviously described [27]. Carbonylated proteins were detected using the Oxyblotdetection kit(Intergen). Briefly, 20
m
gof proteinswere reactedwith2,4-dinitrophenylhydrazine(DNP)for15minat258C. Samples were resolved on 12% SDS-PAGE and DNP-derivatizedproteins were identified by immunoblot using an anti-DNP
antibody. GPx activity was measured spectrophotometrically, using H2O2 as substrate, by means of a glutathione
reductase-coupledreaction,inwhichthedecreaseofNADPHis considered proportionalto GPx-mediatedH2O2 reduction. GPx activity was
thereforeexpressedasnmolofNADPHconsumedmin1mgprot1.
2.8. Fluorescencemicroscopy
Cellswereculturedoncoverslips,fixedwith4% paraformalde-hydeandpermeabilized.Monoclonal anti-ser-139-phosphorylat-ed histone H2A.X (Upstate Biotechnology), and successively probed withanAlexaFluor488-conjugatedsecondary antibody. To visualize nuclei with the cell permeable DNA-specific dye Hoechst33342(Calbiochem-Novabiochem).Imagesweredigitized eitherwithaCoolSnapvideocameraconnectedtoaNikonEclipse TE200fluorescencemicroscope.
2.9. Proteindetermination
ProteinsweredeterminedbythemethodofLowryetal.[28]. 2.10. Datapresentation
Allexperimentsweredoneatleastthreedifferenttimesunless otherwise indicated. The results are presented as meansSD. StatisticalevaluationwasconductedbyANOVA,followedby Bonfer-roni’stest.Differenceswereconsideredtobesignificantatp<0.05. 3. Resultsanddiscussion
3.1. SH-SY5Ycellsshowdifferentsensitivitytodifferentpro-oxidant stimuli
Wepreviouslyidentifiedthatthegastricadenocarcinomacells AGSwerehighlyresistanttoROS-producingdrugs,butsensitiveto treatmentwiththiol-oxidizingchemicals.In ordertogeneralize
this observation, we selected another tumor histotype, the neuroblastomaSH-SY5Y,andtreatedthecellswithH2O2,orwith
diamide, which acts by oxidizing cellular sulfhydryls, without producingROSdirectly.Cellsweretreatedwith100and200
m
M H2O2 ordiamide: concentrationsthat arein therangeof thoseusuallyemployedinstudiesoftoxicology,andthat,also,overlap thosewepreviouslyusedonAGScells[14].After1h-incubation, H2O2anddiamidewereremoved,cellswashedandletgrowingin
fresh medium, and theextent of apoptosisanalyzed cytofluor-ometricallyafter24h-recovery.Itisworthnotingthat,owingto the different cellular metabolism of H2O2 and diamide, the
strengthofoxidativestressproducedupontheadministrationof
Fig.1.Hydrogenperoxide,butnotdiamide,triggerscaspase-dependentapoptosis
inSH-SY5Ycells.(a)SH-SY5Ycellsweretreatedwith100or200mMH2O2or
diamide,inthepresenceorabsenceofthepan-caspaseinhibitorzVADfmkafter
24h.Apoptosis(SubG1)wascytofluorometricallyanalyzeduponpropidiumiodide
staining, withcells incubatedwith DMSO(time 0) usedascontrol. Dataare
expressedas%ofSubG1(apoptotic)cellsandrepresentthemeanSDofn=4
independentexperiments.**p<0.01;***p<0.001(H2O2versuscontrol).yyyp<0.001
(zVADfmkversus+zVADfmk).(b)After18h-treatment,30mgoftotalproteinextract
wasloadedontoeachlaneforthedetectionofthefulllengthandcleavedcaspase9,
caspase3andPARP.Tubulinwasusedasloadingcontrol.Duetothedifferentspecificity
oftheanti-caspase3andanti-PARPantibodiestorecognizethecleavedandfull-length
thetwodrugsisnotcomparableintermsofabsolute concentra-tions.However, datareported inFig.1a indicatedthatSH-SY5Y underwent caspase-dependent apoptosis (subG1) in a dose-dependentmannerwhenincubatedwithH2O2,withthe
percent-ageofapoptosisrangingfrom35%to70%.Bycontrast,theywere muchlesssensitivetodiamidetreatment.Thisdichotomyincell
response was also confirmed by Western blot analyses. In
particular,SH-SY5Ycells treatedwithH2O2showedthe
proteo-lysedformofcaspase9,caspase3andPARPafter18hofrecovery (Fig. 1b). On the contrary, no significant increase in the immunoreactivityoftheseproteinswasobserveduponrecovery fromdiamidetreatment, indicatingthat H2O2,butnot diamide,
inducedtheintrinsicpathwayofapoptosis.Suchdifferenceincell fatewasalsoconfirmedbytheoccurrenceofoxidativedamageto proteinsandDNA,withamarkedincreaseofproteincarbonylsand phospho-H2A.X-positivenuclearfocievidentonlyupontreatment withH2O2(SupplementaryFigure1).
Supplementary material related tothis article found, in the onlineversion,atdoi:10.1016/j.bcp.2012.02.003.
3.2. H2O2anddiamidespecificallymodulateNrf2andp53
transcriptionfactor
Consistentwithourpreviousresults,weinvestigatedwhether thedifferentcellresponsetoH2O2 anddiamiderelieduponthe
activationofp53orNrf2byWesternblotanalysesoftheirnuclear levels.Fig.2a showsthat thenuclearfractionof p53increased time-dependently after 3h-recovery from H2O2 administration,
whereas no significant change occurred at the same time of recoveryfromdiamideincubation.Onthecontrary,Nrf2increased rapidlyafterdiamidetreatmentbuttransientlyaccumulatedalso inresponsetoH2O2,confirmingdatafromtheliterature[29]and
ourpreviousobservationsofadirectsensitivityofNrf2toH2O2
[14].WethenverifiedtheacquisitionofNrf2orp53transcriptional
Fig.2.p53mediatesapoptosisuponH2O2treatment,whereasNrf2isthepro-survivalfactorinvolvedinresponsetodiamide.(a)SH-SY5Ycellsweretreatedwith100mM
H2O2ordiamide.Attheindicatedtimepoints,20mgofnuclearenrichedextractswasloadedontoeachlaneforthedetectionofNrf2andp53.Histone2B(H2B)wasusedas
loadingcontrol.(b)Alternatively,30mgoftotalproteinextractwasloadedontoeachlaneforthedetectionofHO-1andBax.Tubulinwasusedasloadingcontrol.(c)SH-SY5Y
cellsweretransfectedwithsiRNAagainstp53(si-p53)orwithascrambledsequence(siScr).TheefficiencyoftransfectionwasevaluatedbyWesternblotanalysesofp53andis
shownintheinset.After12hfromtransfectioncellsweretreatedwith100mMH2O2.Similarly,cellsweretransfectedwiththedominantnegativemutantofNrf2(DN-Nrf2)
orwithanemptyvector(mock).After48hfromtransfection,cellsweretreatedwith100mMdiamide.After24h,apoptosis(SubG1)wascytofluorometricallyanalyzed.Data
areexpressedas%ofapoptosisandrepresentthemeanSDofn=4independentexperiments.***p<0.001.(d)SH-SY5Ycellsweretreatedwith100mMH2O2ordiamide.Atthe
indicatedtimepoints,cellswereprecipitatedinTCAanddissolvedin20mMTris/HCl,pH8.0containingAMStoallowmodifyingfreethiols.Trx1redoxformswerethenseparated
on4–12%SDS-PAGEinnon-reducingconditions.Reduced(AMS-bound)Trx1[Trx(SH)2]andoxidizedTrx1[Trx(S-S)]areshown.TotalTrx1wasusedascontrol.Densityofreduced
andoxidizedTrx1(reportedbelowtheimmunoblots)wascalculatedusingthesoftwareQuantityone(Bio-Rad)andreportedasarbitraryunits.DataareexpressedasmeansSDof
activitybyWesternblotanalysesoftheexpressionlevelsoftheir target genes, heme-oxygenase 1 (HO-1) and Bax, respectively.
Fig.2bshowsthatHO-1increasedrapidlyduringtherecoveryfrom diamideincubation,whereasBaxaccumulatedonlyafter6–12hof recovery from H2O2 administration, suggesting that survival
responsetodiamidewasmediatedbyNrf2,whereasH2O2-induced
apoptosisreliedonp53activation.Tocorroboratethese hypothe-ses,wetransfectedSH-SY5YcellswithansiRNAagainstp53or, alternatively,withthedominantnegativeformofNrf2(DN-Nrf2) andanalyzedcytofluorometricallytheextentofapoptosis.Fig.2c
shows that p53 knocking down significantly decreased the
percentage of sub-G1 (apoptotic) cells induced by H2O2, while
DN-Nrf2transfectedcellsbecamesensitivetodiamide.
Giventheredox-dependentactivationofp53andNrf2viaTrx1 andKeap1oxidation[14],weevaluatedtheredoxstateofTrx1and Keap1by redoxWestern blot. Fig. 2d indicates that a marked decreasein reduced/oxidizedratioofTrx1occurred onlyduring the recovery from H2O2 incubation, whereas diamide did not
induceanyvisiblechange.Conversely,neitherH2O2nordiamide
altersKeap1redoxstate(datanotshown),suggestingthatother Keap1-independentmechanism(s)shouldberesponsibleforthe activationofNrf2.
3.3. p38MAPKandERK1/2arethemediatorsofcellresponsetoH 2O2
anddiamide
p53activationhasbeenreportedtoberesponsivetop38MAPK
downstreamofTrx1oxidation.Similarly,ithasbeendescribedthat phosphorylationofNrf2byaseriesofkinases(e.g.,ERK1/2)affects itsfateanddistribution[30–32].Inparticular,phosphorylationof Nrf2 on Ser40 is required for Nrf2 to be releasedfrom Keap1,
whereas mutant Nrf2 at Ser40 remained bound to Keap1[32].
However, it isworthwhilementioning thatH2O2 treatmenthas
beenreportedtobroadlyactivateERK,JNK,andp38MAPKinhuman
coloncancerSW620[33]andinSH-SY5Ycells[34],andaprevious studyalsodemonstratedthatbothdiamideandH2O2activateERK
andp38MAPKinvitro[35].Inthelight oftheseobservations,we
analyzedtheactivationstateofMAPKbyWesternblotanalysesof thebasalandphospho-activeformsofERK1/2,JNKandp38MAPK
uponrecoveryfromincubationwithH2O2anddiamide.Fig.3aand
bshowthatERK1/2wastheonlymemberalwaysactivatedupon treatment with 100
m
M diamide, whereas H2O2 induced theactivation ofall theenzymesanalyzed, althoughwithdifferent modalities.Inparticular,ERK1/2appearedphospho-activatedonly at3hoftreatment,andrapidlyreturnedtothecontrollevels,thus
Fig.3.ERKandp38MAPK
aretheupstreamkinasesinvolvedinthemodulationofcellviability.SH-SY5Ycellsweretreatedwithdiamide(a)orH2O2(b).Attheindicatedtime
points,20mgoftotalproteinextractwasloadedontoeachlaneforthedetectionofbasalandphospho-activeformsofERK1/2,JNKandp38MAPK
.Tubulinwasusedasloading
control.(c)SH-SY5YcellsweretreatedwithdiamideorH2O2inthepresenceorabsenceoftheERK1/2inhibitorU0126,JNKinhibitorSP600125,orp38MAPKinhibitor
SB203580.After24h,apoptosis(SubG1)wascytofluorometricallyanalyzed.Ascontrol()cellswereincubatedwithDMSO.Dataareexpressedas%ofapoptosisand
resemblingthekineticsofNrf2nucleartranslocationinresponse to H2O2 exposure. p38MAPK became rapidly and completely
phosphorylatedalready at 3hto be restored only after 24
h-treatment. Conversely, JNK phosphorylation seemed to be
delayedwith respect to p38MAPK,as itreached the maximum valuesonlyat12and24h-treatment.Toverifytheexactroleof
each MAPK in the cell response to the different pro-oxidant insults,wetreatedSH-SY5YcellswithH2O2ordiamide,in the
presenceofthe pharmacologicalinhibitors ofERK1/2,JNK and p38MAPK,namelyU0126,SP600125andSB203580,respectively.
Cytofluorometricanalysesperformedafter24h-recovery show that ERK1/2 inhibition resulted in a significant increase of
Fig.4.ERKandp38MAPKaretheactivatingkinasesofNrf2andp53,andtheirinductionismutuallyexclusive.
SH-SY5Ycellsweretreatedfor3hwith100mMdiamideorH2O2.Twentymgoftotalproteinextract(aandb)wasloadedontoeachlaneforthedetectionofphospho-p38MAPK
andphospho-ERK1/2,Bax,HO-1,whereas30mgofnuclearenrichedextract(candd)wasloadedontoeachlaneforthedetectionofNrf2andp53.TubulinandH2Bwereused
asloadingcontroloftotalandnuclearextract,respectively.
Fig.5.Glutathioneavailabilityaffectsdiamide-mediatedeffects,butnotH2O2toxicity.SH-SY5Ycellsweretreatedwith(a)100mMdiamideinthepresenceorabsenceofBSO
or,alternatively(b)with100mMH2O2inthepresenceorabsenceoftheantioxidantsNACandDMTU,orwithGSH-ethylester(GSHest).After24h,apoptosis(SubG1)was
cytofluorometricallyanalyzed.Dataareexpressedas%ofapoptosisandrepresentthemeanSDofn=4independentexperiments.*p<0.01;**p<0.01;***p<0.001.SH-SY5Y
cellsweretreatedwith100mMdiamide,inthepresenceorabsenceofBSO(c),orH2O2,inthepresenceorabsenceofNACandDMTU(d).Attheindicatedtimepoints,20mgoftotal
proteinextractwasloadedontoeachlaneforthedetectionofphospho-p38MAPK
apoptosiswhen thecells were treatedwith diamide,whereas p38MAPKinhibition,carriedoutuponH2O2incubation,yieldeda
decreaseofsubG1percentages(Fig.3c),indicatingthattheywere themasterregulatorsofcellsurvivalandcelldeathobservedin ourexperimentalconditions.Interestingly,JNKinhibitordidnot resultinanysignificantmodulationofcellresponsetobothH2O2
anddiamide,butalteredpersetheviabilityofSH-SY5Y(Fig.3c) andcell cycleprogression byblockingthecellsinG2/Mphase (data not shown). This evidence perfectly correlates with our previousobservationsperformedinthesamecellline,andargue for an active role of JNK in the replicative capability of neuroblastoma cells [36]. On the basis of these results, we focusedonERK1/2 and p38MAPK andanalyzed whethertheir inhibitioncould impact onsignal transductionleading to cell deathorsurvival.WesternblotanalysesshowninFig.4aindicate that ERK1/2 inhibition induced a decrease in the expression levelofHO-1and,concomitantly,inthenuclearcontentofNrf2 incellstreatedwithdiamide(Fig.4c).Onthecontrary,p38MAPK inhibition resulted in a marked reduction of both Bax level (Fig.4b)andnuclearaccumulationofp53(Fig.4d).Itisworth notingthat,theunresponsivenessofNrf2todiamidecausedby ERK1/2inhibition,wasassociatedwithavisibleaccumulationof p53intothenuclearcompartment(Fig.4c),whereasamarked increase of nuclear Nrf2, upon H2O2 treatment, was clearly
evidentupontheinhibitionofp38MAPK(Fig.4d).
3.4. GSHandGPxaretheredoxmodulatorsofcellresponsetoH2O2
anddiamide
SincewepreviouslyfoundthatchangesofGSHredoxstatewere tightlyassociatedwiththepatternofcellresponse(deathversus resistance)[14],weinvestigatedthisissuealsoinSH-SY5Ycells upon12h-incubationwith1mMBSO,thespecificinhibitorofGSH neo-synthesis,beforeaddingdiamide.Apoptoticextentwasthen evaluated cytofluorometrically after 24h of recovery. Data reported in Fig.5a showthat GSHdepletionrenderedthecells sensitive to diamide with apoptosis values reaching 50%. By contrast,weenrichedtheintracellularantioxidantpoolbymeans of 1h-incubationwith: 10mM of theethyl ester formof GSH (GSHest),5mMNAC,or20mMDMTU.Undertheseconditions, SH-SY5Y cells were protected against H2O2-mediated apoptosis,
althoughatdifferentextents(Fig.5b).Indeed,DMTU,whichacts as direct H2O2 scavenger,completely rescued cells from death,
whereasNACandGSHest,whicharecanonicalthiolantioxidants, partiallycontributedtothepro-survivalresponse,withadecrease inthepercentageofapoptosisofonly45%and29%,withrespectto H2O2-treatedcells.WethenselectedDMTUandNACasthemost
effectiveanti-apoptoticcompoundsandperformedWesternblot analyses of the pro-apoptotic axis p38MAPK/p53. Fig. 5c shows
thatphospho-activep38MAPKandp53increasedinGSH-depleted
cells when incubatedwithdiamide,whereas theydecreased in
Fig.6.GPxactivitybuffersH2O2toxicityindependentlyonGSHconcentration.(a)SH-SY5YcellswereletgrowingfortwoweekswithdifferentconcentrationsofNa2SeO3,and
GPxactivitywasmeasuredspectrophotometrically.AGSwereusedaspositivecontrol.DataareexpressedasnmolofNADPHconsumedmin1mgprot1andrepresentthe
meanSDofn=6independentexperiments.***p<0.001.(b)Aftertwoweek-administrationof25or50nMNa2SeO3,SH-SY5Ycellsweretreatedwith100mMH2O2.After24h,
apoptosis(SubG1)wascytofluorometricallyanalyzed.Dataareexpressedas%ofapoptosisandrepresentthemeanSDofn=4independentexperiments.***p<0.001.(c)SH-SY5Y
cellsweretransfectedwithapCDNA3vectorcontainingtheWTformofGPxorwithanemptyvector(mock).After48hfromtransfectioncellsweretreatedwith100mMH2O2.After
24h,apoptosis(SubG1)wascytofluorometricallyanalyzed.Dataareexpressedas%ofapoptosisandrepresentthemeanSDofn=4independentexperiments.***p<0.001.(d)
SH-SY5Ycellsweretreatedfor3hwith100mMH2O2.ThirtymicrogramsofnuclearenrichedextractwasloadedontoeachlaneforthedetectionofNrf2andp53.H2Bwasusedas
H2O2-treatedcellswhenpre-incubatedwithDMTUandNAC,ina
mannerresemblingtheiranti-apoptoticeffect(Fig.5d).
To comprehend the redox bases underlying the different sensitivitytoH2O2anddiamideinSH-SY5Ycellswithrespectto
whatpreviouslyreportedforAGS,wemeasuredGSHcontentinboth cellslines.Takingintoaccountacellvolumeof10
m
l/mgprotein[37],wefoundthatthemolarconcentrationofGSHcalculatedin SH-SY5Ycellswas significantlyhigherthaninAGS (7.650.78mM versus1.650.35mM,n=10,p<0.001).Thisresultnicelycorrelated withtheresistanceoftheneuroblastomacelllinetodiamidetoxicity; nevertheless,itdidnotexplainSH-SY5YsensitivitytoH2O2.SinceAGS
cellshavebeenpreviouslyreportedtobeequippedwiththeadditional gastrointestinalisoformofGPx(GPx2orGI-GPx)[38],wewonderedto evaluatedifferencesinGPxactivitybetweenAGSandSH-SY5Y.Fig.6a showsthatthebasalGPxactivitymeasuredinAGScellswasthree-fold higherwithrespecttothatevaluatedinSH-SY5Y.However,two week-incubationwith25or50nMNa2SeO3,whichisusuallyemployedto
inducetheexpressionofGPx,increasedperoxidaticactivityina dose-dependentmanner,uptovaluescomparable,orevenhigher,tothose measuredinAGS(Fig.6a).WethentreatedSH-SY5YcellswithH2O2
andanalyzedcytofluorometricallytheextentofapoptosis.Asshownin
Fig.6b,SH-SY5YgrowninthepresenceofNa2SeO3wereresistantto
H2O2-mediated apoptosis, and the rescue of cell viability directly
correlatedwiththeconcentrationofNa2SeO3usedtoup-regulateGPx
activity.Toconfirmthesedata,wealsooverexpressedGPx1,themost abundantformofGPx,whichpreferentiallycatalyzesthereductionof H2O2.Aftertwoday-incubationwithNa2SeO3,necessarytoprovide
seleniumtothenew-synthesizedproteins,GPx1activitywasmeasured tobe40.752.15nmolNADPHminmgoftotalprotein,therefore we treated SH-SY5Y cells with H2O2 and analyzed the extent of
apoptosiscytofluorometrically.Fig.6cshowsthatGPx1overexpressing cellsaretotallyprotectedfromH2O2toxicity.Inlinewiththisresults,
Westernblotanalysesperformedonnuclearextractsindicatedthat Nrf2accumulatedinGPx1-overexpressingcellsduringtherecovery fromH2O2treatment,whereasp53immuno-reactivelevelsremained
unchanged(Fig.6d),confirmingthatsurvival/deathresponsetoH2O2
-mediatedoxidativestressdependedontheamountofH2O2lasting
uponGPx-mediatedscavenging. 4. Conclusions
Thecapabilityofseveraltumorcelllinestofacepro-oxidant
conditions and overcome oxidative damages is among the
principalalterationscausingtumortransformationandresistance tochemotherapeutics.Alargenumberofmoleculescommonly employedtodayincancertherapyinducesselectivetumorkilling by interfering with the maintenance of DNA integrity or by impairing the efficiency of the DNA replication machinery. Moreover,manyofthese drugsinduceoxidativestressas side effectoftheirmechanismofaction.Therefore,theoverexpression ofdetoxifying/antioxidantsystems,suchasthoseup-regulatedby Nrf2isconsistentwiththegenerationofaneoplasticphenotype, whoseaggressivenessdirectlycorrelateswiththecapabilityto evade apoptosisinduced by chemotherapeutic approaches.To corroboratethis hypothesis,ithasbeen veryrecently demon-stratedthatNrf2isinducedbydiverseoncogenes,suchasK-Ras, B-RafandMyc,in ordertodecreaseROS concentrationandto mediateoncogenesis[24].
WepreviouslydemonstratedthatH2O2treatmentinducesKeap1
oxidation and its detachment from Nrf2 in tumor histotypes efficientlyequippedwithGPx(e.g.,gastricorintestinal adenocarci-noma cells). This molecular event does not take place upon treatment with disulfide stressors that, conversely, triggers apoptoticcelldeathviatheredoxactivationoftheTrx1/p38MAPK/
p53-dependentsignalingcascade.Thesepiecesofevidencearguefor two important issues: (i) disulfide stress is not able to induce
oxidation of Keap1 cysteines and, in turn, Nrf2 activation; (ii) alterationofthiolredoxhomeostasiscouldbeavaluabletoolto circumventNrf2-mediatedresistancetoROS-basedchemotherapy. Inthesearchingforpossibleclinicalimplicationsoftheseresults, inthisworkwehavehighlightedtwoimportantconsiderations.The first one deals with the fact that tumor histotypes resistant to disulfidestressorsobviouslyexist.Thesecond,andmoreimportant, isthat,alsointhiscase,resistanceresponseisgovernedbyNrf2. However,aswe previouslyreported[14],no Keap1 oxidationis observedupondisulfidestress.Thiscouldreasonablydependonat leastthreedistinctissues:(i)theredoxpotentialofKeap1reactive cysteines;(ii)thedifferentchemicalstructureandsterichindrance ofH2O2anddiamide;(iii)GSHconcentrationandGPxactivityofthe
cell.Whilethefirsttwopointsareofgeneralapplicationandcould affectallredoxreactionsinvolvingKeap1,thelastoneiscellspecific. Inparticular,inthiswork,wehaveprovidedevidencethathighGSH concentration candampendisulfidestress, but areinefficientto counteractH2O2toxicity,unlesscomplementedwithhighlevelsof
GPxactivity.ThisdependsonthefactthatGSHcanreactdirectly with diamide (rate constant=3102M1s1) [39], whereas
GSH-mediatedreductionofH2O2canoccuralmostexclusivelyif
catalyzedbyGPx.SH-SY5Ycellsperfectlyfitthiscondition,since theyareveryrichinGSH,butshowlowlevelsofGPx.Inagreement withthebiochemicalfeaturesabovementioned,pre-treatmentwith theROSscavengerDMTU,overexpressionofGPx1,oradditionof selenitetotheculturemedia,astooltoinduceGPx1expression, completely abolish H2O2-induced toxicity; but further increase
ofGSH,orthiolenrichmentonlypartiallypreventsthis phenome-non.Onthecontrary,depletionofGSHbyBSOsensitizesSH-SY5Y cells to diamide and activates apoptosis via the p38MAPK
/p53-mediatedsignalingpathway.Ithasbeenexhaustivelyreportedthat BSO enhancesapoptotic response of neuroblastomato different stimulibyincreasingthesteady-stateconcentrationofROS[40,41]. Therefore, although not directly investigated in this work, it is reasonable to hypothesize that also in our conditions, BSO-dependent GSH depletion results in ROS production, which ultimatelyactivatesp38MAPK/p53signalingpathway.
Thesecondimportantconcernarising fromthiswork isthat, although Keap1 oxidation does not occur upon treatmentwith disulfide stressors, nevertheless, the anti-apoptotic response involvesNrf2asprincipalmediator.Inthesearchingforalternative pathways underlying this event, able tocircumvent the redox-dependentdetachmentofKeap1fromNrf2,weidentifiedERK1/2 phosphorylation as the pivotal process responsible for Nrf2 activation.Severalreportsindicatethatalsothispathwayisredox sensitive,butindependentonthedirectoxidationofKeap1[42–44]. Indeed,ithasbeenreportedthatmembersofMAPKfamily,andin particularERK1/2,areabletophosphorylateNrf2onSer40inaway
allowing its detachment fromKeap1 [32,45]. Interestingly,this signalingpathwayhasbeenindicated:(i)toincreaseantioxidant defense,mainlyGSHneo-synthesisandHO-1expression[44,46];(ii) tobeinduceduponstressfulconditions,suchaschemotherapeutic treatments[44,46,47];(iii)tobemutuallyexclusiveofthep38MAPK
-mediated apoptotic pathways [14,46,48]. Interestingly, these observationsareallinaccordancewiththeresultsobtainedinthis work.Inparticular,wehavealsoobservedthatdiamide adminis-trationresultsinasignificantincreaseofGSHlevelsstartingfrom6h of treatment(datanot shown).Thesedatafurther confirmNrf2 activationandultimatelyunderlinetheimportanceofGSHredox bufferagainstdisulfidestressinneuroblastomacells.
Acknowledgments
Thisworkwaspartially supportedby grantsfromMinistero dellaSalute,AIRC(#IG10636),andfromMinisterodell’Universita` edellaRicerca(MIUR).
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