Antileukemia component, dehydroeburicoic acid from Antrodia camphorata induces DNA damage and apoptosis in vitro and in vivo models

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Phytomedicinexxx (2012) xxx–xxx

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

Antileukemia

component,

dehydroeburicoic

acid

from

Antrodia

camphorata

induces

DNA

damage

and

apoptosis

in

vitro

and

in

vivo

models

Ying-Chi

Du

a,1

_,

_Fang-Rong

_Chang

a,b,c,d,1

_,

_Tung-Ying

_Wu

a

_,

_Yu-Ming

_Hsu

a

_,

_Mohamed

_El-Shazly

a,e

_,

Chieh-Fu

Chen

f

_,

_Ping-Jyun

_Sung

g,h

_,

_Yan-Yu

_Lin

a

_,

_Yi-Hsin

_Lin

a

_,

_Yang-Chang

_Wu

a,i,j,k,∗∗

_,

_Mei-Chin

_Lu

a,g,h,∗

a_Graduate_Institute_of_Natural_Products,_College_of_Pharmacy,_Kaohsiung_Medical_University,_Kaohsiung_807,_Taiwan b_Cancer_Center,_Kaohsiung_Medical_University_Hospital,_Kaohsiung_807,_Taiwan

c_Research_and_Development_Center_of_Chinese_Herbal_Medicines_and_New_Drugs,_College_of_Pharmacy,_Kaohsiung_Medical_University,_Kaohsiung_807,_Taiwan d_Department_of_Marine_{Biotechnology}_and_Resources,_National_Sun_Yat-sen_University,_Kaohsiung_804,_Taiwan

e_Department_of_{Pharmacognosy}_and_Natural_Products_Chemistry,_Faculty_of_Pharmacy,_Ain-Shams_University,_Organization_of_African_Unity_Street_11566,_Abassia,_Cairo,_Egypt f_Chih-Ying_Plant_Research_and_Development_Foundation,_Taipei_City_100,_Taiwan

g_Graduate_Institute_of_Marine_{Biotechnology,}_National_Dong_Hwa_University,_Pingtung_944,_Taiwan h_National_Museum_of_Marine_Biology_&_Aquarium,_Pingtung_944,_Taiwan

i_School_of_Chinese_Medicine,_College_of_Chinese_Medicine,_China_Medical_University,_Taichung_404,_Taiwan j_Natural_Medicinal_Products_Research_Center,_China_Medical_University_Hospital,_Taichung_404,_Taiwan k_Center_for_Molecular_Medicine,_China_Medical_University_Hospital,_Taichung_404,_Taiwan

a

r

t

i

c

l

e

i

n

f

o

Keywords: Antrodiacamphorata Dehydroeburicoicacid(DeEA) DNAdamage

Apoptosis

TopoisomeraseIIinhibitor

a

b

s

t

r

a

c

t

Antrodiacamphorata(AC)isanativeTaiwanesemushroomwhichisusedinAsianfolkmedicineasa chemopreventiveagent.Thetriterpenoid-richfraction(FEA)wasobtainedfromtheethanolicextractof ACandcharacterizedbyhighperformanceliquidchromatography(HPLC).FEAcausedDNAdamagein leukemiaHL60cellswhichwascharacterizedbyphosphorylationofH2A.XandChk2.Italsoexhibited apoptoticeffectwhichwascorrelatedtotheenhancementofPARPcleavageandtotheactivationof caspase3.Fivemajortriterpenoids,antcinK(1),antcinC(2),zhankuicacidC(3),zhankuicacidA(4), anddehydroeburicoicacid(5)wereisolatedfromFEA.ThecytotoxicityofFEAmajorcomponents(1–5) wasinvestigatedshowingthatdehydroeburicoicacid(DeEA)wasthemostpotentcytotoxiccomponent. DeEAactivatedDNAdamageandapoptosisbiomarkerssimilartoFEAandalsoinhibitedtopoisomerase II.InHL60cellsxenograftanimalmodel,DeEAtreatmentresultedinamarkeddecreaseoftumorweight andsizewithoutanysigniﬁcantdecreaseinmicebodyweights.Takentogether,ourresultsprovided theﬁrstevidencethatpureACcomponentinhibitedtumorgrowthinvivomodelbackingthetraditional anticanceruseofACinAsiancountries.

Introduction

Antrodiacamphorata(AC),alsoknownasAntrodiacinnamomea orTaiwanofungus camphoratus,is anendemic fungusinTaiwan which only parasitizes in the internal heartwood or the dark humidwoodsurfaceofCinnamomumkanehirai(Bullcamphortree)

∗ Correspondingauthorat:GraduateInstituteofMarineBiotechnology,National DongHwaUniversity,Pingtung944,Taiwan.Tel.:+88688825038;

fax:+88688825087.

∗∗ Correspondingauthorat: School of ChineseMedicine, College ofChinese Medicine,ChinaMedicalUniversity,Taichung404,Taiwan.Tel.:+886422057153; fax:+886422060248.

E-mailaddresses:[email protected](Y.-C.Wu), [email protected](M.-C.Lu).

1_The_ﬁrst_two_authors_contributed_equally_to_this_study.

(Aoet al.2009;Luet al.2009a).MatureACfruitingbodies are

regarded asthecoloredgemsof theforests, rubyin Taiwanese forest,due totheircolorful orangetobrown-redappearance.It takesoneyearforACfruitingbodiestogrowtothesizeofoneEuro forcingthepriceofonekilogramtoreach10,000–28,000D.Due totheslowgrowthrateandthehighpriceofwildfruitingbodies, ACiswidelycultured.

AC,whichisknowninfolkmedicineas“chang-chih”hasalong historyinthepreventionandtreatmentofseverallifethreatening diseasesincludingcancer,inﬂammatory disorders,andhepatitis

(GeethangiliandTzeng2009;Huangetal.2010;Luetal.2009b;

Shenetal.2003;Yehetal.2009).Currently,ACisusedasfood

dietarysupplementforcancerpreventionandhepatoprotectionin severalAsianand Europeancountries.Itsproductsare commer-ciallyavailableinChina,Japan,Korea,Malaysia,andSingapore.It isalsomarketedintheUnitedKingdomunderthenameAntrodia camphorata.

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PHYMED-51211; No.ofPages9

2 Y.-C.Duetal./Phytomedicinexxx (2012) xxx–xxx

OurgrouphasbeendevotedtotheanticancerresearchofAC for many years. In ourprevious report, we demonstrated that the water extract of AC wild fruiting bodies (ACW) promoted thefunctional maturationofdendriticcellsintheexpressionof phenotypiccharacteristics,IL-12productionandchemotaxic

activ-ity (Lu et al.2009b). Moreover,according to1_H _NMR _analysis,

it wassuggested thatthe polysaccharidesof ACWcouldact as apotentadjuvantforcancerimmunotherapythroughpromoting Th1immuneresponse.Wealsoreportedthattheethanolicextract fromthewildfruitingbodiesofAC(EEAC)couldinduceHL60cell apoptosisandpotentiatetheanticancereffectoftrichostatinA(Lu

etal.2009a).FractionationofEEACusingdifferentsolvents

pro-videdthreefractions,amongthemtheethylacetatefraction(FEA) wastheprimarycontributortoEEACcytotoxicity.The aforemen-tionedresultsencouragedustoidentifyFEAmajorcomponentsand studytheircytotoxicactivity.Webelievethatathorough under-standingof thecytotoxicmolecularmechanism inducedbyFEA majorcomponentswillshedlightonthepotentialutilizationof thesecomponentsasfutureanticanceragents.

Materialsandmethods

Assessmentofapoptosis

TheaccumulationofthesubG1populationincancercellswas

determinedbyflowcytometry.Cellswereseededonto6cmdishes and treatedwithor withouttheindicated concentrationofthe testedsamplesfor24h.CellswerethenwashedtwicewithPBSand collectedbycentrifugationat200×gfor5minat4◦C.Cellswere fixedin70%(v/v)ethanolat4◦C for30min.Afterfixation,cells weretreatedwith0.2mlofDNAextractionbuffer(0.2MNa2HPO4

and0.1Mcitricacidbuffer,pH7.8)for30min,centrifuged,and resuspended in 1ml of propidium iodide staining buffer(0.1% TritonX-100,100␮g/mlRNaseA,500␮g/mlofpropidiumiodide inPBS)at37◦Cfor30min.Cytometricanalyseswereperformed usingaﬂowcytometerFACS-Calibur(Becton-Dickinson,SanJose, CA,USA)andCellQuestsoftware.Approximately10,000cellswere countedforeachdetermination.

Theexternalizationofphosphatidylserine(PS)andmembrane integrity wasquantiﬁedusing anAnnexinV-FLOUS stainingkit (RocheDiagnosticsGmbH,Mannheim,Germany).Inshort,106_cells

weregrownin35mmdiameterplatesandlabeledwithAnnexin V-FLOUS(10␮g/ml)andpropidiumiodide(PI)(Sigma–AldrichCorp., St.Louis,MO,USA)(20␮g/ml)priortoharvesting.Afterlabeling, allplateswerewashedwithbindingbufferandharvestedby scrap-ing.Cellswereresuspendedinbindingbufferataconcentrationof 2×105_cells/ml_before_analysis_by_ﬂow_cytometry.

NeutralcometassayforthedetectionofDNAdouble-strand breaks(DSBs)

TheassaywascarriedoutusingaCometAssayTM_Kit_(Trevigen

Inc.,Gaithersburg,MD,USA)followingthemanufacturer’sprotocol fortheneutralcometassay.Brieﬂy,cancercells(2×105_cells/ml)

weretreatedwiththetestedsamplesfor18h.Cellswere com-binedwith1%lowmeltingpointagaroseataratioof1:10(v/v). From themixture,75␮l werepipetted onto CometSlideTM _and

allowedtostandat4◦Cin thedark.The slideswereimmersed inice-coldlysissolutionfor30–60min.Theslideswereplacedin ahorizontalelectrophoresisapparatusandelectrophoresedin1× TBE(90mMTris–HCl,90mMboricacid,and2mMEDTA,pH8.0) at20V for10min.Thesampleswerethen fixedin70% ethanol anddriedbeforebeingstainedwith1:10,000SYBRGreenIto visu-alizecellularDNA.Thefluorescenceimageswereanalyzedusing theTriTekCometImageprogramtocircumscribethe‘head’and the‘tail’regions ofeach cometand theintegratedfluorescence

valuesofeachdeﬁnedareawererecorded.Thecometlengthwas measuredfromthetrailingedgeofthenucleustotheleadingedge ofthetail.ThislengthwasindicativetotheextentofDNAdamage. Calculationswereaveragedperreplicate.

Westernblottinganalysis

Celllysateswerepreparedasdescribedpreviously(Yangetal. 2006), by treating thecells for 30minin RIPA lysis buffer[1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1mM sodium orthovanadate, 100␮g/ml phenylmethylsulfonylﬂuorideand30␮g/mlaprotinin,all chem-icalswereobtainedfromSigma–AldrichCorp.].Thelysateswere centrifugedat20,000× gfor30min,and theprotein concentra-tioninthesupernatantwasdeterminedusingaBCAproteinassay kit (Pierce, Rockford, IL, USA).Equal amounts of proteinswere respectivelyseparatedby7.5%,10%or12%ofSDS-polyacrylamide gelelectrophoresisandthenelectro-transferredtoaPVDF mem-brane.Themembranewasblockedwithasolutioncontaining5% fatfree drymilk TBSTbuffer(20mM Tris–HCl,pH7.4,150mM NaCland0.1%Tween20)for1handwashedwithTBSTbuffer. Pro-teinexpressionwasmonitoredbyimmunoblottingusingspeciﬁc antibodies:Bcl-2andBcl-x(Santa-CruzBiotechnology,SantaCruz, CA,USA);caspases3,7,8and9;Bax,Bid,Bad,p-H2AX,andPARP (CellSignalingTechnologies,Beverly,MA,USA).Caspases3,8,and 9inhibitors(Z-DEVD-FMK,Z-IETD-FMK,andZ-LEHD-KMK)were purchasedfromR&DSystemsInc.(Minneapolis,MN,USA). Anti-mouseandrabbit1gGperoxidase-conjugatedsecondaryantibodies werepurchasedfromPierce.Theseproteinsweredetectedbyan enhancedchemiluminescencekit(Pierce,Rockford,IL,USA). AssayoftopoisomeraseIIinhibitorsandpoisons

Theassaywasperformedasdescribedpreviouslywithminor modiﬁcations(Girietal.2010).Standardrelaxationreaction mix-tures(20␮l)containing50mMTris–HCl(pH8.0),10mMMgCl2,

200mM potassium glutamate, 10mM dithiothreitol, 50␮g/ml bovineserumalbumin,1mMATP,0.3␮gofpHOT1plasmidDNA, two unitsof human topoisomerase II(Topogen,Columbus, OH, USA)andtheindicatedconcentrationsofetoposideandDeEAwere incubatedat37◦Cfor30min.Reactionswereterminatedbyadding 2␮lof10%SDStofacilitatetrappingtheenzymeinacleavage com-plex,followedbytheadditionof2.5␮lofproteinaseK(50␮g/ml) todigesttheboundprotein(incubated37◦Cfor15min)andﬁnally byadding 0.1volofthesampleloadingdye.TheDNAproducts wereanalyzedbyelectrophoresisthroughvertical2%agarosegels at2V/cmin0.5×TAEbuffer.Gelswerestainedwithethidium bro-mideandphotographedusinganEagleEyeIIsystem(Stratagene, LaJolla,CA,USA).

HumanleukemiaHL-60cellsxenograftanimalmodel

Establishmentof nudemice withxenograftswasperformed asdescribedpreviously(Dowdyetal.2006).Six-week-oldfemale immunodeﬁcientathymicmicewerepurchasedfromtheNational LaboratoryAnimaland ResearchCenter(Taipei,Taiwan).Allthe animalswere maintained under standard laboratoryconditions (temperature24–26◦C,12–12hdark–lightcircle)andfedwith lab-oratorydietandwater.Theprotocolsofanimalexperimentswere conformedtotheGuidefortheCareandUseofLaboratoryAnimals, publishedbytheUSNationalInstituteofHealth(NIHpublication no.85-23,revisedin1996).

HL60cells(1×107₎_resuspended_in_0.2_ml_PBS_were_injected_s.c.

intotherightﬂankofeachmouse,andtumorgrowthwas moni-toredeveryday.Fourteendaysaftertumorcellinjection,micewith conﬁrmedtumorgrowthwererandomlydividedintotwogroups.

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Fig.1.Westernplottingandalkalinecometassays.(A)EffectofEEACandFEAonapoptoticandDNAdamaged-relatedproteinsinHL60cells(24h).(B)EffectofFEAon tumorsuppressorgenes,apoptoticandDNAdamaged-relatedproteinsinHL60cells(12hand24h).(C)EffectofFEAontheexpressionofsurvival-relatedproteinsandCdk4 (12hand24h).(D)AssessmentofDNAdamagebycometassay.HL60cellsweretreatedwithEEACorFEAfor18handthensubjectedtothealkalinecometassaytodetect thebrokenDNA(comettails).

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Fig.2. HPLCchromatogramoftheethylacetatefraction(FEA,anactivetriterpenoid-richfraction)fromtheethanolicextractofwildAC.Standardisolates(compounds1–5) areshownforcomparison.

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Fig.3.TheeffectofFEAcompounds2–5onapoptosisinductionandDNAdamageinHL60cells.(A)FACSdotplotsrepresentingHL60cellsstainedwithAnnexinVandPI aftertreatmentwith2–5(72h).(B)Effectof2–5onthepercentageofAnnexinV-positivecellsatdifferenttreatmenttimes(24,48,and72h).(C)Westernblottingdetecting theexpressionofapoptoticandDNAdamaged-relatedproteinscausedby2–5.

DeEA(10␮g/g)wasorallyadministeredtothetreatmentgroup and thecontrolgroup receivedsolventonly.DeEAwas admin-istratedeveryotherdayforﬁveweeks.Animalsweresacriﬁced bycarbondioxide.Thetumorswerecarefullydissectedfromeach

mouse,andtumorweightsweremeasured.Halfofthetumortissue wasﬁxedwithformalinandusedforimmunohistochemical stud-ies,whereastheotherhalfwassnapfrozeninliquidnitrogenand storedat−80◦_C._These_tissues_were_used_to_extract_proteins_for

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Fig.4.ThecytotoxiceffectofDeEAonHL60cells.(A)DeEAandFEAcytotoxicactivityatvariousconcentrations(72h)inMTTassay.(B)EffectofDeEAandFEAon anti-apoptoticandpro-apoptoticproteinsandcaspases3,7,and9(72h).(C)GraphicalrepresentationofthepercentagesofcellsatSubG1,G1,SandG2/MphasesinDeEA-treated

cells.(D)RepresentativeimagesofcometassayshowingabnormalcomettailscausedbychromosomalDNAdoublestrandbreaksinDeEA(2.5,5and10␮g/ml)treatedHL 60cells.(E)DeEAeffectontailmovementinHL60cells.HistogramwasgeneratedbyTriTekCometImageprogram.Valuesareexpressedasmean±SEM.*Signiﬁcantly differentfromcontrolgroupsatp<0.05.(F)EffectofDeEAonthecleavageofPARPandonthephosphorylationofH2A.XandChk2.

westernblottingassay.Tumorsizewasmeasuredthreetimesa weekusingcalipersandtumorvolumeswerecalculatedaccording tothestandardformula:width2_×_length/2.

Statistics

Theresultswereexpressedasmean±standarddeviation(SD). Comparisonineachexperimentwasperformedusinganunpaired Student’st-test,anda pvalueofless than0.05wasconsidered statisticallysigniﬁcant.

Resultsanddiscussion

TheapoptoticandtheDNAdamagingactivitiesofEEACandFEA Ourpreviousstudyrevealedthattheethanolicextractofwild AC(EEAC)anditsactivefraction(FEA,theethylacetatefraction fromEEAC)possessedcytotoxicityagainst leukemiaHL60 cells withIC50valuesmeasuredafter24hof104.82and80.53␮g/ml,

respectively(Luetal.2009a).Inthecurrentstudy,EEACandFEA werepreparedaspreviously reported(seesupplementarydata,

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Y.-C.Duetal./Phytomedicinexxx (2012) xxx–xxx 7 Table1

ComparisonsofpeakareaandheightfortherespectivecomponentsinFEAwithHPLCat254nm.

Compound Peaknumber Retentiontime(min) Area(%) Height(%)

AntcinK 1a 6.004 4.96 7.08 1b 6.175 6.23 9.40 AntcinC 2a 24.805 7.34 4.20 2b 26.733 5.36 4.36 ZhankuicacidC 3a 27.778 6.91 6.59 3b 28.981 8.07 7.59 ZhankuicacidA 4a 46.825 7.17 9.24 4b 47.453 6.37 7.68 Dehydroeburicoicacid 5 78.058 4.58 8.79 Others 43.01 35.07 Total 100.00 100.00

and100␮g/mlofEEACorFEAandsubsequentlycelllysateswere analyzedbywesternblotting.FEAorEEACinduced phosphoryla-tionofH2A.XatSer-139(␥H2A.X)(Fig.1A)whichisconsideredas abiomarkerforDNAdouble-strandbreaks(KuoandYang2008; Mahetal.2010).Additionally,theuseofFEAorEEACenhancedthe cleavageofPARPandtheactivationofcaspase3,whichareboth regardedasmarkersofapoptosis(Fig.1A).Theseresultsimplied thatFEAistheactiveapoptoticfractionofEEACandthusselected forfurtherinvestigation.

TheeffectofusingFEA(100␮g/ml)onproteinexpressionwas also determined. FEA ledto a marked increase in the amount oftumorsuppressorgenes,p21andp27,enhancementofH2A.X and Chk2 phosphorylation, aswellas induction ofPARP cleav-age(Fig.1B).Ontheotherhand,phosphorylationlevelsineachof thesurvivalproteins(Akt,Raf,andPTEN)aswellasthe expres-sionofCdk4 decreasedfollowing treatmentwithFEAina time dependentmanner(Fig.1C).TheDNAdamagingeffectofFEAwas furtherconﬁrmedusingalkalinecometassayresultingincomets withabnormal tailssizes (Fig. 1D). Theaforementioned results conﬁrmedtheDNAdamagingandtheapoptoticactivitiesofFEA implyingitsimportanceasapotentialcytotoxicagent(Kimetal.

2011).

TheapoptoticandtheDNAdamagingactivitiesofFEAmajor components

FEAmajorcomponentswereseparatedbynormalphase chro-matography and puriﬁed by reverse phase high performance liquidchromatography(HPLC)yieldingﬁvemajorconstituents(see

supplementarydata, sectionS4). Theisolated compoundswere

fourergostanes,antcinK(1),antcinC(2),zhankuicacidC(3),and zhankuicacidA(4)aswellasonelanostane,dehydroeburicoicacid

(5)(Chenetal.1995;Shenetal.2003).Theisolationofthesemajor

triterpenesasstandardsledtotheestablishmentofHPLC ﬁnger-print ofFEA(Fig.2)(seesupplementarydatafor detailedHPLC

condition,sectionS5).Accordingtothepublishedreport(Chenetal.

1995),eachACergostanetriterpene(compounds1–4)existsasa pairofstereoisomersduetothepresenceofchiralcenteratC-25. Twopeaksrepresentinganisomericpairofeachergostanecanbe observedintheHPLCﬁngerprint.Forexample,peakpairs1aand 1b,2aand2b,3aand3b,aswellas4aand4bcorrespondingto antcinK(1),antcinC(2),zhankuicacidC(3),andzhankuicacid A(4),respectivelywereobserved(Fig.2B–E).Ontheotherhand, dehydroeburicoicacidwithnochiralcenteratC-25existedonly asasingleisomer,peak5(Fig.2F).PeakareasandheightsofFEA majorcomponents(1–5)at254nmwerecalculatedandcompared asshowninTable1.Theareasofthelabeledpeaks(1–5)accounted for56.99%ofthetotalpeakareasobservedintheHPLCﬁngerprint.

Regardingtheantiproliferativeactivityof1–5againstleukemia HL 60 cells, 1showednoactivity evenat a highconcentration (100␮g/ml). Compounds2–5exhibitedantiproliferative activity againstHL60cellsandwerefurtherinvestigatedusingAnnexin VandPIdoublestainingtechnique.HL60cellsweretreatedby dif-ferentconcentrationsofantcinC(2),zhankuicacidC(3),zhankuic acidA(4),anddehydroeburicoicacid(5)for24,48,and72h.Cells werestainedwithAnnexinV/PIandexaminedwithﬂow cytom-etry.FACSdotplotsrepresentingAnnexinVandPIstainingafter thetreatmentwith2–5areshowninFig.3A.Thequantiﬁcation ofAnnexinV/PIexperimentrevealedthat4and5increasedthe apoptoticpopulationofHL60cellsinadosedependentmanner. Ontheotherhand,only5(10␮g/ml)inducedapoptosisinatime dependentmanner(Fig.3B).

Compounds 4 and 5 induced cleavage of PARP in a dose dependentmanner(Fig.3C).Thetreatmentof HL60 cells with

4 (20␮g/ml) and 5 (10␮g/ml) induced the activation of cas-pase 3 and the phosphorylation of H2A.X. The analytical and thebiologicalassaysclearlyimpliedthat 5(DeEA)isFEAmajor component withthemostpotentapoptotic andDNA damaging activities.

TheapoptoticandtheDNAdamagingactivitiesofDeEA

TofurtherconﬁrmthefactthatDeEAistheleadactive com-poundofFEA(triterpenoid-richfraction)thecytotoxicityofDeEA andFEAagainstHL60cellswascompared(Fig.4A).DeEA exhib-itedmorepotentcytotoxicitycomparedtoFEAwithIC503.39and

22.4␮g/ml,respectively,after72hasindicatedbyMTTassay.The effectofDeEAandFEAontheexpressionofanti-apoptotic (Bcl-2 and Bcl-x) and pro-apoptotic (Bax, Bid,and Bad) proteinsas wellastheactivationofcaspases3,7,and9wasalsoinvestigated (Fig.4B).DeEAwasmorepotentcomparedtoFEAinenhancing theexpressionofpro-apoptoticproteinsBidandBad.AlsoDeEA inducedtheactivationofcaspases3,7,and9andsimilarresults wereobserveduponthetreatmentofFEA.Toanalyzewhetherthe apoptoticinductionofDeEAandFEAinvolvesintrinsicorextrinsic apoptoticpathways,speciﬁcinhibitorsforcaspases3,8,and9were tested.Pretreatmentofcellswithcaspaseinhibitorsdidnotinhibit theapoptoticeffectofDeEAandFEA,suggestingthattheapoptotic effectofDeEAandFEAisindependentoncaspaseactivation(data notshown).

TreatmentofHL60cellswithincreasingdosesofDeEA(0,2.5,5 and10␮g/ml)resultedinG2/Mdistributionratesof21.1%,26.6%, 28.7%and39.5%,respectivelyafter24h(Fig.4C).Similarresults wereobservedafter48and72h.ThusDeEAtreatmentappearedto induceG2/Mphasearrestinadosedependentmanner.

ToclarifytheroleofDeEAasDNAdamagingagent,wetreatedHL 60cellswithvariousconcentrationsofDeEAfor18handanalyzed

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theleveloftailmovementwiththealkalinecometassay.DeEAat 10␮g/mlcausedsevereDNAdamageinHL60cellsasindicatedby abnormaltailsizeincometassay(Fig.4DandE).

ToexaminetheextentofDNAdamagewhichcanleadtothe acti-vationofcellcyclecheckpointsinHL60cells,theeffectofDeEAon PARP,p-H2A.Xandp-Chk2wasstudied(Fig.4F).DeEAat10␮g/ml significantlyincreasedthephosphorylationofChk2andH2A.X,but didnotchangethephosphorylationofChk1(datanotshown). Addi-tionally,DeEAinducedPARPcleavage.Thesefindingsconfirmed theapoptotic and theDNA damagingactivities of DeEAon HL 60cells.

TheinhibitoryactivityofDeEAontopoisomeraseII

TopoisomerasesareessentialenzymesforDNAmetabolismand theirinhibitorsareemergingaspotentialcytotoxicagents. Expo-sureof cellsto DNAtopoisomerase I(topoI) or topoisomerase II(topo II) inhibitorsleads toDNA damage that ofteninvolves

Fig.5.EffectofDeEAontopoisomeraseIImediatedsupercoiledpHOT1plasmid DNArelaxation.Lane1–3:positivecontrol,etoposide(100,200and400␮M);Lane 4:negativecontrolplasmidDNA;Lane5–9:DeEA(0,2.5,5,10,and20␮g/ml);Lane 10:plasmidDNA+topoisomeraseII;Lane11:linearDNA.

Fig.6.EffectofDeEAonHL60cellstumorgrowthinxenograftanimalmodel.FemalenudemicebearingleukemiaHL60tumorsweretreatedwiththesolvent(negative control)orDeEA(10␮g/g)for5weeks.(A)Arepresentativepictureoftumorgrowthinxenograftnudemiceadministratedsolventonly(left)andDeEA(right).(B)Tumor volumesweremeasuredeveryotherday,andresultsareexpressedasmean±SEM.*Significantlydifferentfromcontrolgroupsatp<0.05.(C)Arepresentativepictureof tumortissueasitappearsintherightflankofthemouse.(D)HistogramofthetumorweightfromthecontrolgroupandDeEAtreatedgroup.Valuesareexpressedas mean±SEM.*Significantlydifferentfromcontrolgroupsatp<0.05.(E)EffectofDeEAonPARPandonH2A.Xintumorsamples.Twomicewerepickedrandomlyfromeach group.C1andC2representtwomicefromthecontrolgroup.DeEA1andDeEA2representtwomicefromtheDeEAtreatedgroupchosenrandomly.

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Y.-C.Duetal./Phytomedicinexxx (2012) xxx–xxx 9 theformationofDNAdouble-strandbreaks(Darzynkiewiczetal.

2009; Deng et al. 2009).Mizushina et al. (2004) revealed that

dehydroebriconicacid,fromthesclerotiumofPoriscocosinhibited DNAtopoIIwithnoactivityonDNAtopoI.Thestructureof dehy-droebriconicacidissimilartoDeEAexceptthehydroxylgroupat C-3inDeEAisreplacedbyacarbonylgroupindehydroebriconic acid.

TheinhibitoryactivityofdehydroebriconicacidonDNAtopo IIencouragedustoexaminewhetherDeEAtreatmentcaninduce DNAdamageinleukemiacellsthroughinhibitionoftopoIIornot. Forthis purpose,a cell-freeDNAcleavageassaywasperformed using enzyme-mediated negatively supercoiled pHOT1plasmid DNA.ItwasfoundthatDeEAinducedDNAcleavageinthepresence oftopoII(Lanes6–9)(Fig.5),asindicatedbytheappearanceof lin-earDNA(markedbyL).Theresultsweresimilartothoseobtained uponutilizingetoposide,astandardtopoIIinhibitor(Lanes1–3) (Dasetal.2007).

TheinhibitoryeffectofDeEAontumorgrowthinxenograftanimal model

HL60cellswereinoculatedsubcutaneouslyattherightflankof femaleimmunodeficientathymicmice.After5weeksoftreatment, thetumorgrowthofHL60cellswassignificantlysuppressedby oraladministrationofDeEA(10␮g/g)(Fig.6A).Theaveragetumor sizeonday31inthecontrolgroupwas229.46mm3_,_whereas_the

averagetumorsizeintheDeEA-treatedgroupwas102.75mm3

(Fig.6B).ThetumorsizewassigniﬁcantlylowerintheDeEA-treated groupas compared tothecontrol group(p<0.05) withno sig-niﬁcantdifferenceinthemice bodyweights. Attheend of the treatment,thetumortissuewasisolatedandweighed(Fig.6C). The mean of tumor weights was obviously less in the DeEA-treatedgroupcomparedtothecontrolgroup(p<0.05)(Fig.6D). Thewesternblottinganalysisoftumortissueindicatedthat the levelofPARPwasdecreasedandthephosphorylationofH2A.Xwas increasedinDeEA-treatedgroupcomparedtothecontrolgroup (Fig.6E).

Conclusion

Inthisstudy,weillustratedtheapoptoticandtheDNA damag-ingactivitiesofthetriterpenoid-richfraction(FEA)anditsmajor componentsobtainedfromtheethanolicextractofA.camphorata. FEAinducedPARPcleavageandcaspase3activationindicatingits apoptoticactivity.ItalsoinducedthephosphorylationofH2A.X as a marker of DNA double strandbreak. From the active FEA fraction, dehydroeburicoicacid(DeEA)wasisolated asthelead most active component. DeEAinduced G2/M phase arrest in a dose dependent manner in HL 60 cells. It also induced apop-toticandDNAdamagingmarkersrevealingitsroleasthepotent cytotoxicconstituentofFEA.AdditionallyitinhibitedtopoII sug-gestingthepossibleroleofthisenzymeinthecytotoxicactivity ofDeEA.TheantitumoreffectofDeEAwasfurtherdemonstrated inreducingtumorweightandsizeinxenograftanimalmodel.The presentﬁndingsnotonlysupporttheethnopharmacologicaluse ofA.camphoratabutalsodivulgetheantitumoractivityof dehy-droeburicoicacidasthemajorleadcompoundofthistreasured mushroom.

Conﬂictofinterest

Theauthorsdeclarethattheyhavenoconﬂictsofinterest.

Acknowledgments

ThisworkwassupportedbytheNationalScienceCouncilgrants (NSC98-2324-B-037-001and98-2321-B-037-060)andthe Depart-mentofHealth,ExecutiveYuan,Taiwan(DOH99-TD-C-111-002). ThisworkwasalsosupportedbytheAcademic-Industrial Collab-orative ProjectofKaohsiung Medical Universityand YungPeng BiotechnologyCo.,Ltd.,Kaohsiung(S099009and100A502).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.phymed.2012.03.014.

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