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TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009 1359

Cannabinoid action induces autophagy-mediated cell death through stimulation

of ER stress in human glioma cellsMara Salazar,1,2 Arkaitz Carracedo,1 igo J. Salanueva,1 Sonia Hernndez-Tiedra,1 Mar Lorente,1,2

Ainara Egia,1 Patricia Vzquez,3 Cristina Blzquez,1,2 Sofa Torres,1 Stephane Garca,4 Jonathan Nowak,4 Gian Mara Fimia,5 Mauro Piacentini,5 Francesco Cecconi,6 Pier Paolo Pandolfi,7 Luis Gonzlez-Feria,8 Juan L. Iovanna,4 Manuel Guzmn,1,2 Patricia Boya,3 and Guillermo Velasco1,2

1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain. 2Centro de Investigacin Biomdica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

33D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biolgicas, Consejo Superior de Investigaciones Cientficas (CSIC), Madrid, Spain. 4INSERM U624, Campus de Luminy, Marseille, France.

5National Institute for Infectious Diseases, IRCCS L. Spallanzani, Rome, Italy. 6Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy. 7Cancer Genetics Program,

Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. 8Department of Neurosurgery, University Hospital, Tenerife, Spain.

Autophagycanpromotecellsurvivalorcelldeath,butthemolecularbasisunderlyingitsdualroleincancerremainsobscure.Herewedemonstratethat9-tetrahydrocannabinol(THC),themainactivecomponentofmarijuana,induceshumangliomacelldeaththroughstimulationofautophagy.OurdataindicatethatTHCinducedceramideaccumulationandeukaryotictranslationinitiationfactor2(eIF2)phosphorylationandtherebyactivatedanERstressresponsethatpromotedautophagyviatribbleshomolog3dependent(TRB3-dependent)inhibitionoftheAkt/mammaliantargetofrapamycincomplex1(mTORC1)axis.Wealsoshowedthatautophagyisupstreamofapoptosisincannabinoid-inducedhumanandmousecancercelldeathandthatactivationofthispathwaywasnecessaryfortheantitumoractionofcannabinoidsinvivo.ThesefindingsdescribeamechanismbywhichTHCcanpromotetheautophagicdeathofhumanandmousecancercellsandprovideevidencethatcannabinoidadministrationmaybeaneffectivetherapeuticstrategyfortargetinghumancancers.

IntroductionMacro-autophagy, hereafter referred to as autophagy, is ahighlyconservedcellularprocessinwhichcytoplasmicmaterialsincludingorganellesaresequesteredintodouble-membranevesiclescalledautophagosomesanddeliveredtolysosomesfordegradationorrecycling(1).Inmanycellularsettings,triggeringofautophagyreliesontheinhibitionofmammaliantargetofrapa-mycincomplex1(mTORC1),aneventthatpromotestheactiva-tion(de-inhibition)ofseveralautophagyproteins(Atgs)involvedintheinitialphaseofmembraneisolation(1).Enlargementofthiscomplextoformtheautophagosomerequirestheparticipationof2ubiquitin-likeconjugationsystems.Oneinvolvestheconjuga-tionofATG12toATG5andtheotherofphosphatidylethanol-aminetoLC3/ATG8(1).Thefinaloutcomeoftheactivationoftheautophagyprogramishighlydependentonthecellularcontextandthestrengthanddurationofthestress-inducingsignals(25).Thus,besidesitsroleincellularhomeostasis,autophagycanbeaformofprogrammedcelldeath,designatedtypeIIprogrammedcelldeath,orplayacytoprotectiverole,forexampleinsituations

ofnutrientstarvation(6).Accordingly,autophagyhasbeenpro-posedtoplayanimportantroleinbothtumorprogressionandpromotionofcancercelldeath(24),althoughthemolecularmechanismsresponsibleforthisdualactionofautophagyincan-cerhavenotbeenelucidated.

9-Tetrahydrocannabinol(THC),themainactivecomponentofmarijuana(7),exertsawidevarietyofbiologicaleffectsbymim-ickingendogenoussubstancestheendocannabinoidsthatbindtoandactivatespecificcannabinoidreceptors(8).Oneofthemostexcitingareasofresearchinthecannabinoidfieldisthestudyofthepotentialapplicationofcannabinoidsasantitumoralagents(9).Cannabinoidadministrationhasbeenfoundtocurbthegrowthofseveraltypesoftumorxenograftsinratsandmice(9,10).Basedonthispreclinicalevidence,apilotclinicaltrialhasbeenrecentlyruntoinvestigatetheantitumoralactionofTHConrecurrentgliomas(11).Recentfindingshavealsoshownthatthepro-apoptoticandtumorgrowthinhibitingactivityofcannabi-noidsreliesontheupregulationofthetranscriptionalco-activa-torp8(12)anditstargetthepseudo-kinasetribbleshomolog3(TRB3)(13).However,themechanismsthatpromotetheactiva-tionofthissignalingrouteaswellasthetargetsdownstreamofTRB3thatmediateitstumorcellkillingactionremainelusive.InthisstudywefoundthatERstressevokedupregulationofthep8/TRB3pathwayinducedautophagyviainhibitionoftheAkt/mTORC1axisandthatactivationofautophagypromotedtheapoptoticdeathoftumorcells.Theuncoveringofthispathway,

Conflictofinterest:Theauthorshavedeclaredthatnoconflictofinterestexists.

Nonstandardabbreviationsused:Atg,autophagyprotein;eIF2,eukaryotictranslationinitiationfactor2;MEF,mouseembryonicfibroblast;THC,9-tetrahy-drocannabinol;mTORC1,mammaliantargetofrapamycincomplex1;PDI,proteindisulphideisomerase;TRB3,tribbleshomolog3.

Citationforthisarticle:J. Clin. Invest.119:13591372(2009).doi:10.1172/JCI37948.

Downloaded from http://www.jci.org on March 12, 2015. http://dx.doi.org/10.1172/JCI37948

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1360 TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009


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whichwebelieveisnovel,forpromotingtumorcelldeathmayhavetherapeuticimplicationsinthetreatmentofcancer.

ResultsAutophagy mediates THC-induced cancer cell death.Asafirstapproachtogaininsightintothemorphologicalchangesinducedincan-cercellsbycannabinoidadministration,weperformedelectronmicroscopyanalysisofU87MGhumanastrocytomacells.Inter-estingly,doublemembranevacuolarstructureswiththemorpho-logicalfeaturesofautophagosomeswereobservedinTHC-treatedcells(Figure1,AC).TheconversionofthesolubleformofLC3(LC3-I) to the lipidatedandautophagosome-associated form(LC3-II)isconsideredoneofthehallmarksofautophagy(1),andthusweobservedtheoccurrenceofLC3-positivedotsaswellastheappearanceofLC3-II(Figure1D)incannabinoid-challengedcells.Inaddition,co-incubationwiththelysosomalproteaseinhibitorsE64dandpepstatinA,whichblocksthelaststepsofautophagicdegradation(14),enhancedTHC-inducedaccumulationofLC3-II(Figure 1E), confirming that cannabinoids induce dynamicautophagyinU87MGcells.Furthermore,incubationwiththecan-nabinoidreceptor1(CB1)antagonistSR141716preventedTHC-inducedLC3lipidationandformationofLC3dots(Figure1D),indicatingthatinductionofautophagybycannabinoidsreliesonCB1receptoractivation.Sinceautophagyhasbeenimplicatedinpromotionandinhi-

bitionofcellsurvival,wenextinvestigateditsparticipationinthecancercelldeathinducingactionofTHC.Pharmacologicalinhibitionofautophagyatdifferentlevels(SupplementalFigure1,AC;supplementalmaterialavailableonlinewiththisarticle;doi:10.1172/JCI37948DS1)orselectiveknockdownofATG1(anessentialproteinintheinitiationofautophagy;ref.1)(Figure1,FandG),ATG5(anessentialproteinintheformationoftheautophagosome;ref.1)(SupplementalFigure1,DF),orAMBRA1(arecentlyidentifiedbeclin-1interactingproteinthatregulates

autophagy;ref.15)(SupplementalFigure1,DF)stronglyreducedcannabinoid-inducedautophagyandcelldeath.Moreover,trans-formedAtg5-deficientmouseembryonicfibroblasts(MEFs),whicharedefectiveinautophagy(16),weremoreresistantthantheirwild-typecounterpartstoTHC-inducedcelldeath(Figure1H)anddidnotundergoautophagyuponcannabinoidtreatment(Figure1I).Takentogether,thesefindingsdemonstratethatautophagyplaysaprominentroleinTHC-inducedcancercelldeath.

THC induces autophagy via ER stressdependent upregulation of p8 and TRB3.Inadditiontothepresenceofautophagosomes,electronmicroscopyanalysisofcannabinoid-treatedcellsrevealedthepres-enceofnumerouscellswithdilatedER(Figure2A).Inlinewiththisobservation,immunostainingoftheERluminalmarkerpro-teindisulphideisomerase(PDI)showedastrikingdilationintheERofTHC-treatedU87MGcells(Figure2B),aneventthatwasassociatedwithanincreasedphosphorylationofthesubunitofeukaryotictranslationinitiationfactor2(eIF2),ahallmarkoftheERstressresponse(17)(Figure2C).Inaddition,THC-inducedERdilationandeIF2phosphorylationwerepreventedbypharmaco-logicalblockadeoftheCB1receptor(Figure2,BandC).Time-courseanalysisofPDIandLC3immunostaining,eIF2

phosphorylation,andLC3lipidationofcannabinoid-treatedcellsrevealedthatERstressoccurredearlierthanautophagy(Figure2,DandE).Ofinterest,cannabinoidadministrationproducedsimi-laractivationofERstressandautophagy,aswellascelldeath,inotherhumanastrocytomacelllines(SupplementalFigure2,AF),aprimarycultureofhumangliomacells(SupplementalFigure2,GI),andseveralhumancancercelllinesofdifferentorigin,includingpancreaticcancer(SupplementalFigure2,JL),breastcancer,andhepatoma(datanotshown).However,neitherERdila-tionnoreIF2 phosphorylationorautophagywasevidentinnor-mal,nontransformedprimaryastrocytes(SupplementalFigure3),whichareresistanttocannabinoid-inducedcelldeath(13).WenextinvestigatedwhetheractivationofERstressisinvolved

intheinductionofautophagyinresponsetocannabinoidtreat-mentofcancercells.WehavepreviouslyshownthatTHC-inducedaccumulationofdenovosynthesizedceramide,aneventthatoccursintheER(18),leadstoupregulationofthestress-regulatedproteinp8anditsERstressrelateddownstreamtargets,ATF4,CHOP,andTRB3,toinducecancercelldeath(13).Ofimportance,incubationwithISP-1(aselectiveinhibitorofserinepalmitoyl-transferase,theenzymethatcatalyzesthefirststepofsphingo-lipidbiosynthesis;ref.18)preventedceramideaccumulation(Sup-plementalFigure4A);THC-inducedERdilation(SupplementalFigure4B);eIF2phosphorylation(Figure3A);p8,ATF4,CHOP,andTRB3upregulation(SupplementalFigure4C);andautophagy(Figure3B),supportingthatceramideaccumulationisinvolvedincannabinoid-triggeredERstressandautophagy.WealsoverifiedbymeansofRNAinterferencethatCaCMKKwhichhadbeenpreviouslyimplicatedinactivatingautophagyinresponsetoERstressassociatedcalciumrelease(19)wasnotinvolvedinTHC-inducedautophagyandcelldeath(datanotshown).Asphosphor-ylationofeIF2 onSer51attenuatesgeneralproteinsynthesiswhileenhancingtheexpressionofseveralERstressresponsegenes(17),weusedcellsderivedfromeIF2S51AknockinmicetotestwhethereIF2phosphorylationregulatestheexpressionofp8anditsdownstreamtargets.Inagreementwiththishypothesis,THCtreatment(whichpromotedceramideaccumulationinbothwild-typeandeIF2S51AimmortalizedMEFs;SupplementalFigure5A)triggeredp8,ATF4,CHOP,andTRB3upregulation(Figure

Figure 1Inhibition of autophagy prevents THC-induced cancer cell death. (AC) Effect of THC on U87MG cell morphology. Representative elec-tron microscopy photomicrographs are shown (6 h). Scale bars: 500 nm. Note the presence of early (A, open arrows, and B) and late (A, filled arrows, and C) autophagosomes in THC-treated but not vehicle-treated (veh-treated) cells. (D) Top: Effect of SR141716 (SR1; 1 M) and THC on LC3 immunostaining (green) in U87MG cells (18 h; n = 3). The percentage of cells with LC3 dots relative to the total cell number is shown in the corner of each panel (mean SD). Scale bar: 20 m. Bottom: Effect of SR1 and THC on LC3 lipidation in U87MG cells (18 h; n = 3). (E) Effect of E64d (10 M) and pepstatin A (PA; 10 g/ml) on THC-induced LC3 lipidation in U87MG cells (18 h; n = 3). (F and G) Effect of THC treatment and transfection with control siRNAs (siC) or ATG1-selective siRNAs (siATG1) on cell viability (F; mean SD; n = 3), LC3 immunostaining (G, left panels; 18 h; percentage of cells with LC3 dots relative to the total number of cells cotransfected with a red fluo-rescent control siRNA, mean SD; n = 3; scale bar: 20 m), and LC3 lipidation (G, right panel; 18 h; n = 3) in U87MG cells. (H and I) Effect of THC on cell viability (H; mean SD; n = 3), LC3 immunostaining (I, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 3; scale bar: 20 m), and LC3 lipidation (I, right panel; 18 h; n = 3) in Atg5+/+ and Atg5/ RasV12/T-large antigen MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (D) and Atg5+/+ (H and I) cells and compared with siC-transfected, THC-treated U87MG cells (F and G). THC concentration was 6 M.


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3C)aswellasautophagy(SupplementalFigure5B)inwild-typecellsbutnotintheireIF2S51Acounterparts.Wesubsequentlyaskedwhetherp8anditsdownstreamtar-

getsregulateautophagy.Knockdownofp8orTRB3preventedTHC-inducedautophagy(Figure3,DandE)butnotERdilation(SupplementalFigure4D)inU87MGcells.Furthermore,THCinducedautophagy inp8+/+butnotp8-deficienttransformedMEFs(Figure3FandSupplementalFigure5C).Altogether,thesefindingsrevealthatTHCinducesautophagyofcancercellsvia

activationofanERstresstriggeredsignalingroutethatinvolvesstimulationofceramidesynthesisdenovo,eIF2phosphoryla-tion,andp8andTRB3upregulation.

THC inhibits Akt and mTORC1 via TRB3.InhibitionofmTORC1isconsideredakeystepintheearlytriggeringofautophagy(6).Wethereforetestedwhethercannabinoid-inducedupregulationofthep8pathwayleadstoautophagyviainhibitionofthiscom-plex.THCtreatmentofU87MGcellsreducedthephosphorylationofp70S6kinase(awell-establishedmTORC1substrate)andthe

Figure 2ER stress precedes autophagy in cannabinoid action. (A) Effect of THC on U87MG cell morphology. Note the presence of the dilated ER in THC- but not vehicle-treated cells (6 h). Arrows point to the ER. Scale bars: 500 nm. (B) Effect of SR1 (1 M) and THC on PDI immunostaining (red) in U87MG cells (8 h; n = 3). The percentage of cells with PDI dots relative to the total cell number is shown in the corner of each panel (mean SD). Scale bar: 20 m. (C) Effect of SR1 (1 M) on THC-induced eIF2 phosphorylation of U87MG cells (3 h; OD relative to vehicle-treated cells, mean SD; n = 3). (D) Effect of THC on PDI (red) and LC3 (green) immunostaining in U87MG cells (n = 3). The percentage of cells with PDI or LC3 dots relative to total cell number at each time point (mean SD) is shown. Scale bar: 20 m. (E) Effect of THC on eIF2 phosphorylation and LC3 lipidation in U87MG cells (n = 3). **P < 0.01 compared with THC-treated (B) or vehicle-treated (C and D) cells.


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Figure 3THC induces autophagy via ER stressevoked p8 and TRB3 upregulation. (A and B) Effect of ISP-1 (1 M) on THC-induced eIF2 phosphoryla-tion (A; 3 h; n = 3) and LC3 immunostaining (B, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 3; scale bar: 20 m) in U87MG cells. sip8, p8-selective siRNA; siTRB3, TRB3-selective siRNA. (C) Effect of THC on p8, ATF4, CHOP, and TRB3 mRNA levels of eIF2 WT and eIF2 S51A MEFs as determined by real-time quantitative PCR (8 h; n = 3). Numbers indicate the mean fold increase SD relative to vehicle-treated eIF2 WT MEFs. (D) Top: Analysis of p8 and TRB3 mRNA levels. Results from a representative RT-PCR experiment are shown. The numbers indicate gene expression levels as determined by real-time quantitative PCR (mean fold change SD relative to siC-transfected cells; n = 5). Bottom: Effect of THC on LC3 immunostaining (green) of U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 4). The percentage of cells with LC3 dots relative to cells cotransfected with a red fluorescent control siRNA is shown in each panel (mean SD). Scale bar: 20 m. (E) Effect of THC on LC3 lipidation in U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 6). (F) Effect of THC on LC3 lipidation (top; 18 h; n = 5) and LC3 immunostaining (bottom; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 4; scale bar: 40 m) in p8+/+ or p8/ MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (B), eIF2 WT (C), or p8+/+ (F) cells and compared with siC-transfected, THC-treated U87MG cells (D).


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Figure 4THC inhibits the Akt/mTORC1 pathway via TRB3. (A) Effect of THC on p70S6K and S6 phosphorylation of U87MG cells (n = 6). (B) Effect of THC on cell viability (left panel; 24 h; mean SD; n = 6) and LC3 lipidation (right panel; 18 h; n = 4) in Tsc2+/+ and Tsc2/ MEFs. (C) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation of U87MG cells (18 h; OD relative to vehicle-treated cells, mean SD; n = 7). (D) Effect of THC on cell viability (left panel; 24 h; mean SD; n = 4) and LC3 lipidation (right panel; 18 h; n = 4) of pBABE and myristoylated Akt (myr-Akt) MEFs. (E) Effect of THC on Akt co-immunoprecipitation with TRB3 in U87MG cell extracts (8 h; OD relative to vehicle-treated cells, mean SD; n = 9; input: TRB3). (F and G) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation and LC3 lipidation (G only) of siC- and siTRB3-transfected (F; 18 h; OD relative to vehicle-treated siC-transfected U87MG cells, mean SD; n = 7; upper panel shows an analysis of TRB3 mRNA levels) and EGFP (Ad-EGFP) or rat TRB3 (Ad-TRB3) adenoviral vectorinfected (G; 18 h; OD relative to vehicle-treated Ad-EGFPinfected U87MG cells, mean SD; n = 4; upper panel shows an analysis of rTRB3 mRNA levels) U87MG cells. (H) Effect of THC on Akt, p70S6K, and S6 phosphorylation of p8+/+ and p8/ MEFs (n = 7). *P < 0.05 and **P < 0.01 compared with THC-treated Tsc2+/+ (B) and pBABE (D) MEFs and compared with vehicle-treated (C and E), vehicle-treated siC-transfected (F), or Ad-EGFPinfected (G) U87MG cells.


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ribosomalproteinS6(awell-establishedp70S6kinasesubstrate)(Figure4,AandC),indicatingthatmTORC1isinhibitedincan-nabinoid-challengedcells.Inaddition,thecannabinoid-induceddecreaseinp70S6kinaseandS6phosphorylation,autophagy,andcelldeathwerenotevidentinTsc2/cells,inwhichmTORC1isconstitutivelyactive(20)(Figure4BandSupplementalFigure6,AandB),furthersupportingamajorroleformTORC1inhibitionintheinductionofautophagiccelldeathbycannabinoids.TheproteinkinaseAktpositivelyregulatestheactivityofthe

mTORC1complexbyphosphorylatingandinhibitingTSC2andPRAS40(awell-establishedAktsubstratewithinthemTORC1complex).Thus,AktinhibitiondecreasesmTORC1activityandpromotesautophagy(20).Inlinewiththisidea,THCdecreasedthephosphorylationofAkt,TSC2,andPRAS40aswellasp70S6kinaseandS6(Figure4C).ThisinhibitionoftheAkt/mTORC1pathwaywasabrogatedbyincubationwithaCB1receptorantago-nist(SupplementalFigure6C)oraceramidesynthesisinhibitor(SupplementalFigure6D).Likewise,cellsoverexpressingamyris-toylated(constitutivelyactive)formofAktwereresistanttoTHC-inducedmTORC1inhibition,autophagy,andcelldeath(Figure4DandSupplementalFigure6,EandF),furthersupportingthatTHCinducesautophagyviaAktinhibition.SinceTRB3hasbeenshowntodirectlyinteractwithandinhibit

Akt(21,22),weinvestigatedwhetherupregulationofTRB3wasresponsible forTHC-inducedAkt/mTORC1 inhibition. Sev-eralobservationssupportthatthisisindeedthecase:(a)THCincreasedtheamountofAktcoimmunoprecipitatedwithTRB3fromU87MGextracts(Figure4E),(b)knockdownofTRB3pre-ventedtheeffectofTHConAkt,TSC2,PRAS-40,p70S6kinase,andS6phosphorylation(Figure4F),and(c)TRB3overexpressiondecreasedAkt,TSC2,PRAS40,p70S6kinase,andS6phosphoryla-tion,enhancedtheinhibitoryeffectofTHConthephosphoryla-tionoftheseproteins,andpromotedautophagy(Figure4G).Inlinewiththeseobservations,THCfailedtoinhibitAkt,p70S6kinase,andS6phosphorylationofeIF2S51Aknockinorp8-deficientMEFs,inwhichTRB3didnotbecomeupregulateduponcannabinoidtreatment(Figure4HandSupplementalFigure6,GandH).Altogether,thesedatademonstratethatupregulationofp8andTRB3induceautophagyoftumorcellsviainhibitionoftheAkt/mTORC1pathway.

THC-induced autophagy promotes the apoptotic death of cancer cells.While analyzing themechanismof cannabinoid cell-killingaction,weobservedthatincubationwiththepan-caspaseinhibi-torZVAD-fmkpreventedcelldeathtothesameextentasgenetic(Figure5A)orpharmacological(SupplementalFigure7)inhibi-tionofautophagy.Furthermore,Bax/Bakdoubleknockout(DKO)immortalizedMEFs,whichareprotectedagainstmitochondrialapoptosis(23),wereresistanttoTHC-inducedcelldeathandapoptosis(Figure5B)butunderwenteIF2phosphorylationandautophagy(Figure5C)uponTHCtreatment.Wethereforeinvestigatedwhethercannabinoid-inducedautophagypromotedtheapoptoticdeathofcancercells.Time-courseanalysisofLC3andactivecaspase-3immunostaininginU87MGcellsrevealedthatautophagyprecededtheappearanceofapoptoticfeaturesinTHC-treatedcells(Figure5D).Inaddition,selectiveknockdownofATG1(Figure5D)aswellasofAMBRA1orATG5(Supplemen-talFigure8)preventedTHC-inducedcaspase-3activation.More-over,unliketheirwild-typecounterparts,Atg5-deficientimmor-talizedMEFsdidnotundergophosphatidylserinetranslocationtotheouterleafletoftheplasmamembrane(Figure5E),loss

ofmitochondrialmembranepotential(Figure5F),orincreasedproductionofreactiveoxygenspecies(SupplementalFigure9)inresponsetocannabinoidtreatment.Thesefindingsindicatethatactivationoftheautophagy-mediatedcelldeathpathwayoccursupstreamofapoptosisincannabinoidantitumoralaction.

Activation of autophagy is necessary for cannabinoid antitumoral action in vivo.Todeterminetheinvivorelevanceofourfindings,wefirstinvestigatedwhetherTHCpromotestheactivationoftheabove-describedautophagy-mediatedcelldeathpathwayinU87MGcellderivedtumorxenografts,inwhichwehaverecentlyshownthatcannabinoidtreatmentreducestumorgrowth(specifically,THCadministrationfor14daysdecreasedtumorgrowthby50%;ref.13).Analysisofthesetumorsrevealedthatcannabinoidadminis-trationincreasesTRB3expressionanddecreasesS6phosphoryla-tion(Figure6A).Likewise,formationofLC3dotsaswellasincreaseinLC3-IIandactivecaspase-3immunostainingwereobservedinTHC-treated,butnotvehicle-treated,tumors(Figure6B).Tofurther investigatewhetheractivationofthep8pathway

mediatescannabinoidantitumoralaction,wealsoanalyzedtumorsderivedfromp8+/+andp8/RasV12/E1A-transformedMEFs(inthiscase,THCadministrationfor8daysdecreasedby45%thegrowthofp8+/+tumorsbuthadnosignificanteffectonp8/tumors;ref.13).THCtreatmentincreasedTRB3expression,decreasedS6phos-phorylation,andincreasedautophagyaswellasTUNELandactivecaspase-3immunostaininginp8+/+butnotp8/tumors(Figure6CandSupplementalFigure10).Moreover,THCtreatmentenhancedthenumberofcellswithLC3dotsandTUNEL-positivenucleiinp8+/+butnotinp8/tumors(Figure6C).Inordertoverifytheimportanceofautophagyforcannabinoid

antitumoralaction,wenextgeneratedtumorswithAtg5+/+andAtg5/RasV12/T-largeantigentransformedMEFs.THCadminis-trationreducedbymorethan80%thegrowthoftumorsderivedfromwild-typecellsbuthadnosignificanteffectonthosetumorsgeneratedbyautophagy-deficientcells(Figure7A).Furthermore,cannabinoidadministrationincreasedautophagy,TUNEL(Fig-ure7B),andactivecaspase-3immunostaining(SupplementalFig-ure11)inAtg5+/+butnotAtg5/tumors.Likewise,cannabinoidadministrationincreasedthenumberofcellswithLC3dotsandTUNEL-positivenucleiinAtg5+/+butnotAtg5/tumors(Figure7B).Takentogether,thesefindingsdemonstratethatactivationoftheautophagy-mediatedcelldeathpathwayisindispensableforcannabinoidantitumoralaction.Finally,weanalyzedthetumorsof2patientsenrolledinaclinical

trialaimedatinvestigatingtheeffectofTHConrecurrentglioblas-tomamultiforme.ThepatientsweresubjectedtointracranialTHCadministration,andbiopsiesweretakenbeforeandafterthetreat-ment(11).Inthe2patients,cannabinoidinoculationincreasedTRB3immunostaininganddecreasedS6phosphorylation(Figure8A).Interestingly,thenumberofcellswithautophagicphenotype(Figure8B)aswellaswithactivecaspase-3immunostaining(Fig-ure8C)wasincreasedinthetumorsamplesobtainedafterTHCtreatment.Althoughthesestudieswereonlyconductedinspeci-mensfrom2patients,theyareinlinewiththepreclinicalevidenceshownaboveandsuggestthatcannabinoidadministrationmightalsotriggerautophagy-mediatedcelldeathinhumantumors.

DiscussionInthisstudyweshowthatcannabinoids,anewfamilyofpotentialantitumoralagents,induceautophagyofcancercellsandthatthisprocessmediatesthecelldeathpromotingactivityofthesecom-


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Figure 5Autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death. (A) Effect of THC and the pan-caspase inhibitor ZVAD (10 M) on the viability of Atg5+/+ and Atg5/ MEFs (36 h; percentage of viable cells relative to the corresponding Atg5+/+ vehicle-treated cells, mean SD; n = 3). (B) Effect of THC on the apoptosis of Bax/Bak WT and Bax/Bak DKO MEFs as determined by cytofluorometric analysis of Annexin V/propidium iodide (PI) (24 h; mean SD; n = 3). The mean SD percentage of Annexin Vpositive/PI-positive and Annexin Vpositive, PI-nega-tive cells is shown in the upper and lower corners, respectively. (C) Effect of THC on eIF2 phosphorylation (3 h; n = 3) and LC3 lipidation (18 h; n = 4) of Bax/Bak WT and DKO MEFs. (D) Left: Effect of THC on autophagy and apoptosis of U87MG cells transfected with siC or siATG1. Green bars, cells with LC3 dots; red bars, active caspase-3positive cells; white bars, cells with both LC3 dots and active caspase-3 staining. Data correspond to the percentage of cells with LC3 dots (green bars), active caspase-3positive cells (red bars), and cells with LC3 dots and active caspse-3 staining (white bars) relative to the total number of transfected cells at each time point (mean SD; n = 3). Right: Representative photomicrographs (36 h; scale bar: 20 m). (E and F) Effect of THC on apoptosis (E; 24 h; n = 3) and loss of mitochondrial membrane potential as determined by DiOC6(3) staining (F; 24 h; n = 4) of Atg5+/+ and Atg5/ MEFs. In E, the mean SD percentage of Annexin Vpositive/PI-positive and Annexin Vpositive, PI-negative cells is shown in the upper and lower corners, respectively. **P < 0.01 compared with THC-treated Atg5+/+ (A, E, and F) and Bax/Bak WT (B) MEFs and from THC-treated, siC-transfected cells (D).


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Figure 6THC activates the autophagic cell death pathway in vivo. (A) Effect of peritumoral THC administration on TRB3 and p-S6 immunostaining in U87MG tumors. TRB3- or p-S6stained area normalized to the total number of nuclei in each section; numbers indicate the mean fold change SD; 18 sections were counted for each of 3 dissected tumors for each condi-tion. Scale bar: 50 m. (B) Left: Effect of peritumoral THC administration on LC3 and active caspase-3 immunostaining in U87MG tumors. Arrows point to cells with LC3 dots. The numbers indicate the percentage of active caspase-3posi-tive cells relative to the total number of nuclei in each sec-tion SD. Ten sections were counted for each of 3 dissected tumors for each condition. Scale bars: 20 m. Right: Effect of peritumoral THC administration on LC3 lipidation in U87MG tumors. Representative samples from 1 vehicle-treated and 1 THC-treated tumor are shown. Numbers indicate the LC3-I and LC3-II OD values relative to vehicle-treated tumors (mean SD). n = 3. (C) Left: Effect of THC administration on LC3 immunostaining (green) and TUNEL (red) in RasV12/E1A p8+/+ and p8/ tumor xenografts. Arrows point to cells with LC3 dots and TUNEL-positive nuclei. Right: Bar graph shows the percentage of TUNEL-positive nuclei or cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean SD). Eighteen sections were counted from 3 dissected tumors for each condition. Scale bars: 50 m. Inset shows the magnification of 1 selected cell (arrows point to LC3 dots; scale bar: 10 m). *P < 0.05 and **P < 0.01 compared with vehicle-treated tumors.


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pounds.Severalobservationsstronglysupportthisidea:(a)THCinducedautophagyandcelldeathindifferenttypesofcancercellsbutnotinnontransformedastrocytes,whichareresistanttocan-nabinoidkillingaction,(b)pharmacologicalorgeneticinhibitionofautophagypreventedTHC-inducedcelldeath,(c)autophagy-deficienttumorswereresistanttoTHCgrowth-inhibitingaction,and(d)THCadministrationactivatedtheautophagiccelldeathpathwayin3differentmodelsoftumorxenograftsaswellasin2humantumorsamples.Dependingonthecellularcontextandthestrengthandduration

ofthetriggeringstimulus,autophagyisinvolvedinthepromotionorinhibitionofcancercellsurvival(4,5,24,25).However,themolec-ularbasesofthisdualroleofautophagyincancerremainunknown.

Datapresentedheredemonstratethatinductionofautophagybycannabinoidsleadstocancercelldeathandidentifythesignalingrouteresponsiblefortheactivationofthiscellularprocess.Thus,ourfindingssuggestthatTHCviaactivationoftheCB1recep-torandstimulationofceramidesynthesisdenovoactivatesanearlyERstressresponsethatleadstoincreasedphosphorylationofeIF2onSer51.ExperimentsperformedwitheIF2S51Amutantcellshaveshownthatphosphorylationofthisresidue,whichisknowntoattenuategeneralproteintranslationwhileenhancingtheexpressionofseveralgenesrelatedwiththeERstressresponse(17),isrequiredfortheupregulationofthestressproteinp8anditsERstressrelateddownstreamtargetsATF4,CHOP,andTRB3aswellasfortheinductionofautophagybycannabinoids.Furthermore,

Figure 7Autophagy is essential for cannabinoid antitumoral action. (A) Effect of peritumoral THC administration on the growth of Atg5+/+ (upper panel) and Atg5/ (lower panel) RasV12/T-large antigen MEF tumor xenografts generated in nude mice (mean SD; n = 7 for each condition). Photo-graphs show representative images of vehicle- and THC-treated tumors. (B) Left: Effect of THC administration on LC3 immunostaining (green) and apoptosis as determined by TUNEL (red) in Atg5+/+ and Atg5/ MEF tumor xenografts. Representative images from 1 vehicle-treated and 1 THC-treated Atg5+/+ and Atg5/ tumors are shown. Right: Bar graphs show the percentage of TUNEL-positive nuclei and cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean SD). Eighteen sections were counted from 3 dissected tumors for each condition (vehicle-treated and THC-treated). Scale bar: 50 m. (C) Schematic of the proposed mechanism of THC-induced cell death (see text for details). **P < 0.01 compared with vehicle-treated tumors.


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wedemonstratethattheupregulationofp8andTRB3,whichhasbeenpreviouslyimplicatedincannabinoid-evokedcelldeath(13),isacrucialeventinthetriggeringofautophagy.Ceramideaccumula-tionhasbeenproposedtoinduceERstress(26,27)andautoph-

agy(28),andeIF2phosphorylationhasbeenimplicatedintheinductionofautophagyinresponsetodifferentsituations(2931).However,themolecularmechanismsresponsiblefortheseactionshavenotbeenclarified.Findingspresentedherenowsuggestthat

Figure 8THC administration promotes autophagy in glioblastomas of 2 patients. Analysis of different parameters in 2 patients with glioblastoma mul-tiforme before and after intracranial THC treatment (it was estimated that doses of 610 M were reached at the site of administration). (A) TRB3 and p-S6 immunostaining. Representative photomicrographs are shown. Numbers indicate the TRB3- or p-S6stained area normalized to the total number of nuclei in each section (mean fold change SD) relative to the corresponding pre-treatment sample. Fifteen sections were counted for each tumor and each condition (before and after treatment). Scale bar: 50 m. (B) Representative photomicrographs of LC3 diamino-benzidine immunostaining. The mean percentage of cells with LC3 dots SD relative to the total number of nuclei in each section is noted in the corner of each panel. Ten sections were counted from each biopsy for each condition. Arrows point to cells with LC3 dots. Scale bar: 20 m. (C) Representative photomicrographs of active caspase-3 diaminobenzidine immunostaining. Numbers indicate the percentage of cells with active caspase-3 staining SD relative to the total number of nuclei in each section. Ten sections were counted from each biopsy for each condition. Arrows point to cells with active caspase-3 staining. Scale bar: 20 m. *P < 0.05 and **P < 0.01 compared with before treatment.


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upregulationofthep8-TRB3pathwayconstitutesamechanismbywhichdenovosynthesizedceramideandeIF2phosphorylationpromoteautophagy,thusidentifyingwhatwebelieveisanovelcon-nectionbetweenERstressandautophagy.Ourdataalsodemonstratethattheautophagy-promotingactiv-

ityofthep8-regulatedpathwayisbasedonitsabilitytoinhibittheAkt/mTORC1axis.RegulationofmTORC1largelyreliesontheactivityoftheprosurvivalkinaseAkt,whoseinhibitionleadstomTORC1inactivationand,inturn,toautophagy(20).Ourfind-ingsrevealthatTHCupregulatesTRB3,promotingitsinteractionwithAktandleadingtodecreasedphosphorylationofthiskinaseaswellasofitsdirectsubstratesTSC2andPRAS40,whichtrig-gersmTORC1inhibitionandinductionofautophagy.TRB3hasbeenpreviouslyshowntoinhibitAkt(21,22),althoughtheprecisecontributionofthispseudo-kinasetotheregulationofAktactiv-ityindifferentcellularcontextsisunclear(32).Herewedemon-stratethatTRB3inhibitionoftheAkt/mTORC1axisisessentialforcannabinoid-inducedautophagyofcancercells.Moreover,weshowthatthispathwayisessentialforcannabinoidantitumoralaction.Thus,THCadministrationleadstoTRB3upregulation,mTORC1inhibition,inductionofautophagy,andreductionoftumorgrowthindifferentmodelsoftumorxenografts,butnotinp8-deficienttumorsthataredefectiveintheupregulationofthep8/TRB3pathway.Furthermore,activationofthispathwaywasalsoevidentin2gliomapatientsthathadbeentreatedwithTHC.TheseresultsthusuncoveraroleforTRB3thatmaybeofgreatimportanceintheregulationofcancercelldeath.Autophagyhasbeenproposedtoprotectfromapoptosis,act

asanapoptosis-alternativepathwaytoinducecelldeath,oracttogetherwithapoptosisasacombinedmechanismforcelldeath(6,33).However,verylittleisknownabouttheroleoftheinterplaybetweenthese2cellularprocessesinthecontroloftumorgrowthinresponsetoanticanceragents.Ourresultsnowclearlydemon-stratethatinductionofautophagyisinvolvedinthemechanismbywhichcannabinoidspromotetheactivationofthemitochon-drialpro-apoptoticpathway.Thus,neithertumorsinwhichthep8-regulatedpathwayhasbeenablated(andinwhich,therefore,THCtreatmentdoesnotinduceautophagy)nortumorsintrin-sicallydeficientinautophagyundergoapoptosisinresponsetoTHC,andsotheyareresistanttoTHCantitumoralaction.Thesefindingsrevealthatautophagyisrequiredfortheactivationofapoptosisinresponsetocannabinoidtreatmentinvivo.ItisworthnotingthattheconcentrationsofTHCusedinthis

studyareinthesamerangeasthoseadministeredintracraniallytothepatientsinwhichweobservedactivationoftheautophagy-mediatedcelldeathpathway(11)andcouldbethusconsideredclinicallyrelevant.Ofinterest,intraperitonealadministrationofTHCtoU87MGtumorxenograftsproducesasimilardecreaseintumorgrowth(thatoccursinconcertwithincreasedautophagyandapoptosis)tothatobservedwhenthecannabinoidisadminis-teredperitumorally(ourunpublishedobservations).Consideringthatnosignsoftoxicitywereobservedintheclinicaltrialpatients(11)orintumor-bearinganimalstreatedintracranially,peritumor-ally,orintraperitoneallywithTHC(refs.34and35anddatanotshown),andthatnooverttoxiceffectshavebeenreportedinotherclinicaltrialsofcannabinoiduseincancerpatientsforvariousapplications(e.g.,inhibitionofnausea,vomiting,andpain)andusingdifferentroutesofadministration(e.g.,oral,oro-mucosal)(9,36),ourfindingssupportthatsafe,therapeuticallyefficaciousdosesofTHCmaybereachedincancerpatients.

Insummary,inthisstudyweidentifywhatwebelieveisanewroutethatlinkstheERstressresponsetotheactivationofautoph-agyandpromotestheapoptoticdeathoftumorcells(Figure7C).Theidentificationofthispathwaywillhelptounderstandthemoleculareventsthatleadtoactivationofautophagy-mediatedcelldeathbyanticancerdrugsandmaycontributetothedesignofnewtherapeuticstrategiesforinhibitingtumorgrowth.

MethodsCell culture and viability.Corticalastrocyteswerepreparedfrom24-hour-oldmiceaspreviouslydescribed(13).PrimaryculturesofbraintumorcellswerepreparedandculturedasdescribedintheSupplementalMethods.U87MG,T98G,U373MG,andMiaPaCa2cells,p8+/+andp8/RasV12/E1AMEFs,Atg5+/+andAtg5/T-largeantigenMEFs(providedbyNoboruMizushima,TokyoMedicalandDentalUniversity,Tokyo,Japan),Bax/Bakwild-typeandBax/BakDKOT-largeantigenMEFs(providedbyLucaScorrano,DulbeccoTelethonInstitute,Milan,Italy,andPatriziaAgosti-nis,CatholicUniversityofLeuven,Leuven,Belgium),eIF2S51SWTandeIF2S51AT-largeantigenMEFs(providedbyRichardKaufman,Uni-versityofMichigan,AnnArbor,Michigan,USA,andCesardeHaroandJuanJ.Berlanga,CentrodeBiologaMolecularSeveroOchoa,AutonomaUniversity,Madrid,Spain),Tsc2+/+andTsc2/p53/MEFs,emptyvector(pBABE)andpBABE-myr-AktMEFs,andAtg5+/+andAtg5/RasV12/T-largeantigenMEFswereculturedinDMEMcontaining10%FBSandtrans-ferredtomediumcontaining0.5%FBS(exceptRasV12/E1A-transformedMEFs,whichweretransferredtomediumcontaining2%FBS)18hbeforeperformingthedifferenttreatments.p8+/+andp8/RasV12/E1AMEFsaswellasAtg5+/+andAtg5/RasV12/T-largeantigenMEFscorrespondtoapolyclonalmixofatleast20differentselectedclones.Unlessotherwiseindicated,THCwasusedatafinalconcentrationof5M.CellviabilitywasdeterminedbytheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-liumbromide]test(Sigma-Aldrich).

Flow cytometry.Briefly,cells(approximately5105cellsperassay)weretrypsinized,dividedin2tubes,washed,andcollectedbycentrifugationat1,500gfor5min.Onealiquotwasincubatedfor10minat37CwithAnnexinVFITC(BDBiosciences).Propidiumiodide(1g/ml)wasaddedjustbeforecytofluorometricanalysis.Theotheraliquotwassimultane-ouslylabeledwith3,3-dihexyloxacarbocyanineiodide(DiOC6[3],40nM;Invitrogen)andhydroethidium(5M;Invitrogen)for10minutesat37C,followedbycytofluorometricanalysis.Cells(10,000)wererecordedineachanalysis.FluorescenceintensitywasanalyzedinanEPICSXLflowcytometer(BeckmanCoulter).

Western blot.Westernblotanalysiswasperformedfollowingstandardprocedures.AlistoftheantibodiesusedcanbefoundinSupplemen-talMethods.DensitometricanalysiswasperformedwithQuantityOnesoftware(Bio-Rad).

Transfections.U87MGcells (75%confluent)were transfectedwithsiRNAduplexesusingtheDharmaFECT1Transfectionreagent(Dhar-macon).Cellsweretrypsinizedandseeded24haftertransfection,atadensityof5,000cells/cm2.Transfectionefficiencywasgreaterthan70%asmonitoredwithacontrolfluorescent(red)siRNA(siGLORISC-FreesiRNA;Dharmacon).Inimmunofluorescenceexperiments,controlandselectivesiRNAswereusedina1:5ratio,andcellswithredspotswerescoredastransfected.

Infections with adenoviral vectors.U87MGcells(75%confluent)weretrans-ducedfor1hwithsupernatantsobtainedfromHEK293cellsinfectedwithadenoviralvectorscarryingEGFP(providedbyJavierG.Castro,HospitalInfantilUniversitarioNioJess,Madrid,Spain),ratHA-taggedTRB3(donatedbyPatrickIynedjian,UniversityofGeneva,Geneva,Switzerland)(32),orhumanEGFP-LC3(providedbyAvivaTolkovskyandChristoph


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Goemans,UniversityofCambridge,Cambridge,UnitedKingdom).Infec-tionefficiencywasgreaterthan80%asdeterminedbyEGFPfluorescence.

RNA interference.Double-strandedRNAduplexeswerepurchasedfromDharmacon.AlistofsequencescanbefoundintheSupplementalMethods.

RT-PCR analysis. RNAwasisolatedusingTrizolReagent(Invitrogen).cDNAwasobtainedwithTranscriptorReverse transcriptase (RocheAppliedScience).PrimersandamplificationconditionscanbefoundintheSupplementalMethods.

Real-time quantitative PCR.cDNAwasobtainedusingTranscriptor(RocheAppliedScience).Real-timequantitativePCRassayswereperformedusingtheFastStartUniversalProbeMastermixwithRox(RocheAppliedSci-ence),andprobeswereobtainedfromtheUniversalProbeLibrarySet(RocheAppliedScience).PrimersequencescanbefoundintheSupple-mentalMethods.Amplificationswererunina7900HT-FastReal-TimePCRSystem(AppliedBiosystems).Eachvaluewasadjustedbyusing18SRNAlevelsasareference.

Immunoprecipitation.U87MGcellswerelysedinHEPESlysisbuffer(seeSupplementalMethodsforbuffercomposition).Lysate(14mg)waspre-clearedbyincubatingwith520lofproteinGSepharoseconjugatedtopre-immuneIgG.Thelysateextractswerethenincubatedwith520lofproteinGSepharoseconjugatedto520goftheanti-TRB3anti-bodyorpre-immuneIgG.TRB3antibody(aminoterminalend,ab50516;Abcam)wascovalentlyconjugatedtoproteinGSepharoseusingdimethylpimelimidate. Immunoprecipitationswerecarriedoutfor1hat4Conarotatorywheel.Theimmunoprecipitateswerewashed4timeswithHEPESlysisbuffer,followedby2washeswithHEPESkinasebuffer.Theimmunoprecipitateswereresuspendedin30lofsamplebuffer(notcon-taining2-mercaptoethanol)andfilteredthrougha0.22-mSpin-Xfilter,and2-mercaptoethanolwasaddedtoaconcentrationof1%(vol/vol).Sam-plesweresubjectedtoelectrophoresisandimmunoblotanalysis.

Ceramide levels. Ceramidelevelsweredeterminedaspreviouslydescribed(37).Confocal laser scanning microscopy. Standardprotocols for immuno-

fluorescencemicroscopywereused(seeSupplementalMethodsfortheantibodiesused).ToquantifythepercentageofcellswithLC3orPDIdots,atleast200cellsperconditionwerecountedinrandomlyselectedfields.Inallcases,onlythosecellswith4ormoreprominentdotsofeitherLC3orPDIwerescoredpositively.

In vivo treatments.TumorsderivedfromU87MGcellsandp8+/+andp8/MEFswere inducedandtreatedaspreviouslydescribed(13).TumorsderivedfromAtg5+/+orAtg5/RasV12/T-largeantigenMEFs(seeSupplemen-talMethodsfortheprocedureusedtogeneratethesecells)wereinducedinnudemicebysubcutaneousinjectionof107cellsinPBSsupplementedwith0.1%glucose.Tumorswereallowedtogrowuntilanaveragevolumeof200250mm3,andanimalswereassignedrandomlytothedifferentgroups.Atthispoint,vehicleorTHC(15mg/kg/d)in100lofPBSsupplementedwith5mg/mlBSAwasadministereddailyinasingleperitumoralinjection.Tumorsweremeasuredwithanexternalcaliper,andvolumewascalculatedas(4/3)(width/2)2(length/2).AllproceduresinvolvinganimalswereperformedwiththeapprovaloftheComplutenseUniversityAnimalExperi-mentationCommitteeaccordingtoSpanishofficialregulations.

Human tumor samples.Tumorbiopsieswereobtainedfrom2recurrentglioblastomamultiformepatientswhohadbeentreatedwithTHC.Thecharacteristicsofthepatientsandtheclinicalstudyhavebeendescribedindetailelsewhere(11).Briefly,THCdissolvedin30mlofphysiologicalsalinesolutionplus0.5%(wt/vol)humanserumalbuminwasadministeredintratumorallytothepatients.Patient1receivedatotalof1.46mgofTHCfor30days,whilepatient2receivedatotalof1.29mgofTHCfor26days(itwasestimatedthatdosesof610MTHCwerereachedatthesiteofadministration;ref.11).Sampleswerefixedinformalin,embeddedinpar-affin,andusedforimmunomicroscopy.

Immunomicroscopy of tumor samples.Samples fromtumorxenograftsweredissected,Tissue-Tek(Sakura)embedded,frozen,and,beforethestainingprocedureswereperformed,fixedinacetonefor10minatroomtemperature.Samplesfromhumantumorsweresubjectedtodeparaf-finization,rehydration,andantigenretrievalbeforethestainingproce-dureswereperformed.Standardprotocolsforimmunofluorescenceorimmunohistochemistrymicroscopywereused(seeSupplementalMeth-ods).NucleiwerecounterstainedwithTOTO-3iodide(U87MGandhumantumorsamples;Invitrogen)orHoechst33342(MEFtumors;Invitrogen).FluorescenceimageswereacquiredusingMetamorph-Offline6.2software(UniversalImaging)andZeissAxioplan2Microscope.

TUNEL.Tumorsampleswerefixed,blocked,andpermeabilized,andTUNELwasperformedaspreviouslydescribed(13).

Electron microscopy.Ultrastructuralanalysisofvehicle-andTHC-treatedcellswasassessedbyconventionalembeddingintheepoxy-resinEML-812(TaabLaboratories).Ultrathin(20-to30-nm-thick)sectionsofthesam-pleswereobtainedusingaLeica-Reichert-Jungultramicrotomeandthenstainedwithsaturateduranylacetateleadcitratebystandardprocedures.UltrathinsectionswereanalyzedinaJEOL1200-EXIItransmissionelec-tronmicroscopeoperatingat100kV.

Statistics.StatisticalanalysiswasperformedbyANOVAwithapost-hocanalysisusingtheStudent-Neuman-Keulstest.Differenceswereconsid-eredsignificantwhenthePvaluewaslessthan0.05.

AcknowledgmentsThisworkwassupportedbygrantsfromtheSpanishMinistryofEducationandScience(MEC)(HF2005/0021,toG.Velasco;SAF2006/00918, toM.Guzmn;andBFU2006-00508, toP.Boya),Santander-ComplutensePR34/07-15856,toG.Velasco),ComunidaddeMadrid (S-SAL/0261/2006, toM.Guzmn),andLaLiguecontreleCancerandCanceropolePACA(toJ.L.Iovanna).M.Salazarwas the recipientofa fellowship fromtheMEC.A.CarracedowastherecipientoffellowshipsfromGobiernoVasco,theFederationofEuropeanBiochemicalSoci-eties,andtheEuropeanMolecularBiologyOrganization.M.LorenteandP.BoyahaveaJuandelaCiervaandaRamnyCajalcontractfromtheMEC,respectively.S.Hernndez-TiedrahasatechniciancontractfromtheSpanishMinistryofEduca-tionandtheFondoSocialEuropeo.TheauthorsthankDarioAlessi (UniversityofDundee,Dundee,UnitedKingdom)fordonatinganti-PRAS40antibodiesandfortechnicalsupportforimmunoprecipitationexperiments;GemmaFabris,JosefinaCasas,andEvaDalmau(InstitutodeInvestigacionesQumicasyAmbientales,Barcelona,Spain)foranalyzingceramidesam-ples;JosLizcano,JosBayascas,MaraM.Caffarel,andPatriziaAgostinisfortheirexperimentalsuggestions;andothermem-bersofourlaboratoryfortheircontinualsupport.

Received forpublicationNovember3,2008, andaccepted inrevisedformFebruary11,2009.

Addresscorrespondenceto:GuillermoVelasco,DepartmentofBio-chemistryandMolecularBiologyI,SchoolofBiology,ComplutenseUniversity,c/JosAntonioNovaiss/n,28040Madrid,Spain.Phone:34-913944668;Fax:34-913944672;E-mail:[email protected].

ArkaitzCarracedoandAinaraEgiaspresentaddress is:Can-cerGeneticsProgram,BethIsraelDeaconessCancerCenterandDepartmentofMedicine,BethIsraelDeaconessMedicalCenter,HarvardMedicalSchool,Boston,Massachusetts,USA.


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