The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

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ContentslistsavailableatSciVerseScienceDirect

Phytomedicine

jo u r n al h om e p a g e :w w w . e l s e v i e r . d e / p h y m e d

The

novel

phloroglucinol

derivative

BFP

induces

apoptosis

of

glioma

cancer

through

reactive

oxygen

species

and

endoplasmic

reticulum

stress

pathways

Dah-Yuu

Lu

a,∗

_,

_Chih-Shiang

_Chang

b

_,

_Wei-Lan

_Yeh

c

_,

_Chih-Hsin

_Tang

d

_,

_Chi-Wai

_Cheung

f

_,

Yuk-Man

Leung

a

_,

_Ju-Fang

_Liu

b,e

_,

_Kar-Lok

_Wong

f,g,h,∗∗

a_Graduate_Institute_of_Neural_and_Cognitive_Sciences,_China_Medical_University,_Taichung,_Taiwan b_Graduate_Institute_of_{Pharmaceutical}_Chemistry,_China_Medical_University,_Taichung,_Taiwan

c_Cancer_Research_Center,_Department_of_Medical_Research,_Changhua_Christian_Hospital,_Changhua,_Taiwan d_Department_of_{Pharmacology,}_China_Medical_University,_Taichung,_Taiwan

e_Central_Laboratory,_Shin_Kong_Wu_Ho-Su_Memorial_Hospital,_Taipei,_Taiwan

f_Department_of_{Anesthesiology,}_Li_Ka_Shing_Faculty_of_Medicine,_University_of_Hong_Kong,_Hong_Kong,_China g_Department_of_{Anesthesiology,}_China_Medical_University,_Taichung_40402,_Taiwan

h_Department_of_{Anesthesiology,}_Taishan_Medical_University,_Taian,_Shandong,_China

a

r

t

i

c

l

e

i

n

f

o

Keywords: Prenyl-phloroglucinolderivatives ROS Apoptosis Glioma ERstress

a

b

s

t

r

a

c

t

Prenyl-phloroglucinolderivativesfromhopplantshavebeenshowntohaveanticanceractivities.This

studyistheﬁrsttoinvestigatetheanticancereffectsofthenewphloroglucinolderivative

(2,4-bis(4-ﬂuorophenylacetyl)phloroglucinol;BFP).BFPinducedcelldeathandanti-proliferationinthreeglioma,

U251,U87andC6cells,butnotinprimaryhumanastrocytes.BFP-inducedconcentration-dependently

celldeathingliomacellswasdeterminedbyMTTandSRBassay.Moreover,BFP-inducedapoptoticcell

deathingliomacellswasmeasuredbyHochest33258stainingandﬂuorescence-activatedcellsorter

(FACS)ofpropidineiodine(PI)analysis.TreatmentofU251humangliomacellswithBFPwasalsofound

toinducereactiveoxygenspecies(ROS)generation,whichwasdetectedbyaﬂuorescencedyeusedFACS

analysis.TreatmentofBFPalsoincreasedanumberofsignatureendoplasmicreticulum(ER)stress

mark-ersglucose-regulatedprotein(GRP)-78,GRP-94,IRE1,phosphorylationofeukaryoticinitiationfactor-2␣

(eIF-2␣)andup-regulationofCAAT/enhancer-bindingproteinhomologousprotein(CHOP).Moreover,

treatmentofBFPalsoincreasedthedown-streamcaspaseactivation,suchaspro-caspase-7and

pro-caspase-12degradation,suggestingtheinductionofERstress.Furthermore,BFPalsoinducedcaspase-9

andcaspase-3activationaswellasup-regulationofcleavedPARPexpression.Treatmentofantioxidants,

orpre-transfectionofcellswithGRP78orCHOPsiRNAreducedBFP-mediatedapoptotic-relatedprotein

expression.Takentogether,thepresentstudyprovidesevidencestosupportthatROSgeneration,GRP78

andCHOPactivationaremediatingtheBFP-inducedhumangliomacellapoptosis.

Introduction

Glioblastomasareoneofthemostlethaltypesofprimary cen-tralnervous systemtumors, and theirbiological features make successfultreatmentverydifﬁcult.Moreover,glioblastomas gen-erallyproverefractorytotreatmentbysurgery,irradiation,and conventional chemotherapy. Their abnormal biological features leadtouncontrolledgrowth,invasiveness,andangiogenesis,and

∗ Correspondingauthorat:No.91,Hsueh-ShihRoad,Taichung,Taiwan. Tel.:+886422053366x8206;fax:+886422071507.

∗∗ Correspondingauthorat:No.2,YudeRd.,NorthDist.,Taichung,Taiwan. Tel.:+886422052121x3550.

E-mailaddresses:[email protected](D.-Y.Lu), [email protected](K.-L.Wong).

ultimatelyfacilitatecellproliferationandsurvival(Ambergeretal. 1998;Griscellietal.2000;Kleihuesetal.1995;Kouletal.2006; Stuppetal.2005).Thesedysregulatedpathwaysprovidethebasis fordesigningmolecular-targetedtherapyfortreatmentofgliomas. Endoplasmic reticulum (ER) is anorganelle in thesecretory pathways,andservesasacentralroleinlipidsynthesis,protein folding and modiﬁcation.However,proteinfolding in theERis impairedunderavarietyoftoxicinsults,includinghypoxia, fail-ure ofprotein synthesis,protein misfolding,and Ca2+_overload,

andcanresultinERstress-relatedevents(Abcouweretal.2002; SoboloffandBerger2002).Thereisincreasingevidencethat ER stressplaysacrucialroleintheregulationofapoptosis.Ithasbeen reportedthatERstresstriggersseveralspeciﬁcsignalingpathways, suchasER-associatedproteindegradationandtheunfolded pro-teinresponse(UPR)(Feldmanetal.2005;Moenneretal.2007). TheUPRinducestheexpressionofER-residentchaperones,suchas 0944-7113/$–seefrontmatter © 2012 Elsevier GmbH. All rights reserved.

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1094 D.-Y.Luetal./Phytomedicine19 (2012) 1093–1100

GRP(glucose-regulatedprotein)-78andGRP-94(Lee2001).GRPs arethemostabundantglycoproteinsin theERandplay critical rolesinERregulation.TheprotectivefunctionsofGRPproteinhave alsobeenobservedinresistancetoradiationincancercells(Kubota etal.2005).Ontheotherhand,severalpro-apoptoticfactorslike CHOP/GADD153,andpro-apoptoticBcl-2familymemberslikeBax, have beenalsoshown to beinvolved in ERstress-induced cell death.CHOP/GADD153isapparentlyapro-apoptotictranscription factorinducedduringERstress(Hardingetal.2002;McCullough etal. 2001;Rao et al.2004; Wanget al.1996).Theeukaryotic translationinitiationfactor2alpha(eIF2␣)phosphorylationis a highlyconservedpointofmoleculethatadaptscellstoERstress (Moenneretal.2007;Weketal.2006).Itprovidesstressresistance byarrestingproteintranslationandinductionofstress-inducible cytoprotectiveproteins.Furthermore,ROSgenerationappearsto betriggeredbytheactivationofthemitochondrial-dependentcell deathpathwaythrough thepro-apoptotic Bcl-2proteinsBaxor Bak,andisfurthertransformedsequentiallyintomoretoxicROS, likehydrogenperoxidereactiveoxygenspecies,whichwith conse-quenceinducecelldeath(Feigetal.,1994;RoosandKaina,2006). Numerousnaturalproductsarerecognizedtobecancer preven-tiveagentsorantineoplasticagents(Hsiaetal.2004;Loetal.2011; Wengetal.2008).Therearevariouskindsofnatural phlorogluci-nolderivativesthathavebeenidentiﬁedaspossessinganticancer activity.For example,prenyl-phloroglucinolderivativehumulon fromhopplants caninducecell apoptosisin humanmalignant glioblastomacells.Moreover,ithasalsobeenreportedthat prenyl-phloroglucinolderivativelupulonfromhopplantshasapoptotic effectoncoloncancer(Lamyetal.2007,2008,2011).Bullatenone isatriketonephloroglucinol,themaincytotoxiccomponent com-pound in Lophomyrtus bullata (Larsen et al. 2005). Thouvenol, thealkylphloroglucinol,isolatedfromProtorhusthouvenotii,has shown cytotoxicity in ovarian cancer cells (Cao et al. 2004). Recently, we also reported that phloroglucinol derivates have anti-canceractivitiesinhumancoloncancer(Huangetal.2011) andchondrosarcoma(Liuetal.2011).However,theanti-cancer activityof phloroglucinolderivatives in humanglioma remains unclear.

Inthisstudy,wesynthesizedthenewphloroglucinol deriva-tive 2,4-bis(4-ﬂuorophenylacetyl)phloroglucinol (BFP) (Fig. 1A) andinvestigateditsanticanceractivityinhumangliomacells.Our dataindicatethatBFPreducestumorgrowthandsurvivalofhuman gliomacells.

Materialsandmethods

Materials

Thephloroglucinol derivative(Fig.1; chemical purity≥95%) wassynthesizedattheGraduateInstituteofPharmaceutical Chem-istry,ChinaMedicalUniversity(Taichung,Taiwan)followingthe generalprocedure. Fetalbovine serum (FBS), Dulbecco’s modi-fied Eagle’s medium (DMEM), and OPTI-MEM were purchased fromGibcoBRL(InvitrogenLifeTechnologies,Carlsbad,CA). Pri-maryantibodiesagainstcleavedcaspase3 andphosphorylation ofeIF-2␣werepurchased fromCell SignalingTechnology (Dan-vers,MA).Primaryantibodiesspecificforcalpain1,IRE1,GRP78, GRP94, PARP, pro-caspase3, pro-caspase 9, pro-caspase 7 and ␤-actin were purchased from SantaCruz Biotechnology (Santa Cruz, CA, USA). Primary antibody against pro-caspase-12 was purchasedfrom BDBioscience (SanJose, CA).Propidium iodide (PI),2,7-dichlorodihydrofluorescein diacetate(H2DCFDA),

stau-rosporineandotherchemicalswereobtainedfromSigma–Aldrich (St.Louis,MO).

Cellculture

C6cellsoriginatedfromaratbrainglioma.U87andU251cells originatedfromahumanbrainglioma.Allcelllineswerepurchased fromtheAmericanTypeCultureCollection(Manassas,VA).C6cells weremaintainedwithF12medium(InvitrogenLifeTechnologies, Carlsbad,CA),whileU87andU251cellsweremaintainedin75cm2

ﬂaskswithDMEM.

Human astrocytes were purchased from Sciencell Research Laboratories (isolated from human cerebral cortex, Cat# 1800, Carlsbad,CA)andwereculturedinhumanastrocytemedium (Sci-encell,Cat#1801)onpoly-l-lysinecoatedtissueculturedishes. Mediawaschangedeverythreedaysandcellswerepassagedonce aweekata1:5ratio.

All cells were cultured in medium supplemented with 10% heat-inactivatedfetalbovineserum(FBS),100U/mlpenicillin,and 100mg/mlstreptomycinat37◦C,incubatedinahumidiﬁed atmo-sphereconsistingof5%CO2and95%air.

SulforhodamineBassay(SRB)

TheSRBassayisbasedonthemeasurementofcellularprotein contentandtheprocedurewasfollowedaccordingtoourprevious report(Huangetal.2011).AftertreatmentwithBFPfor24h,cells wereﬁxedwith10%trichloroaceticacidandstainedbySRBat0.4% (w/v)in1%aceticacidfor30min.UnboundSRBwaswashedout by1%aceticacidandSRB-boundcellsweresolubilizedwith10mM Trizmabase.Theabsorbancewasreadatawavelengthof515nm usingamicroplatereader(Bio-Tek,Winooski,VT).

MTTassay

Cellviabilitywasdeterminedby 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment withBFPfor24or48h, mediawereremovedand washedwith PBS.MTT(0.5mg/ml)wasthenaddedtoeachwellandthe mix-turewasincubatedfor2hat37◦C.MTTreagentwasthenreplaced withDMSO(100␮lperwell)todissolveformazancrystals.Afterthe mixturewasshakenatroomtemperaturefor10min,absorbance wasdetermined at 550nm using a microplate reader (Bio-Tek, Winooski,VT).

Hoechst33258staining

Hoechst33258 is a DNA-binding fluorescent dye and being usedtodetermineapoptoticnuclei.CellswereincubatedwithBFP forindicatedtimeperiods,andthenstainedwithHoechst33258 (1␮g/ml)for10min.Resultsweredeterminedbyvisual observa-tionofnuclearmorphologythroughfluorescencemicroscopy. Quantificationofapoptosisbyflowcytometry

CellsweretreatedwithvariousconcentrationsofBFPfor24h and then washedtwice withPBS.For apoptosis determination, cells wereﬁxedby70% ethanolat roomtemperatureand then re-suspendedinPBScontaining50␮g/mlpropidiumiodide(PI), 100␮g/mlRNaseAand0.1%TritonX-100for30min.Theapoptotic nucleiweredeterminedbyﬂow cytometry.Cellswere immedi-atelyanalyzedusingFACScanandtheCellquestprogram(Becton Dickinson,LincolnPark,NJ).

Westernblotanalysis

Theprotocolofwholecellproteinlysiswasfollowedaccording toourpreviousreport(Huangetal.2011).Brieﬂy,cellsweretreated

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Fig.1.BFPinducescancercelldeathandanti-proliferationingliomacells.(A)Thestructureofanovelphloroglucinolderivative2,4-bis(4-ﬂuorophenylacetyl)phloroglucinol; BFP.(B)Cellswereincubatedwithvariousconcentrationsofstaurosporine(5,10,50or100nM),andcellviabilitywasexaminedbyMTTassayafter24or48htreatment. CellsweretreatedwithvariousconcentrationsofBFP(1,3,10or30␮M),andcellviabilitywasexaminedbyMTTassayandSRBassayafter24or48htreatmentinU251(C andD),U87(EandF)andC6(GandH).Resultsareexpressedasthemeans±S.E.M.ofatleastthreeindependentexperiments.*p<0.05comparedwiththevehicletreatment group.

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withBFPforvarioustimeperiodsand thenlysedwith radioim-munoprecipitationassaybuffer. Proteinsampleswereseparated bysodiumdodecylsulphate-polyacrylamidegelsandtransferred topolyvinyldiﬂuoride(PVDF)membranes.Theblotswereprobed withprimaryantibodyfor 1hatroomtemperature and subse-quently incubated with a secondary antibody for 1h at room temperature.Theblotswerevisualizedbyenhanced chemilumi-nescenceusingKodakX-OMATLSﬁlm(EastmanKodak,Rochester, NY).Theblotsweresubsequentlystrippedthroughincubationin strippingbufferandreprobedfor␤-actinasaloadingcontrol.

siRNAtransfection

ThesiRNAs against humanGRP78, CHOP and control siRNA werepurchasedcommerciallyfromSantaCruzBiotechnology.Cells weretransientlytransfectedwiththesiRNAbyLipofectamine2000 (LF2000;Invitrogen,Carlsbad,CA)accordingtothemanufacturer’s instructions.Brieﬂy,thesiRNA(ataﬁnalconcentrationof100nM) andLF2000werepremixedinOPTI-mediumfor20minandthen appliedtothecells.Anequalvolumeofmediumcontaining20%FBS wasadded6hlater.Aftertransfectionfor24h,LF2000-containing mediumwasreplacedwithfreshserum-freemediumandtreated withBFPforanother24h.

ReverseTranscriptase-PCR(RT-PCR)

TotalRNAwasextractedfromcellsusingaTRIzolkit(MDBio Inc.,Taipei,Taiwan).Reversetranscriptionreactionwasperformed using1␮goftotalRNAwhichconvertedintocDNAusingtheRT kit(Promega,Madison,WI),thenampliﬁedusingoligonucleotide primers:

GRP78: 5-GCTCGACTCGAATTCCAAAG-3 and 5 -TTTGTCAGG-GGTCTTTCACC-3;

GRP94:5-CAGTTTTGGATCTTGCTGTGG-3 and 5 -CAGCTGTAGA-TTCCTTTGC-3;

GAPDH:5-TGGGCTACACTGAGCACCAG-3and5 -GGGTGTCGCTG-TTGAAGTCA-3.

Each PCR cycle was carried out for 30s at 95◦C, 1min at 55◦C,and1minat65◦C.PCRproductswerethenseparated elec-trophoretically in a 2% agarose gel and stained withethidium bromide.Thebandintensitywasquantiﬁedwithadensitometric scannerandpresentedastherelativelevelofGAPDH.

Reactiveoxygenspecies(ROS)assay

TheproductionofROSwasassessedspectrofluorimetricallyby oxidationofspecificprobes2,7-dichlorodihydrofluorescein diac-etate(H2DCFDA).Cellswereplatedat6well-platesandexposedto

BFPforanother2h.ThecellswereincubatedwithDHE(10␮M)or H2DCFDA(10␮M)for30minat37◦C.Theﬂuorescenceintensity

wasmeasuredwithexcitationﬁlterof488and525nmemission wavelengthsusingtheﬂowcytometry.

Statistics

The values given are means±S.E.M. The signiﬁcance of dif-ferencebetweentheexperimentalgroupandcontrolgroupwas assessedbytheStudent’st-test.Thedifferencewassigniﬁcantif thepvaluewas<0.05.

Results

BFPinducescellapoptosisinhumangliomacells

As shown in Fig. 1B, staurosporine induced glioma cell death in a concentration-dependent manner. Staurosporine were used as a positive control to induce cell apoptosis (IC50≈30nM). To investigate the cytotoxicity of 2,4-bis(4-fluorophenylacetyl)phloroglucinol (BFP, Fig.1A)in glioma cells, weexaminedtheeffectsoncellviabilityinthreedifferentglioma cells,U251,U87andC6.Cellswereincubatedwithvarious con-centrations of BFP (1, 3, 10 or 30␮M), and the cell viability was examined by MTT assay and SRB assay for 24 or 48h. BFPsignificantlyinducedcelldeathinallthree typesofglioma cells (Fig. 1C–H). Furthermore, the SRB assay determined that BFP-induced cell death for 24 and 48h in glioma cells U251 (IC50<1␮M),U87(IC50<3␮M)andC6(IC50<10␮M),butnotin primaryhumanastrocytes(IC50>30␮M;datanotshown).Next, wefurtherconfirmedthatwhetherBFPinducescelldeaththrough anapoptoticmechanism.Nuclear shrinkage,chromatin conden-sation,andnuclearfragmentationarehallmarksofcellapoptosis. AsshowninFig.2A,treatmentwithBFP(3_␮M)for4hinduced nuclearshrinkageandnuclearcondensation,andfor8hobviously revealednuclearcondensationand fragmentation.Furthermore, BFP-inducedcellapoptosiswasexaminedbyevaluatingthe sub-G1groupusingpropidiumiodide(PI)whichwasanalyzedbyflow cytometry.BFP inducedconcentration-dependent sub-G1 arrest inU251humangliomacells(Fig.2B).TodeterminewhetherBFP inducesapoptosisbytriggeringthemitochondrialapoptotic path-way,wemeasuredthechangeintheexpressionof Bcl-2family proteins.TreatmentofU251cellswithBFPinducedBaxprotein up-regulationsignificantlybutdidnotaffectBcl-2proteinexpression (Fig.2C).TreatmentofBFPalsoincreasedprocaspase-3 degrada-tionandcaspase-3cleavedformexpressioninU251cells(Fig.2D). Upstream procaspase-9is alsodegraded and cleaved-caspase-9 increasedupon BFPtreatmentin U251 cells (Fig. 2D). Notably, BFP also increased cleaved-PARP expression time-dependently (Fig.2E).

BFPinducesreactiveoxygenspeciesgenerationandERstressin U251humangliomacells

Ithasbeenreportedthatoxidativestresshasbeenimplicated inthepro-apoptoticactivitiesofcancertherapy(Lauetal.2008; MatesandSanchez-Jimenez2000).Therefore,wenextexamined whethertheROS accumulationisinvolved inBFP-induced apo-ptosis. BFPinduced an increase in intracellular H2O2 levels,as

shownbyH2DCF-DAstainingwhichwereobservedbya

ﬂuores-cencemicroscope(Fig.3A)andanalyzedbyFACSdetectionassay (Fig.3B).ERstressisgenerallycharacterizedbyup-regulationof IRE1,GRP78, GRP94,calpain 1 and CHOP,and phosphorylation of eukaryoticinitiation factor-2␣(eIF2␣). BFPexposure caused a signiﬁcant increase in theexpression of GRP 78 and GRP 94 proteinlevels(Fig.4A)andmRNAlevels(Fig.4B).Wenext deter-minedwhethertheactivityofcalpain(calcium-dependent thiol proteases)wouldbeinducedbyBFPingliomacells.Asshownin Fig.4C,BFPalsoincreasedcalpain1expressioninU251cells. More-over,BFPalsoinducedIRE1(Fig.4D),CHOPexpressionandthe phosphorylationofeIF2_␣atSer51(Fig.4E)inU251cells.Oneof thehallmarksoftheapoptoticprocessisthecaspasesactivation, whichrepresentsbothinitiatorsand executorsofdeathsignals. BFPalsotriggeredthepro-caspase-7andpro-caspase-12 degrada-tioninatime-dependentmanner(Fig.4F).Theseresultsindicate thattheoccurrenceof ERstressisinvolved inBFP-inducedcell death.

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Fig.2.BFPinducescellapoptosisinU251humangliomacells.(A)CellsweretreatedwithBFPforindicatedtimeperiods.Hocheststainingwasvisualizedbyﬂorescence imaging.BFPtreatmentinducednuclearshrinkage(indicatedinarrowhead),condensationandfragmentation(indicatedinarrows).(B)Thepercentageofapoptoticcells wasanalyzedbyﬂowcytometryofPIstainingaftertreatmentwithvariousconcentrationsofBFPfor24h.Resultsareexpressedasthemeans±S.E.M.(n=3–4).Cellswere incubatedwithBFP(3␮M)fordifferenttimeperiods,levelsofapoptoticproteins(C–E)wereexaminedbyWesternblotanalysis.Resultsaretherepresentativeofthree independentexperiments.

BFP-inducedcaspase-3andcaspase-9activationaremediatedby ROSgeneration,GRP78andCHOPexpression

TheseresultssuggestthatBFPtriggersERstressandinduces cancer cell apoptosis in U251 cancer cells. We next fur-ther determined whether the ROS generation is involved in

BFP-induced ER stress in glioma cells. As shown in Fig. 5A, BFP-induced IRE1 increase, and procaspase-3 and procaspase-9 degradation were reversed by treatment with two dif-ferent antioxidants apocynin (10␮M) and N-acetylcysteine (NAC,10mM).Furthermore,pre-transfectionwithsiRNAagainst GRP78 signiﬁcantly reduced BRP-induced ER stress proteins

Fig.3.BFPincreasesreactiveoxygenspeciesreleaseinU251humangliomacells.CellswereincubatedwithBFP(3␮M)forindicatedtimeperiods.TheproductionofROS wasexaminedbyﬂuorescenceimage(A)andquantitativedbyﬂowcytometry(B).Resultsareexpressedasthemean±S.E.M.ofthreeindependentexperiments.*,p<0.05 comparedwiththecontrolgroup.

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Fig.4. BFPincreasesERstress-relatedproteinsactivationinU251humangliomacells.CellswereincubatedwithBFP(3␮M)forindicatedtimeperiods.ERstress-related proteinexpressionswereexaminedbyWesternblotanalysis.Resultsaretherepresentativeofthreeindependentexperiments.CellswereincubatedwithBFPforindicated timeperiods,mRNAexpressionofGRP78andGRP94wereexaminedbyRT-PCRanalysis(B).Resultsaretherepresentativeofthreeindependentexperiments.

expression such as IRE1 and CHOP expression (Fig. 5B). Moreover,pre-transfectionwithGRP78siRNAalsoreduced BFP-mediated cell apoptosis proteins expression, such as cleaved PARP, procaspase-9 degradation and cleaved caspase-3 up-regulation (Fig. 5C). On the other hands, pre-transfection with siRNA against CHOP also reduced BFP-mediated cell apoptosis (Fig.5D).

Discussion

Apoptosishasbeendescribedasmultiplepathwaysconverging fromnumerousdifferentinitiatingeventsandinsults.Increasing evidenceshowsthatefﬁcacyofantitumoragentsandcancer pre-ventiveagentsisrelatedtotheintrinsicpropensityofthetarget tumorcellstorespondtotheseagentsbyapoptosis.Inthisstudy,

Fig.5. InvolvementofROSgeneration,GRP78andCHOPexpressioninBFP-inducedcaspase-9andcaspase-3activationinU251gliomacells.(A–D)Levelsofapoptoticand ERstress-relatedproteinswereexaminedbyWesternblotanalysis.Resultsaretherepresentativeofatleastthreeindependentexperiments.

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wedemonstratedthatthemolecularmechanismbywhichBFP trig-geredhumangliomacellsapoptosis.Ourresultsdemonstratedthat BFPinhibitedcancercellgrowthandproliferationofthree differ-entgliomacells,suchasU251,U87andC6cells.BFPalsoinduced nuclearshrinkage(4h),andnuclearcondensationand fragmenta-tion(8and24h)aswellassubG1arrest,thelastphaseofapoptosis, in U251 cancer cells. In conclusion, these observations suggest that BFPinduces cancercell death-mediated apoptosis.Indeed, treatmentwithBFPcaused theactivationof caspasesincluding caspase-9andcaspase-3,associatedwiththedegradationofPARP, whichprecededtheonsetofapoptosis.

Our previousstudy(Huang et al.2011; Liu etal. 2011)and presencedatademonstratedthatphloroglucinolderivativesinduce humancancercelldeath(IC50<10␮M)butnotinprimaryhuman cells(IC50>30␮M).Thefundamentalﬁndinginthisstudyprovides importantevidencetosupporttheinvolvementofERstressinthe inductionofapoptosisbyBFPinU251gliomacells.Thefollowing experimentalevidenceinthepresentstudyrevealsthatthe induc-tionofERstress-relatedproteinsmaybeinvolvedinBFPinduced apoptosis: (i)BFPinduced IRE1,GRP78,and GRP94expression; (ii)BFPincreasedCHOPexpressionwhich isone ofthehighest induciblegenesduringERstress;(iii)suppressionofGRP78and CHOPgenesbyspeciﬁcsiRNAattenuatedBFP-inducedcaspase-9 andcaspase-3activation;(iv)BFPalsoinducedphosphorylationof eIF-2␣.Takentogether,BFPinducesup-regulationofIRE1,GRP78, GRP94andCHOPaswellasphosphorylationofeIF-2a,allofwhich aremediatingERstress.

Bcl-2familyproteinsplayanimportantroleincancercells apo-ptosis(Cotter2009;Leberetal.2010).TheBcl-2familycanregulate mitochondrialmembranepermeabilization.Baxproteinmediates mitochondrial membrane permeabilization. Treatment of U251 cellswithBFPinducedBaxproteinlevelincreasebutdidnotaffect Bcl2withinaperiodof4handprolongedBaxexpressionat24h, whichmayleadtoanincreaseinthepro-apoptotic/anti-apoptotic Bcl-2ratio.Ontheotherhand,themitochondrialapoptotic path-wayhasbeendescribedasanimportantdownstreamsignalofROS inapoptoticcelldeath(Iwamaruetal.2007;Zuetal.2005).High levelsofROScanalsoinduceapoptosisbytriggering mitochon-drialpermeabilitytransitionporeopening,releaseofpro-apoptotic factorsandactivationofcaspase-9andcaspase-3(Iwamaruetal. 2007;Zuetal.2005).IthasbeenreportedthatinhibitionofROSbya ROSscavengerpreventscamptothecin-inducedapoptosis(Wenzel etal.2004).Here,wealsofoundthatFBPincreasedROS gener-ationinhumangliomacells.Treatmentwithantioxidantssuchas NACandapomycinbothreducedBFP-inducedIRE1expression, pro-caspase-9andpro-caspase-3degradation.Inagreementwiththese observations,wenotedthatthemitochondrialdysfunctionandROS generationmaybeinvolvedinBFP-inducedcellapoptosisofhuman gliomacells.

OurresultsshowedthatROSgeneration,up-regulationof pro-apoptoticproteinandactivationofcaspases maybeinvolvedin BFP-inducedapoptoticcelldeathinhumanglioma,withitsability tocauseERstress.Inconclusion,thenovelphloroglucinol deriva-tiveBFP-induced humanglioma celldeath is mediatedby ROS generation,whichsubsequentlyinducesGPR78andCHOP expres-sion,increasescaspasesactivity,suchascaspase-9andcaspase-3, resultinginapoptosis.Presentstudyonthemolecularbasiswill provide valuable strategies of target signal transducers for the developmentofeffectiveanti-tumortherapy.

Acknowledgments

ThisworkwassupportedbygrantsfromtheNationalScience Council(NSC100-2320-B-039-010andNSC100-2627-B-039-005), China Medical University (CMU99-COL-26-1 & 2), and Taiwan

DepartmentofHealthClinicalTrialandResearchCenterof Excel-lence(DOH100-TD-B-111-004).

References

Abcouwer,S.F.,Marjon,P.L.,Loper,R.K.,VanderJagt,D.L.,2002.ResponseofVEGF expressiontoaminoaciddeprivationandinducersofendoplasmicreticulum stress.InvestigativeOphthalmologyandVisualScience43,2791–2798. Amberger,V.R.,Hensel,T.,Ogata,N.,Schwab,M.E.,1998.Spreadingandmigration

ofhumangliomaandratC6cellsoncentralnervoussystemmyelininvitrois correlatedwithtumormalignancyandinvolvesametalloproteolyticactivity. CancerResearch58,149–158.

Cao, S.,Schilling, J.K.,Randrianasolo,A., Andriantsiferana, R.,Rasamison,V.E., Kingston,D.G.,2004.NewcytotoxicalkylphloroglucinolsfromProtorhus thou-venotii.PlantaMedica70,683–685.

Cotter,T.G.,2009.Apoptosisandcancer:thegenesisofaresearchﬁeld.Nature ReviewsCancer9,501–507.

Feig,D.I.,Sowers,L.C.,Loeb,L.A.,1994.Reversechemicalmutagenesis: identiﬁca-tionofthemutageniclesionsresultingfromreactiveoxygenspecies-mediated damagetoDNA.ProceedingsoftheNationalAcademyofSciencesoftheUnited StatesofAmerica91,6609–6613.

Feldman,D.E.,Chauhan,V.,Koong,A.C.,2005.Theunfoldedproteinresponse:anovel componentofthehypoxicstressresponseintumors.MolecularCancerResearch 3,597–605.

Griscelli,F.,Li,H.,Cheong,C.,Opolon,P.,Bennaceur-Griscelli,A.,Vassal,G.,Soria, J.,Soria,C.,Lu,H.,Perricaudet,M.,Yeh,P.,2000.Combinedeffectsof radiother-apyandangiostatingenetherapyingliomatumormodel.Proceedingsofthe NationalAcademyofSciencesoftheUnitedStatesofAmerica97,6698–6703. Harding,H.P.,Calfon,M.,Urano,F.,Novoa,I.,Ron,D.,2002.Transcriptionaland

trans-lationalcontrolintheMammalianunfoldedproteinresponse.AnnualReview ofCellandDevelopmentalBiology18,575–599.

Hsia,T.C.,Yang,J.H.,Lin,H.J.,Yu,C.S.,Yu,F.S.,Chung,J.G.,2004.Paclitaxelinhibits N-acetyltransferaseactivityandgeneexpressioninhumanstomachtumorcells (SC-M1).ResearchCommunicationsinMolecularPathologyandPharmacology 115-116,21–38.

Huang,S.M.,Cheung,C.W.,Chang,C.S.,Tang,C.H.,Liu,J.F.,Lin,Y.H.,Chen,J.H.,Ko,S.H., Wong,K.L.,Lu,D.Y.,2011.PhloroglucinolderivativeMCPPinducescellapoptosis inhumancoloncancer.JournalofCellularBiochemistry112,643–652. Iwamaru,A.,Iwado,E.,Kondo,S.,Newman,R.A.,Vera,B.,Rodriguez,A.D.,Kondo,

Y.,2007.Eupalmerinacetate,anovelanticanceragentfromCaribbean gor-gonianoctocorals,inducesapoptosisinmalignantgliomacellsviathec-Jun NH2-terminalkinasepathway.MolecularCancerTherapeutics6,184–192. Kleihues,P.,Soylemezoglu,F.,Schauble,B.,Scheithauer,B.W.,Burger,P.C.,1995.

Histopathology,classiﬁcation,andgradingofgliomas.Glia15,211–221. Koul,D.,Shen,R.,Bergh,S.,Sheng,X.,Shishodia,S.,Lafortune,T.A.,Lu,Y.,deGroot,

J.F.,Mills,G.B.,Yung,W.K.,2006.InhibitionofAktsurvivalpathwaybya small-moleculeinhibitorinhumanglioblastoma.MolecularCancerTherapeutics5, 637–644.

Kubota,H.,Suzuki,T.,Lu,J.,Takahashi,S.,Sugita,K.,Sekiya,S.,Suzuki,N.,2005. IncreasedexpressionofGRP94proteinisassociatedwithdecreasedsensitivity toX-raysincervicalcancercelllines.InternationalJournalofRadiationBiology 81,701–709.

Lamy,V.,Bousserouel,S.,Gosse,F.,Minker,C.,Lobstein,A.,Raul,F.,2011.Lupulone triggersp38MAPK-controlledactivationofp53andoftheTRAILreceptor apop-toticpathwayinhumancoloncancer-derivedmetastaticcells.OncologyReports 26,109–114.

Lamy,V.,Roussi,S.,Chaabi,M.,Gosse,F.,Lobstein,A.,Raul,F.,2008.Lupulone,a hopbitteracid,activatesdifferentdeathpathwaysinvolvingapoptotic TRAIL-receptors,inhumancolontumorcellsandintheirderivedmetastaticcells. Apoptosis13,1232–1242.

Lamy,V.,Roussi,S.,Chaabi,M.,Gosse,F.,Schall,N.,Lobstein,A.,Raul,F.,2007. Chemopreventiveeffectsoflupulone,ahop{beta}-acid,onhumancolon cancer-derivedmetastaticSW620cellsandinaratmodelofcoloncarcinogenesis. Carcinogenesis28,1575–1581.

Larsen, L., Benn, M.H., Parvez, M., Perry, N.B., 2005. A cytotoxic triketone-phloroglucinol-bullatenone hybrid from Lophomyrtus bullata. Organic & BiomolecularChemistry3,3236–3241.

Lau,A.T.,Wang,Y.,Chiu,J.F.,2008.Reactiveoxygenspecies:currentknowledgeand applicationsincancerresearchandtherapeutic.JournalofCellularBiochemistry 104,657–667.

Leber,B.,Lin,J.,Andrews,D.W.,2010.Stillembeddedtogetherbindingtomembranes regulatesBcl-2proteininteractions.Oncogene29,5221–5230.

Lee,A.S.,2001.Theglucose-regulatedproteins:stressinductionandclinical appli-cations.TrendsinBiochemicalSciences26,504–510.

Liu,J.F.,Yang,W.H.,Fong,Y.C.,Kuo,S.C.,Chang,C.S.,Tang,C.H.,2011.BFPP,a phloroglucinolderivative,inducescellapoptosisinhumanchondrosarcoma cellsthroughendoplasmic reticulumstress.BiochemicalPharmacology 79, 1410–1417.

Lo,C.,Lai,T.Y.,Yang,J.S.,Yang,J.H.,Ma,Y.S.,Weng,S.W.,Lin,H.Y.,Chen,H.Y.,Lin, J.G.,Chung,J.G.,2011.GallicacidinhibitsthemigrationandinvasionofA375.S2 humanmelanomacellsthroughtheinhibitionofmatrixmetalloproteinase-2 andRas.MelanomaResearch21,267–273.

Mates,J.M.,Sanchez-Jimenez,F.M.,2000.Roleofreactiveoxygenspeciesin apopto-sis:implicationsforcancertherapy.InternationalJournalofBiochemistryand CellBiology32,157–170.

(8)

McCullough,K.D.,Martindale,J.L.,Klotz,L.O.,Aw,T.Y.,Holbrook,N.J.,2001.Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 andperturbingthecellularredoxstate.MolecularandCellularBiology21, 1249–1259.

Moenner,M.,Pluquet,O.,Bouchecareilh,M.,Chevet,E.,2007.Integrated endoplas-micreticulumstressresponsesincancer.CancerResearch67,10631–10634. Rao,R.V.,Ellerby,H.M.,Bredesen,D.E.,2004.Couplingendoplasmicreticulumstress

tothecelldeathprogram.CellDeathandDifferentiation11,372–380. Roos,W.P.,Kaina,B.,2006.DNAdamage-inducedcelldeathbyapoptosis.Trendsin

MolecularMedicine12,440–450.

Soboloff,J.,Berger,S.A.,2002.SustainedERCa2+_depletion_suppresses_protein syn-thesisandinducesactivation-enhancedcelldeathinmastcells.Journalof BiologicalChemistry277,13812–13820.

Stupp,R.,Mason,W.P.,vandenBent,M.J.,Weller,M.,Fisher,B.,Taphoorn,M.J., Belanger,K.,Brandes,A.A.,Marosi,C.,Bogdahn,U.,Curschmann,J.,Janzer,R.C., Ludwin,S.K.,Gorlia,T.,Allgeier,A.,Lacombe,D.,Cairncross,J.G.,Eisenhauer,E., Mirimanoff,R.O.,2005.Radiotherapyplusconcomitantandadjuvant temozolo-mideforglioblastoma.NewEnglandJournalofMedicine352,987–996.

Wang,X.Z.,Lawson,B.,Brewer,J.W.,Zinszner,H.,Sanjay,A.,Mi,L.J.,Boorstein,R., Kreibich,G.,Hendershot,L.M.,Ron,D.,1996.Signalsfromthestressed endoplas-micreticuluminduceC/EBP-homologousprotein(CHOP/GADD153).Molecular andCellularBiology16,4273–4280.

Wek,R.C.,Jiang,H.Y.,Anthony,T.G.,2006.Copingwithstress:eIF2kinasesand translationalcontrol.BiochemicalSocietyTransactions34,7–11.

Weng,J.R.,Tsai,C.H.,Kulp,S.K.,Chen,C.S.,2008.Indole-3-carbinolasa chemopre-ventiveandanti-canceragent.CancerLetters262,153–163.

Wenzel,U.,Nickel,A.,Kuntz,S.,Daniel,H.,2004.Ascorbicacidsuppresses drug-inducedapoptosisinhumancoloncancercellsbyscavengingmitochondrial superoxideanions.Carcinogenesis25,703–712.

Zu, K., Hawthorn, L., Ip, C., 2005. Up-regulation of c-Jun-NH2-kinase path-way contributes to the inductionof mitochondria-mediatedapoptosis by alpha-tocopherylsuccinateinhumanprostatecancercells.MolecularCancer Therapeutics4,43–50.