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Research article
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.
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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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