（113）

MOLECULARMECHANISMOFARSENICTRIOXIDE−INDUCEDAPOPTOSISINHL−60ANDRLCELLLINESJIEBINZHUl）andSHINOBUNAKAMURA2）1）α肋血γ7ね坤血調わぬ乃励0ゐ紗，助，MZ〃紺α【加血g戒かG和血地＆如OJ〆几ね励dJ＆滋乃Cg2）功匝血如〆α脚払協肋お肌 肋適血脚卸血町物融肋血 励馴砂
ReceivedOctober17，2003

AbsiYtlCt：Recently，ithasbeenreportedthatarsenictrioxide（As203）isaneffective

anticancer agent for acute promyelocyticleukemia仏PL）．In the present study，WeexaminedtheanticancereffectsofAs203atlowconcentrationわ．25〜2．OFLM）ontwohunan

leukemia／lymphomacelllines，HL−60andRL，invitro．WefoundthatAs203inhibitedthe
growth ofHL−60andRL similar to the reportedAPL cellline，NB4・Typicalapoptosis
was observedin morphologlCalstudy and DNAfragmentation assay，aS Wellas a cell
cycle arrest at subGl・To address the mechanism ofAs203−induced apoptosis，We also
examinedtheeffectofAs2030ntheCD95／CD95Lpathwayandbc1−2proteinexpression．
The results showed that the CD95／CD95L expressions were upregulated；meanWhile，
caspase8and caspase3were activated．However，the bcト2protein expression was
downregulated．Using antilニD95monoclonal antibody to block the CD95

pathway，

As203−inducedapoptosiswasameliorated．ThesedatasuggestthatinHL−60andRLcell
lines the CD95／CD95L pathway and downregulation of bc1−2protein expression are
invoIvedinAs203−inducedapoptosis．
Keywords：arSenictrioxide，HL−60，RL，apOptOSis，CD95／CD95Lpathway

INTRODUCTION
Arsenictrioxide（As203）hasbeenreportedtobeveryeffectiveinthetreatmentofacutepromyelocyticleukemia 仏．pL），eVenin patients relapsed after all−tranS retinoic acid
仏TRAトinduced and chemotherapy−1naintained complete remission and without・SeVere
myelosuppressionl）．RecentstudiesshowedthatAs203inducedapoptosisinsomecancercell
lines，SuChas：ChronicB−1ymphocyticleukemia2），aCutemyeloidleukemia3・4），T−Cellleukemia5），

megakaryocyticleukemia6），multiplemyeloma7・8），1ymphoma9），neurOblastomalO），humansmallcell
lung canCerll），human OVarian

CarCinoma12），and human breast cancer13）・The molecularmechanismofAs203treatmentiscontroversial．ThemodulationofPML／RARαfusionprotein

and downregulation ofbc1−2expressionin NB4，APL cellline with t（15；17）translocation，
inducedbyAs203WaSrepOrtedl）．Butrecentstudiesshowedthattheantiproliferativeeffectof
As203maybeindependentonchangesofPML／RARafusionprotein3・4・14），andbc1−2expression・

ThesereportssuggestedthatitmightbeviamodulationoftheglutathioneredoxsysteminNB4
cells9）andviamicrotubuleinhibitioninmyeloidleukemiacells，K5624）．
Current evidence suggests that the mechanism ofsome anticancer agentsinvolves theactivationofthe CD95／CD95Lpathway15L18）．In thepresent study，Weinvestigatedanticancer
molecular mechanism of As2030n tWOleukemia／lymphoma celllines，HL−60and RL，andAs203inducesapoptosisinHL−60andRL（114）

provide evidence that As203induces apoptosis throughthe CD95／CD95L pathway anddownregulationofbc1−2proteinexpression．

MATERIALS ANDMETHODS
l．Celllines
Twohumanleukemia／1ymphomacelllineswereused．HL−60，aCutemyeloidleukemiaQ：he
French−American−Britishclassification，M2）cellline，WaSpurChasedfromtheJapaneseCancer
ResearchResourcesBank，andt（14；18）B−CelllymphomaRLcelllineoverexpressingbc1−2was

obtainedfromtheAmericanTypeCultureCollection択ockville，MD，USA）．Theywerecultured
in RPMI1640medium supplemented withlOO U／ml penicillin，100FLg／ml streptomycin，1mmol／LL−glutamine，and10％fetalbovineseruminahumidifiedatmosphereof95％air／5％
CO2at37℃．
2．Reagent

As203（Wako，Japan）was dissoIvedin distilledwateratastocksolutionofO．01Mand
dilutedtoworkingconcentrationwithphosphate−bufferedsaline住｝BS）beforeusC．

3．Cellgrowthandviabilityassay
Cell growth and viability were determined by MTT（3−［4，5−dimethylthiazo1−2−yl］−2，5−diphenyltetrazoliumbromide）test19）．Briefly，thecells（2×105／ml）Wereharvestedwithor
withoutdifferentconcentrationわ．25−2．OFLM）ofAs203for1−5days，reSpeCtively．Atassaytime，
thecellswerewashedtwicewithcoldPBSand200plofthecellsuspensionwereculturedin96
mult卜welldish，then20FLlofMTTsolutionwereaddedtoeachwellandkeptat37℃for2
hours．Afterthedishwascentrifugedat400gfor5minutes，themediumwasdiscardedand200
FLlofDMSO bimethylsulfoxide）wasaddedtoeachwelltodissoIveMTTfomaZanS．Then
opticaldensitiesweremeasuredbyELISAscanat550−650nm＄LT−Labinstruments，Austria）．
4．MorphologicalobservationbyGiemsastain
Aftertreatmentwithorwithoutl．OFLMofAs203for3days，100iLlofcellsuspensionwere
spunontoslidesbyCytospin＄handon，800rpm，8minutes）andstainedwithGiemsasolution．
Themorphologicchangesofcellswereobservedandphotographedwithlightmicroscopy．
5．Cellcycleanalysisandquantificationofapoptoticcells
Cell cycle was analyzed by flow cytometry釘CM，Becton Dickinson）．As described
previous1y20），atOtaloflO6cellswithorwithoutl．OFLMofAs203treatmentfordifferentdays
werewashedtwicewithPBSandcentrifuged（200×g），thenthecellpelletwasresuspendedinl．5mlhypotonicfluorochomesolution［propidiumiodide任〉I）50Fg／mlinO．1％sodiumcitrateplusO．1％TritonX−100（Sigma）］inthedarkovernightandexaminedbyFCM．Apoptosiswas
quantifiedbymeasuringsubGlfraction．

6．DetectionofCD95，CD95Landbc1−2expression
CD95and CD95L expressions were assessed by FCM．After treatment with or without

J．ZHUgfd（115）

1．0−2．OpMAs203forlday，1050fcellswerewashedtwicewithcoldPBSandincubatedwith5．O
FLlofFITC−COnjugatedantilnOuSeIgGl（MBL，Japan），anti−humanCD95monoclonalantibody（MoAb）（MBL，Japan）oranti−humanCD95LMoAb釘ujisawa，Japan）at4℃inthedarkfor30
minutes．ThenthecellswerewashedthreetimeswithcoldPBSandmeasuredbyFCM．
Forthedetectionofbc1−2proteinexpression21），aftertreatmentwithorwithoutl，OpMAs203
for2days，pellet of5×106

cells was resuspendedin2mlof2％paraformaldehyde．AfterincubationforlOminutesonice，100FLlof1％Triton−Ⅹ100wasadded．Tenminuteslater，Cells
WereWaShedtwicewithcoldPBSandincubatedwith5FLlofFITC−COnjugatedantirmouseIgGl（MBL，Japan）oranti−bcト2MoAb軒ujisawa，Japan）for30minutes．Afterwashingthreetimes，
thecellswereanalyzedbyFCM．
7．Caspase8andcaspase3activityassay
Caspase8and caspase3activitieswere determinedby fluorometricprotease assay kits

（MBL，Japan）．Briefly，aftercellswereincubatedwithorwithoutl．OFLMofAs203for2days，pelletoflO6cellswereresuspendedin50FLlofchilledcelllysisbufferandincubatedoniceforlO
minutes．50FLlofthelmMIETD−AFC住．le一石10−Thr−Asp−7一aminoT4trifluoromethylcoumarin）

substrate for caspase

8

0r thel mM DEVD−AFC

仏splilo−Val−Asp−7−amin0−4

trifluoromethylcoumarin）substrateforcaspase3wasaddedtotheculturedcellsandincubated

at37℃for2hours．Thensampleswereexaminedbythefluorometerwitha385nmexcitation
filteranda538nmemissionfilter．

8．BlockingoftheCD95pathwaybyanti−CD95IgGMoAb
Anti−｛D95IgGMoAb（MBL，Japan）wasusedtoblockCD95pathway．Briefly，atOtalof4×

105cellswithorwithoutl．OFLg／mlofanti−｛D95MoAbwereincubatedfor3hoursat37℃．Thenthecellsweretreatedwithorwithoutl．OpMofAs203for4days．Finallytheapoptoticcellswere
determinedbyPIstaining．

9．Statisticalanalysis
Resultswereexpressedasmean±standarddeviation6D）．statisticalsignificanceb＜0．05）
WaSaSSeSSeduslngStudent

sttest．

RESULTSl．EffectofAs2030nCellgrowthinhibition
CellproliferationwasassessedbyMTTassay．Time−dependentanddose−dependentcell
growthinhibitionsattheconcentrationofAs203わ．25−2．OFLM）areshowninFig．1．As203at
1．0−2．OiLMsignificantlyinhibited thegrowthofbothcelllinesthroughoutcourse伍ay1−5）．
These results showed that As203

WaS a pOtentinhibitor of the proliferation ofleukemia／1ymphomacells．
2．MorphologicalchangeSandcellcyclearreSt

After the treatment ofAs203（1．OiLM）for3days，typicalapoptotic changes，SuCh as
chromatincondensation，Cellularshrinkageandapoptoticbodieswereobserved酔ig．2A）．To
investigatewhethertheinhibitionofcellgrowthisduetocellcyclearrest，WeeXaminedtheAs203inducesapoptosisinHL−60andRL

（116）

HL．

0

や

COntrOl

8

O
O

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e叫7em的白○や As103；0．25ルM
ヰ呵 Aち03：0．5恥M
や Aち0，：1・0ルM
ヰ Aちq；2．0ルM

0

1

2

3

4

5

4

5

億me（days）

B 2・0

0

1

2

3
time（days）

Fig．1．AsD3inducescellgrowthinhibitioninHLL60仏）andRL（B）withvariousconcentrationsfor5days・
Viability of cells was measured by MTT assay．The data are shown as mean±SDfrom three
independentexperiments．

distributionofcellularDNAcontentsbyFCM．FCManalysisshowedadistinctsubGlpeak
fromcellstreatedwithl．OFLMAs203for4daysQig．2B）．
3．UpregulationofCD95／CD95Lexpression

TounderstandmechanismsresponsibleforAs203−inducedapoptosis，theexpressionsof
CD95andCD95LweremeasuredduringthetreatmentofAs2030nHL−60andRLcells・Both
CD95andCD95LproteinsorlglnallyexpressedontwocelllineswereupregulatedbyAs203

J．ZHUelaL

S

l

■t三d

m

賢J

0

el

HL−

t
n
O
C

、専●

札鵬I

B
con伽I Aち03；lJ上M▲：恕 dayl
I

写

亀
■■■一U

day2

01
■■
OU

8M O︐

day4

羞

Fig．2．ApoptoticchangesofcellmorphologyandcellcyclebyAs203．
仏）ApoptoticchangesshowedaftertreatmentwithlpMAs203for3daysinHL−60andRLbyoriginal
magnification（×400）．03）cellcyclearrestedatsubGlafterlpMAs203treatmentfor4daysinHL−60

andRLbyFCM．

Tablel．Activationofcaspases8and3byAs203．

Con廿01
M
RL

201．7土5＄
165．3士55

A亀q
386．5士＄3．2

コ9．9土31．8

391．名士62．2

257，1土75．1

112．5±9．62（比．3士50．9

AfterHL−60andRLincubatedwithorwithoutlL▲MAs203for2days，

CaSPaSeS8and3wereactivated．Thedataareshownasmean±SDfrom
threeindependentexperiments．＊（P＜0．05）Wereconsideredtorepresent
asignincantdifferencebetweentreatmentandcontrol．

treatmentforoneday釘ig．3）．
4．Activationofcaspase8andcaspase3
WestudiedwhetherAs203affectsthedownstreamofCD95／CD95L，includingcaspase8and
CaSpaSe3．Fluorometricproteaseassayshowedthatthefunctionsofcaspase8andcaspase3
increasedsignificantlyinHL−60andRLincubatedwithl．OFLMAs203for2days（rablel）．

As203inducesapoptosisinHL・60andRL

（118）

A
爵lR−翳00縛■︳■00

H

目一山かmou鑓Ⅰがl B■ untr朋血瘍日

Aち03；1〟M

6
−
L

叫．

ロンI辱

Fig．3．UpregulationofCD95／CD95LexpressionbyAs203．仏，B）HL−60culturedwithorwithoutlFLMAs203
for24hours．虹，D）RLculturedwithorwithoutlFLMAs203inCD95expressionand2FLMAs203in
CD95Lexpressionfor24hours．

5．InhibitionofAs203−inducedapoptosisbyanti−CD95MoAb
InordertoconfirmWhetherAs203inducesapoptosisviaCD95／CD95Lpathwayonly，aS
showninFig．4，CD95／95Lpathwaywasblockedbyanti−tD95MoAb・As203−inducedapoptosis
decreasedsignificantlylnnumberinHL−60andRL；however，anti−tD95MoAbdidnotblock
As2037inducedapoptosiscompletely．
6．Downregulationofbc1−2expression
Westudiedwhetherbcト2proteinregulatesAs203−inducedapoptosisinHL−60andRL・As

showninFig．5，bothcelllineshigh1yexpressedbc1−2proteinorlglnally・Aftertreatmentwith
l．OFLMAs203for2days，bc1−2wasdownre糾llatedsignificantly・
DISCUSSION

Recently，As203isaveryeffectiveagentinthetreatmentofpatientswithAPL22・23），butthemolecularmechanismofitsactionhasnotbeenwellunderstood．ManytargetSOfAs203have
beenreportedindifferentpapers，forexample，PML／RARαfusionproteinl），glutathioneredox

system9）andbcト21−3・8・10）・Inthepresentstudy，WeteStedanticancereffectsofAs2030nOther
leukemiacelllines，HL−60andRLatlowconcentrationsofO・25−2・OFLM，Whichwasusedtokeep
thesameconcentrationswithinvivoplasmaleve125）．Time−anddose−dependentinhibitionsof
cellularproliferationweresimilartothatinAPLcellline，NB41）．Inaddition，themorphologiC（119）J．ZHUgfαエeontroI

antiCD95
A5203：1〟M

′

0

鶉
】「

0

0
5
4

0

︵辞︶S叫等lded司

anliCD95＋AちOJ：1〟M

っ

0

J

0
2

0
1

0

0

1

2

3

4

3

4

time（days）

0

1

2

time（days）
Fig．4．EffectofliLMAs2030nblockingtheCD95／CD95Lpathwaybyanti−tD95MoAb・UsingPIstaining，
apoptoticcellsofHL−60andRLweredeterminedfor4daysbyFCM．Thedataareshownasmean±
SDfromthreeindependentexperiments．＊Q｝＜0．05）showssignificantdifference．

changesofapoptoticdeath，forexamplecellularshrinkage，Chromaticcondensation，apOptOtic
bodiesandcellcyclearrestinsubGIwerefound．ThesefindingssuggestthatAs203inducescell
killingviaapoptosis．

CD95andCD95LactivateasignalcascadeviaFasrassociateddeathdomain軒ADD）protein

As203inducesapoptosisinHL−60andRL（120）

QQN Ou1 0Nl
宕
月⊆古U

A

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〇

虻レ2

○ロN

●●

Oul
O∞

﹄iざ∪

ONl

⊂I

す

く⊃
1

BCL−2
Fig．5．Downregulationofbc1−2proteinexpressionbyFCM．HL−60andRLculturedwithorwithoutl・OFLM
As203for2days．

tocleaveprocaspase8tocaspase8，thencaspase8cleavesprocaspase3tocaspase3asfinal
StepOfcelldeath24￣28）．CurrentevidencesuggeststhatthemechanismofsomeanticanceragentsisinvoIvedin theactivationoftheCD95／CD95Lpathwaywhichisakey signalpathway toregulate

apoptosis15￣18）．We

studiedwhetherthispathwayisinvoIvedin

As203−induced

apoptosisinHL−60cellsandRLcells．TheCD95andCD95Lproteinsoriginallyexpressedon
twocelllineswereupregulatedbyAs203．ThesefindingssuggestthattheCD95／CD95Lpathway
maybeinvoIvedintheapoptosisofthecelllinesinducedbyAs203．Tofurthersupportthis
hypothesis，We determined activities ofcaspase8and caspase3，Which are downstream of
CD95／CD95L．Theresultsshowedthatcaspase8andcaspase3wereactivatedbyAs203．Itis
also reported that caspase

8and casapse

3play animportant rolein the process of（121）J．ZHUgfαエ

As203−inducedapoptosisofleukemiacelllines8・10）．TheseindicatethattheCD95／CD95Lpathway
isinvoIvedinAs203−inducedapoptosisofHL−60andRL．TofurtherconfirmWhethercellkillingbyAs203WaSOnlythroughtheCD95／CD95Lpathway，WeblockedtheCD95pathwaybyusing
anti一｛D95

MoAb．As203−induced

apoptosis

wassuppressed．From

thisresultitwasdemonstrated that the CD95／CD95L pathwayis animportant molecular mechanismin
As203−induced

apoptosisin

HL−60andRL．However，anti一｛D95

MoAb

did

not

block

As203−inducedapoptosiscompletely．ItindicatesthatAs203−inducedapoptosisisnotonlyvia
theCD95／CD95Lpathwaybutalsoviaotherpathwayswhichremaintobedetemined．
WealsostudiedthemitochondriapathwaywhichhasbeenreportedasaCD95−independent
deathpathwayandplayingacentralroleinapoptoticactionofAs2031A3・8・10）．Itiswellknownthatbc1−2proteinisanimportantfactorofthemitochondriapathway．Inthepresentstudy，bclr2
expressionin HL−60and RL cells was downregulated by As203．This result suggests that
downregulation ofbc1−2expressionmaybeanotherimportantmechanism ofAs203−induced
apoptosisinHL−60andRL
ThemolecularmechanismofAs203−inducedapoptosisisverycomplex．Weconcludethat
As203inducesgrowthinhibition，CellcyclearrestandapoptosisviatheCD95／CD95Lpathway

anddownregulationofbc1−2expressioninHL−60andRLcelllines．As203mightbeusefulinthe
treatmentofleukemiaandlymphomaotherthanAPL．
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