Cell cycle kinetics, apoptosis rates, DNA damage and TP53 geneexpression in bladder cancer cells treated with allyl isothiocyanate(mustard essential oil).

ContentslistsavailableatScienceDirect

Mutation

Research/Fundamental

and

Molecular

Mechanisms

of

Mutagenesis

jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / m o l m u t

C o m mun i ty a d dr e s s :w w w . e l s e v i e r . c o m / l o c a t e / m u t r e s

Cell

cycle

kinetics,

apoptosis

rates,

DNA

damage

and

TP53

gene

expression

in

bladder

cancer

cells

treated

with

allyl

isothiocyanate

(mustard

essential

oil)

André

Luiz

Ventura

Savio

_,

_Glenda

_Nicioli

_da

_Silva

_,

_Elaine

_Aparecida

_de

_Camargo

_,

Daisy

Maria

Fávero

Salvadori

a_{UNESP—UniversidadeEstadualPaulista,FaculdadedeMedicinadeBotucatu,DepartamentodePatologia,Botucatu,SP18618-970,Brazil} b_{UFOP–UniversidadeFederaldeOuroPreto,EscoladeFarmácia,DepartamentodeAnálisesClínicas,OuroPreto,MG35400-000,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25September2013

Receivedinrevisedform19February2014 Accepted28February2014

Availableonline10March2014

Keywords: Allylisothiocyanate Apoptosis Bladdercancer Cellcycle Genotoxicity TP53

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Allylisothiocyanate(AITC)ispresentinplantsofthecruciferousfamilyandisabundantinmustardseed.

Duetoitshighbioavailabilityinurineafteringestion,AITChasbeenconsideredapromisingantineoplastic

agentagainstbladdercancer.BecauseTP53mutationsarethemostcommonalterationsinbladdercancer

cellsandarefrequentlydetectedininsitucarcinomas,inthisstudy,weinvestigatedwhethertheAITC

effectsinbladdercancercellsaredependentontheTP53status.Twobladdertransitionalcarcinomacell

lineswereused:RT4,withwild-typeTP53;andT24,mutatedTP53gene.AITCwastestedatconcentrations

of0.005,0.0625,0.0725,0.0825,0.0925,0.125and0.25␮Mincytotoxicity,cellandclonogenicsurvival

assays,cometandmicronucleusassaysandforitseffectsoncellcycleandapoptosisbyflowcytometryand

onTP53geneexpression.ThedatashowedincreasedprimaryDNAdamageinbothcelllines;however,

lowerconcentrationsofAITCwereabletoinducegenotoxicityinthemutantcellsfortheTP53gene.

Furthermore,theresultsdemonstratedincreasedapoptosisandnecrosisratesinthewild-typecells,but

notinmutatedTP53cells,andcellcyclearrestintheG2phaseformutatedcellsafterAITCtreatment.

NosignificantdifferencesweredetectedinTP53geneexpressioninthetwocelllines.Inconclusion,

AITCcausedcellcyclearrest,increasedapoptosisratesandvaryinggenotoxicitydependentontheTP53

status.However,wecannotruleoutthepossibilitythatthosedifferencescouldreflectotherintrinsic

geneticalterationsintheexaminedcelllines,whichmayalsocarrymutationsingenesotherthanTP53.

Therefore,furtherstudiesusingothermoleculartargetsneedtobeperformedtobetterunderstandthe

mechanismsbywhichAITCmayexertitsantineoplasticpropertiesagainsttumorcells.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Bladdercanceristhefourthmostcommonneoplasmdiagnosed inmenandtheninthmostcommoninwomen intheWestern world[1]. Approximately90%of malignant bladdertumors are representedbyurothelialcellcarcinomas(UCC),whichpresentas smallpapillaeorinvasivelesions[2].Duetothehighrecurrence rates,progressiontomuscleinvasivediseaseandincreased aggres-siveness,bladdercancerisconsideredahighlymorbiddisease[3]. Themajorityofcasesareassociatedwithcigarettesmokingand occupationalexposuretoaromaticamines[4].Ithasbeenreported thattobaccometabolitesexcretedintotheurineofsmokersare

∗ Correspondingauthor.Tel.:+551438807263;fax:+551438807263. E-mailaddress:[email protected](A.L.V.Savio).

responsibleforapproximately50%ofbladdertumors.Furthermore, smokershaveafour-foldhigherincidenceofbladdercancerthan non-smokers[5].

TherapiesforUCCincludesurgicalprocedures(partialorradical cystectomy),radioandchemotherapy.However,currently,natural substancesfoundinfruits,vegetablesandessentialoilshavebeen investigatedasalternativeapproachesfortreatingdiseasessuchas cancer[6–8].Allylisothiocyanate(AITC),knownasmustard essen-tialoil,isabundantinmustardseedandwasabi(horseradish)andis anaturalcompoundwithchemopreventivepotential.An epidemi-ologicalstudyhasdemonstratedthatrawcruciferousvegetables containingAITCmayreducetheriskofbladdercancer[9].Infact,it wasshownthatapproximately80%ofAITCoraldosesare selec-tivelydeliveredtothebladdertissue throughurinaryexcretion andcanpotentlyinhibitcancerdevelopmentandmuscleinvasion

[10,11].Furthermore,ithasbeensuggestedthatthis compound

http://dx.doi.org/10.1016/j.mrfmmm.2014.02.006

haspromisingactivityfortreatingandpreventingbladdercancer becauseofitscapacityforinterferingwithmitosis,increasing ubi-quitinationandtubulindegradationandinducingapoptosis[12].

TP53mutationsarethemostcommonalterationsinbladder can-cercellsandarefrequentlydetectedininsitucarcinomas(CIS)and inadvanceddiseasestates[13–15].Thesemutationsarerelatedto cellulartransformation,malignancyandthehighrecurrencerate ofurinarybladdercancers[13,16].TP53isacriticalgeneintheG1 checkpointandisevolvedinthetumorresponsetoseveral anti-cancerdrugs[17–19].Itsencodedprotein,p53,canactivateG1cell cyclearrestinresponsetoDNAdamage,extendingthetime avail-ableforDNArepairbeforeentryintotheSphase,andcanplayan importantroleintheapoptosispathways[20,21].Therefore,TP53 mutationscanaffectthep53DNA-bindingactivity,abolishingthe transcriptionalactivationofTP53targetgenesandapoptosis[22].

Numerousstudieshaveinvestigatedtherelationshipbetween p53 and/or TP53 mutationsand theresponse toantineoplastic drugs,oncebothproteinandgenearerelatedtotheDNA dam-ageresponsepathway[17,18,23].Recently,itwasdemonstrated that multiple myeloma cell death caused by the exposure to gemcitabineandclofarabineisp53-dependent[19].Therefore,to investigatetheantineoplasticpotentialof AITC forbladder car-cinoma, weused two celllines withdifferentTP53 status:one carryingthewild-typegene(RT4)andtheothercarryingamutated TP53gene (T24).Thecytotoxicity,mutagenicity,apoptosisrates, cellcycle alterationsand toxicogenomic activitiesofAITC were evaluatedinthetwocelllines.

2. Materialsandmethods

2.1. Celllinesandtestcompound

The humanurothelial carcinoma celllines RT4 (from a low gradetumorwiththewildtypeTP53gene)andT24(froman inva-sivetumorwiththeTP53alleleencodinganin-framedeletionof tyrosine126)werepurchasedfromtheCell BankoftheFederal UniversityofRiodeJaneiro,Brazil,andmaintainedaspreviously describedbydaSilvaetal.[24].Allylisothiocyanate(AITC)was pur-chasedfromSigma-Aldrich(USA)andwasdilutedinto2%Tween20 priortouse.Thetestcompoundconcentrationswereselectedbased onthedataobtainedintheclonogenicsurvivalassay.Alltreatments withAITCwereperformedfor3h,asdescribedbyZhangetal.[25].

2.2. Cytotoxicityandcellproliferation

Cytotoxicity and cellproliferation rates wereassessed using theCellProliferationKitII(XTT)(ROCHEDiagnostics;Mannheim, Germany).Briefly,cells wereseededinto12-wellcultureplates (12×104_and8×104 _{cells/wellforcytotoxicityandcellsurvival,}

respectively).After24h,thecellsweretreatedwithAITCat con-centrationsof 0.005, 0.0625,0.0725, 0.0825, 0.0925,0.125 and 0.25␮Mfor3h.UntreatedcellsandcellstreatedonlywithTween 20 werecultured ascontrols.Threehoursafter incubation,the cellswerewashedwithHank’ssolution(0.4gKCl,0.06gKH2PO4,

0.04gNa2HPO4,0.35gNaHCO3,1gglucoseand8gNaClin1L·H2O).

Afterwashing,50␮LofXTTtestsolution(1mLXTTlabeling solu-tion/20_␮Lofelectron-couplingreagent)wasaddedtoeachwell, andtheabsorbancewasmeasuredat492and690nmafter90min (absorbanceresultsareproportionaltothepercentage ofviable cells).Forevaluatingcytotoxicityandcellproliferation,complete freshmediumwasadded,andthecellswereincubatedat37◦Cfor 21and69h,respectively.Then,a50-␮LaliquotofXTTsolutionwas addedtoeachwell,andtheabsorbancewasmeasuredafter90min. Bothtestswereconductedintriplicate.

2.3. Clonogenicsurvival

Aclonogenicassaywasusedforevaluatingthelong-termeffects ofAITC. Forclonogenicability, cellswereplatedata densityof 1×106 _cells/25cm3 _{culture flask; 24hlater, they weretreated}

withAITCatconcentrationsof0.005,0.0625,0.0725,0.0825and 0.0925␮M for 3h. Cultures were rinsed with Hank’s solution, trypsinized,andapproximately1000cellswereplatedinto25-cm3

cultureflasksandallowedtogrowfor15daystoformcolonies. ThecellswereGiemsastained,andthenumberofcolonieswith 50ormorecellswascounted.Theexperimentswereperformedin triplicate.

2.4. Cometassay

Initially,8×104_{cellswereseededinto12-wellplatesfor24h.}

Then, cells were treated withAITC at concentrations of 0.005, 0.0625,0.0725,0.0825and0.0925␮Mfor3h.Methyl methane-sulfonate(0.006M,5min,37◦C;Sigma-Aldrich,Inc.;St.Louis,MO, USA)wasusedaspositivecontrol.Thecometassaywasconducted basedonthemethodsdescribedinSinghetal.[26]andTiceetal.

[27].Briefly,10␮Lofcellswasaddedto100␮Lof0.5% low-melting-pointagaroseat37◦C.Thismixturewaslayeredontopre-coated slides with 1.5% standard agarose and covered with a cover-slip.Afteragarosesolidificationat4◦C,thecoverslipwasgently removed.Then,theslideswereimmersedintolysissolution(2.5M NaCl,100mMEDTA,10mMTris–HClatpH10,1%sodium sarcosi-nate,1%TritonX-100and10%DMSO)overnight,at4◦C,whichwas followedbyanincubationstepinalkalinebuffer(0.3mMNaOHand 1mMEDTA;pH>13)for20min,toallowDNAunwindand alkali-labilesiteexpression.Electrophoresiswasconductedinthesame alkalinebufferat4◦C,for20min,at25V(0.86V/cm)and300mA. Afterelectrophoresis,theslideswereneutralizedin0.4MTris–HCl (pH7.5)for15min,fixedwithabsoluteethanolandstoredatroom temperatureuntilanalysis.Allstepswereconductedinthedark topreventadditionalDNAdamage.Theslideswerestainedwith SYBRGold(1:10,000;Invitrogen;GrandIsland,NY,USA), immedi-atelybeforeanalysis.Cellviabilitywasassessedusingthetrypan blueexclusiontest(0.4%trypanblue,Sigma-Aldrich,Inc.;St.Louis, MO,USA)exclusiontest(viabilitywasneverlowerthan90%).A totalof150randomlyselectednucleoidspertreatmentwere ana-lyzedunder400×magnificationwithafluorescencemicroscope connectedtoanimageanalysissystem(CometAssayIV, Percep-tiveInstruments;Suffolk,Haverhill,UK).Tailintensity(%DNAin tail)wasusedtoestimateDNAdamage.Theslideswereprepared induplicatefromthreeindependentexperiments.

2.5. Cytokinesis-blockmicronucleusassay

TheMNassaywasperformedbasedonthetechniquedescribed by Fenech [28]. Briefly, 1×106 _{cells were seeded into a dish}

(100mm×20mm). Twenty-four hourslater, cells were treated withAITCatconcentrationsof0.005,0.0625,0.0725,0.0825and 0.0925␮M,for3h.Then,cytochalasinB(3␮g/mL)wasaddedand thecellswereincubatedat37◦Cand5%CO2for28(T24)or44(RT4)

hours.Attheendoftheincubationsteps,cellswerecollectedand centrifugedat800rpmfor5min.Thesupernatantwasdiscarded, and5mLofice-coldhypotonicsolution(0.075MKCL)wasadded. Aftercellfixation,theslideswerestainedwith5%Giemsasolution andanalyzedunderalightmicroscopeat1000×magnification.One thousandbinucleatedcellswereanalyzedineachslide. Doxoru-bicinwasusedasapositivecontrolataconcentrationof0.4␮g/mL for2h,andalltreatmentswereperformedintriplicate.

Fig.1.Percentagesofproliferation(logarithmicscale)inRT4andT24celllinesimmediately(A)andthreedays(B)aftertreatmentwithallylisothiocyanate(AITC).*_p<0.05

inrelationtotheAITCvehiclecontrol(2%Tween20%).Eachpointrepresentsthemeanvalueobtainedfromthreeindependentexperiments.

2.6. Cellcyclekineticsandapoptosisdetection

Forcellcyclekinetics,2×105 _{cellswereseededinto12-well}

plates;24hlater,thecellsweretreatedwithAITCat concentra-tionsof 0.005,0.0625, 0.0725 and 0.0825␮M for 3hand then washedwithHank’ssolution.Freshmediumwasadded,andthe cellswereincubatedagainat37◦Cfor21h.Afterwards,cellswere detachedusingtrypsin-EDTA,resuspendedintofreshmediumand centrifugedat1000rpmfor10min.Thepelletwasresuspended in200␮LofHFS(50␮g/mLpropidiumiodide,0.1%sodiumcitrate and0.1%TritonX-100),placedoniceandprotectedfromthelight foratleast 30min.Thepercentage ofcells intheG0/G1, Sand G2/MphasesweremeasuredusingGUAVACytosoftversion4.2.1 software.Thecellcycleanalyseswereperformedintriplicate.

Aquantitativeassessmentforapoptosiswasperformedusing aGuavaAnnexinreagent(MerckMillipore).AnnexinVwasused fordetectingtheexternalizationofphosphatidylserinetothecell surfaceand 7-AADas an indicatorofcell membrane structural integrity.Briefly, 2_×105 _{cellswere seededinto12-wellculture}

plates.After24h,thecellsweretreatedwithAITCatconcentrations of0.005,0.0625,0.0725and0.0825␮Mfor3h.Afterwards,cells werewashedwithHank’ssolution,andfreshmediumwasadded. UntreatedcellsandcellstreatedonlywithTween20werecultured ascontrols.Cellswerecollected21and45hafterincubation, resus-pendedinto100␮LGuavaNexinreagentfor20mininthedark andimmediatelyanalyzedintheGuavaeasyCyteflowcytometer (Millipore)usingtheGuavaSystemsoftware.Datafrom5000cells werecollectedineachdatafile.Cellularstatuswasdefinedas fol-lows:unstainedcellswereclassifiedas‘alive’;cellsstainedonlyby annexinVwereclassifiedas‘earlyapoptotic’;cellsstainedbyboth annexinVand7-AADwereclassifiedas‘lateapoptotic’;andcells stainedonlyby7-AADwereclassifiedas‘dead’.

2.7. TP53expression

TotalRNAfromthecultured T24and RT4cells wasisolated usingthe RNeasy MiniKit (Qiagen) accordingto the manufac-turer’s instructions. Reverse transcription wasperformed using 6␮Lofrandom hexamerprimers(10×), 6␮Lofreactionbuffer (10×),2.5␮LofdNTPs(25×)and3␮LofMultiScribe(50U/mL,High Capacity;AppliedBiosystems).Afterincubationat25◦Cfor10min and37◦Cfor2h,thecDNAwasstoredat4◦Candat−20◦_C.

Differ-entialexpressionofTP53inthetwocelllineswasassayedusing theTaqMansystem(Applied Biosystems;Foster City,CA,USA). Eachtubecontained2␮LofcDNAtemplate,5␮LofMasterMix TaqMan2×(AppliedBiosystems)and0.5␮Lof20×primers/probe (Assays-on-Demand gene expression products; Applied Biosys-tems).␤-Actinwasusedasahousekeepinggene.Thereactionwas performedusingthefollowingthermalcyclerconditions:94◦Cfor

10minfollowedby40cyclesat94◦Cfor30sand60◦Cfor1min. Fluorescencedatawerecollectedduringeachannealing/extension step.ThereactionswereperformedusinganAppliedBiosystems 7500FASTReal-TimePCRSystemandSDSsoftware,version1.2.3 (SequenceDetectionSystems1.2.3,7500Real-TimePCRSystems, AppliedBiosystems).ForeveryPCRsample,anegative(no tem-plate) control wasprocessed as a routine control.Assays were performedintriplicate.Relativegeneexpressiondatawere ana-lyzedusingthe2−CTmethod[29].

2.8. Statisticalanalysis

StatisticalanalyseswereperformedusingSASsoftware,v.9.2 (StatisticalAnalysisSystem,SASInstitute;Cary,NC,USA).Forthe cytotoxicity,cometandcellproliferationassays,datawere ana-lyzed using ANOVA and Tukey’s test; for the cell proliferation assay (nonparametric distribution—T24 cells), theanalysis was performedusingagammadistributionfollowedbyamultiple com-parisonstestusingtheDIFFoptionoftheGENMODprocedure;for clonogenicsurvivalandapoptosisandcellcycleanalyses,aPoisson distributionandafactorialanalysisbasedonabinomial distribu-tionwereperformed,respectively;forthemicronucleustest,data wereanalyzed usingthe Poisson(RT4 cells) and binomial(T24 cells)distributions.Geneexpressionvalueswereanalyzedby one-wayANOVAfollowedbyTukey’stest.Theresultswereconsidered statisticallysignificantwhenp<0.05.

3. Results

3.1. Cytotoxicity,cellproliferationandclonogenicsurvivalassays Thedatarevealedasignificantdecreaseofcellproliferationonly inRT4 cells3hafter treatmentwith0.25␮MAITC (Fig.1A).In contrast,significantdecreasesweredetectedforbothRT4(0.125 and0.25␮MAITC)andT24(0.25␮M)cellsthreedaysafter treat-ments(Fig.1B).Nocytotoxicitywasvisualized24aftertreatments ineithercellline(Fig.2).Theclonogenicsurvivalassayrevealed significantdecreasesincellcolonies(RT4andT24)afterAITC treat-mentatconcentrationsof0.0625,0.0725,0.0825and0.0925␮M (Fig.3).

3.2. Cometandmicronucleusassays

AllconcentrationsofAITCincreasedprimaryDNAdamagein T24cells;inRT4cells,increaseswereobservedforthethreehighest concentrations(0.0725,0.0825and0.0925␮M)(Table1).No signif-icantdifferencesweredetectedinthefrequencyofmicronucleus betweennegativecontrolandtreatedcells(Table2).

Table1

DNAdamage(tailintensity)inbladdercarcinomacell-lines(RT4andT24)treatedwithallylisothiocyanate(AITC).

Cellline Negativecontrola _ControlTweenb _{Positivecontrol}c _AITC(␮M)

0.005 0.0625 0.0725 0.0825 0.0925 RT4 14.462±1.024 12.055±1.229 80.361±6.127* _{18.085±3.118 19.871±2.018 24.923±6.330}* _{27.400±3.264}* _{30.472±3.820}*

T24 5.807±1.800 5.063±0.218 89.430b±3.906* _{14.807±1.900}* _{13.903±1.790}* _{15.767±1.825}* _{13.933±2.268}* _{15.557±0.659}*

a_Notreatment.

b_{Cellstreatedwith2%Tween20(vehiclecontrol).} c _{Cellstreatedwithmethylmethanesulfonate(0.006M).} *_{p<0.05comparedtotheTweencontrol.}

Table2

Frequenciesofmicronucleated(‰MNC)RT4andT24cellsaftertreatmentwithallylisothiocyanate(AITC). Cellline Negativecontrola

ControlTweenb _{Positivecontrol}c _AITC(␮M)

0.005 0.0625 0.0725 0.0825 0.0925

T24 0.08 0.05 0.38* _{0.16 0.10 0.16 0.10 0.10}

RT4 0 0 0.30* _{0 0 0 0 0.03}

a_Notreatment.

b_{Cellstreatedwith2%Tween20(vehiclecontrol).}

c _{Cellstreatedwithdoxorubicin(0.4␮g/mL).Thevaluesareexpressedin‰MNC(frequencyofmicronucleatedcells/1000cellsanalyzed).} *_{p<0.05comparedtotheTweencontrol.}

Fig.2.Percentagesofproliferation(logarithmicscale)inRT4andT24celllines24h aftertreatmentwithallylisothiocyanate(AITC).*_{p<0.05inrelationtotheAITC}

vehiclecontrol(Tween20%).Eachpointrepresentsthemeanvalueobtainedfrom threeindependentexperiments.

Fig.3. Percentagesofcellcolonies(logarithmicscale)10(T24cells)and15(RT4 cells)daysaftertreatmentwithallylisothiocyanate(AITC).p<0.05(*RT4;+T24). Eachpointrepresentsthemeanvalueobtainedfromthreeindependent experi-ments.

3.3. Cellcycleanalysis

SignificantdecreasesinthenumbersofRT4cellsintheSphase weredetectedafterAITCtreatmentat0.005,0.0625,0.0725and 0.0825␮M.Moreover,aslightincrease,butnotsignificant,inG1 phasecellswasalsoobserved.ForT24cells,significantdecreases (p<0.05) in the numbers of cells in the G1phase (0.0725 and 0.0825␮M)andtheSphase(0.0625and0.0825␮M)accompanied byanincreasednumberofcellsintheG2phaseweredetected, suggestingG2/Mcellcyclearrest(Table3).

3.4. Apoptosis

Increasednecrosis(0.0625,0.0725,0.0825and0.0925␮M)and earlyapoptosis(0.0625,0.0725,0.0825and0.0925␮M)rateswere observedinRT4cells48haftertreatmentwithAITC.ForT24cells, anincreasednecrosisratewasdetectedonly24haftertreatment withAITCat0.0625␮M(Table4).

Table3

Cellcyclekineticsinbladdercarcinomacelllines(RT4andT24)treatedwithallyl isothiocyanate(AITC). RT4 T24 G1(%) Control 40.90±8.84 48.07±1.25 Tween20 51.41±12.30 49.38±0.68 0.005␮M 58.58±6.91 42.29±3.89 0.0625␮M 59.00±3.02 42.34±5.95 0.0725␮M 40.57±3.09 30.47±4.47* 0.0825␮M 39.10±2.37 14.94±2.82* S(%) Control 22.75±17.80 18.22±1.23 Tween20 26.14±25.75 22.70±1.30 0.005␮M 8.75±1.72* _23.05±1.73 0.0625␮M 5.03±0.82* _13.80±1.44* 0.0725␮M 8.05±2.12* _16.89±3.58 0.0825␮M 6.24±1.50* _5.72±2.16* G2(%) Control 24.94±1.06 24.89±1.97 Tween20 20.88±7.48 21.14±4.60 0.005␮M 21.87±2.09 27.96±4.88 0.0625␮M 17.28±1.83 34.48±5.81* 0.0725␮M 21.89±7.54 39.44±13.49* 0.0825␮M 18.64±3.97 64.38±0.70*

PercentageofcellsintheG1,SandG2/Mmitoticphasesareexpressedasthe mean±standarddeviationfromtriplicate.

Table4

Earlyapoptosis,lateapoptosisandnecrosisrates(%)inRT4andT24cellstreated withallylisothiocyanate(AITC).

AITC RT4 T24 24h 48h 24h 48h Necrosis (%) Control 11±2 14±1 2±0 2±1 Tween 9±1 24±1 1±0 3±2 0.005␮M 5±1 24±4 4±1 2±0 0.0625␮M 11±1 11±3* _5±0* _2±0 0.0725␮M 11±2 6±0,5* _{4±1 2±0} 0.0825␮M 10±1 8±1* _{4±0 2±0} 0.0925␮M 7±1 6±1* _{4±2 2±2}

Lateapoptosis(%) Control 22±4 17±2 8±1 12±3 Tween 19±4 9±2 14±1 12±4 0.005␮M 7±1* _{7±2 15±2 15±1} 0.0625␮M 18±1 18±1* _29±1* _18±2 0.0725␮M 34±11* _35±2* _20±5* _26±1* 0.0825␮M 20±0 15±1* _{19±1 21±8}* 0.0925␮M 18±3 43±0* _23±2* _19±2*

Earlyapoptosis(%) Control 1±0 5±4 0±0 5±3 Tween 6±2 6±1 1±0 7±1 0.005␮M 3±1 7±3 1±0 8±2 0.0625␮M 3±0 10±2* _{1±0 9±1} 0.0725␮M 4±0 13±2* _{1±0 11±2} 0.0825␮M 2±0* _14±2* _{1±0 10±1} 0.0925␮M 3±0 10±3* _{1±0 12±2}

Percentageofcellsinnecrosis,lateapoptosisandearlyapoptosisareexpressedas mean±standarddeviationobtainedfromthreeindependentexperimentsusingthe AnnexinVassay(flowcytometry).

* _{p<0.05comparedtotheTweencontrol.}

3.5. TP53expression

The data showed no significant differences for TP53 mRNA expressionaftertreatmentwithAITCineithercellline(Fig.4).

4. Discussion

Epidemiologicalstudieshaveshownthatcruciferousvegetables mayactonlung,breast,prostate,pancreasandbladdercancers, mainlybecause of their highconcentrations of isothiocyanates (ITCs)[30].EachvegetablemayhaveonedifferentITC:forinstance, phenethyl isothiocyanate (PEITC), derived from gluconasturtiin hydrolysis,isfoundinwasabiandwatercress;benzyl isothiocy-anate(BITC), derived from glucotropaeolin hydrolysis, is found incabbage;andtheallylisothiocyanate(AITC),whichisderived fromsigrinahydrolysis,isfoundinmustardandhorseradish[31]. ITCs may act by inhibiting cytochrome P450 isoforms, which

Fig.4.RelativeTP53mRNAlevelsinRT4andT24celllinesaftertreatmentwith allylisothiocyanate(AITC).Therelativeexpressionvaluescorrespondtofold-change values.1_Notreatment;2_{cellstreatedwith2%Tween20(vehiclecontrol);␤-actin}

(endogenousRNAcontrol).

modulatephaseIIenzymesandpreventDNAadductscausedby somecarcinogens[30].Mi[32]hasobservedsignificantinhibition oftheproteasomeactivityinseveralcelllines,includingcervical (HeLa),lung(A549),colon(HT29)andbreastcancer(MCF-7)cells treatedwithBITCandPEITC.Additionally,thissameauthor demon-stratedthatthesecompoundscauserapidaccumulationofp53and NF-kB(IkB)inhibition,apoptosisandG2/Mphasearrest[32].In vitrogrowthinhibitionofUM-UC3humanbladdercarcinomacells wasdetectedaftertreatmentwithAITC[33].Furthermore,AITCis markedlylesstoxictonormalcellsthantocancercells,suggesting itsselectiveactivity[6,34].ThehighbioavailabilityofAITCinurine hasbeensuggestedasanimportantfeatureforbladdercancer ther-apy[11].Infact,someauthorshavedescribedhigherlevelsofAITC inurinarybladdertissuesthaninotherorgansafteradministration

[33,35].

Therefore, based onthe chemotherapeuticpotential of AITC and considering the fact that chemotherapy exposure induces importantadverseeffectssuchashighsystemictoxicity,lackof selectivity, and collaborate to resistance of these tumors after prolongedtreatment, we tested theeffectof this compound in wild-typeandmutatedTP53genecells.Thecellularresponsedue toTP53mutationsafterdifferenttreatmentsispoorlyunderstood, astheresponsedependsonacomplexsignalingcascade.Hofseth etal.[36]reportedthatamutantp53impairedtheDNAdamage response andrendered thetumorcells more resistantto drug-inducedapoptosis. Inthis way,it hasbeenalready shown that afunctionalTP53/p53pathwayimprovesgemcitabine cytotoxic-ity[37].Moreover,apreviousstudyfromourgroupdemonstrated higherapoptosisratesinwild-typecellscomparedtomutantcells fortheTP53geneaftercisplatinandgemcitabinetreatments[24].

Herein,weobservedthatAITCinduceddecreasedcell prolifer-ationthreedaysaftertreatmentatthehighestconcentrationsand nocytotoxicity24haftertreatmentinbothcelllines.Theseresults provideevidencethattheAITCconcentrationsusedledtotheloss ofreproductiveintegrity,i.e.,decreased cellproliferation capac-ity,mostlikelybecauseofsustainedlethaldamage.Incontrast, someauthorshavereportedcytotoxiceffectsofAITC(percentages ofviablecells)inhumanbrainmalignantgliomacells(GBM8401), thoughatconcentrations higherthat 1␮M[38].It isimportant torememberthattheclonogeniccellsurvivalassaydetermines theabilityofacelltoproliferateindefinitely,therebyretainingits reproductiveabilitytoformalargecolony.Thisassayiswidelyused toexaminetheeffectsofagentswithpotentialapplicationinthe clinic[39].Therefore,thelowsurvivalobservedinthisassaycould merelybethecontrolofthelysisratesforcellsthathadalready losttheirreproductivepotentialat24hand/orthreedays[40]). OurpresentstudyalsorevealedincreasedamountsofprimaryDNA damage(cometassay)inbothwild-typeandmutantcellsforthe TP53genetreatedwithAITC.Interestingly,inmutatedcells,even thetwolowestconcentrationsofAITCwereabletoinduce signifi-cantdamage.Therefore,itseemsthattheTP53backgroundmustbe consideredwithregardtothegenotoxicpotentialofAITC. Never-theless,althoughAITCwasabletoreachDNA,nomutageniceffect wasdetectedbythemicronucleusassay.Thesefindingssuggest thatAITC,althoughgenotoxic,isnotaneugenicorclastogenicin thetwocelllines.SimilarresultswerepreviouslyreportedbyLamy etal.[41]testingotherITCs.MTPITC,MTBITCandMTPeITCwere genotoxic,butnotmutagenic,inHepG2cells.Additionally,ithas beenreportedthatAITCisunabletoinduceeitherchromosome aberrationsorsisterchromatidexchanges,evenathighlycytotoxic doses[42].Theauthorssuggestedthat eithertheDNAwas effi-cientlyrepairedorfactorsthattriggerapoptosismightbeactive. Actually,severalmoleculareventsareinvolvedinDNArepair.

RegardingthetoxicogenomiceffectofAITC,ourresultsrevealed nochangeinTP53geneexpressionineithercellline.Recently,it wasdemonstratedthatPEITCmightdepletemutantp53without

causingchangesinp53mRNAexpression[43].Oneexplanationfor thisfindingwasapossiblemechanisminvolvingprotein modifica-tionviacovalentbindingtotheITCfunctionalgroup,makingthe mutantp53cellssignificantlymoresensitivetoPEITC-induced apo-ptosisthanthewild-typecells.Similarly,wesuggestthatAITCcan actattranslationaland/orpost-translationallevels,asweobserved increasedearlyapoptosisratesinthewild-typecellsbut notin mutantcellsfortheTP53gene,suggestingthatAITC-inducedDNA damage triggers apoptosis, most likely through p53 pathways. Kumaretal.[44]havediscussedthatdecreasedBCL-2andincreased BAXexpression,aswellasCAD(caspase-activatedDNase) activa-tionbycaspase-3,mightalsoexplainAITCinduced-apoptosis.

Whentheeffectof AITC onthecell cyclewasanalyzed, we detectedactivitiesdependentontheTP53status.Whileadecreased numberofcellsundergoingtheS-phasewasobservedforthe wild-typeTP53cells,asignificantdecreaseinthenumberofcellsatG1 andSphaseswasfoundforthemutantcells,inparallelwithan increasedpercentageofcellsatG2/Mphase(G2/Marrest).G2/M cellcyclearrestwasalsoobservedinhumangliomacellsafter treat-mentwithAITC [39].Moreover,AITCisabletobindtocysteine residuesand␣-and␤-tubulinsinabladdercancercellline, pro-motingtheirdegradationandubiquitinationandinducingcellcycle arrestinmitosis[12].Studiesusingcolonandprostatecancercell linestreatedwithsulforaphane(whichalsobelongstothe isothio-cyanatefamily)demonstratedaG2/Mcellcyclearrest,lossofBcl-2 geneexpressionandincreasedcaspaseactivity[45,46].

In conclusion, AITC caused cell cycle arrest and increased apoptosisrates andgenotoxicity dependentontheTP53status. However,wecannotruleoutthepossibilitythatthesedifferences couldreflectotherintrinsicgeneticalterationsintheexaminedcell lines,whichmayalsocarrymutationsingenesotherthanTP53. Therefore,furtherstudiesusingothermoleculartargetsneedtobe performedtobetterunderstandthemechanismsbywhichAITC mayexertitsantineoplasticpropertiesagainsttumorcells.

Authorcontributions

Alloftheauthorsreviewedthemanuscript.ALVSconductedall oftheexperiments,interpretedthedataandwrotethemanuscript. GNSsuggestedtheexperimentaldesign,conductedtheflow cytom-etryexperimentsandinterpretedthedata.EACperformedtheflow cytometryexperiments,micronucleustestandcometassay.DMFS wastheadvisor,contributedtotheexperimentaldesignanddata interpretationaswellascriticallyreadthemanuscript.

Funding

ThisstudywassupportedbyFAPESP(scholarship)andCNPq.

Conflictofintereststatement

Theauthorsdeclarethattheyhavenoconflictofinterests.

Acknowledgments

TheauthorsaregratefultoDr.JoséEduardoCorrenteandDr. JoãoPaulodeCastroMarcondesforthestatisticalanalyses.

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