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Arsenic Trioxide(As2O3)Inhibits Murine WEHI-3Leukemia in BALB/c Mice In VivoTung-Yuan Lai,1,2Jen-Jyh Lin,3,4Wen-Wen Huang,5Shu-Chu Kuo,6Yen-Fang Wen,7I-Cheng Lai,8Chin-Chung Lin,9Jai-Sing Yang,10*Jing-Gung Chung5,11*1School of Post-Baccalaureate Chinese Medicine,China Medical University,Taichung 404,Taiwan2Department of Chinese Internal Medicine,China Medical University Hospital,Taichung 404,Taiwan3Chinese Medicine,China Medical University,Taichung 404,Taiwan4Division of Cardiology,China Medical University Hospital,Taichung 404,Taiwan5Department of Biological Science and Technology,China Medical University,Taichung 404,Taiwan6Graduate Institute of Pharmaceutical Chemistry,China Medical University,Taichung 404,Taiwan7Medicinal Chemistry Laboratory,Biomedical Engineering Research Laboratories,Industrial Technology Research Institute,Hsinchu 300,Taiwan8Hope Sun Cancer Center,Chang-Hua Hospital,Department of Health,Executive Yuan,Changhua 513,Taiwan9Department of Chinese Medicine,Fong-Yuan Hospital,Department of Health,Executive Yuan,Taichung 420,Taiwan10Department of Pharmacology,China Medical University,Taichung 404,Taiwan11Department of Biotechnology,Asia University,Wufeng,Taichung 413,TaiwanReceived 26 April 2010;revised 5 August 2010;accepted 10 August 2010ABSTRACT:Arsenic trioxide(As2O3)is used clinically to treat acute promyelocytic leukemia(APL)and has activ-ity in vitro for induction of apoptosis in several solid tumor cell lines.To investigate the potential therapeutic appli-cation of As2O3for leukemia,we analyzed the effects of As2O3on the WEHI-3 cells-induced orthotopic leukemiaanimal model in vivo in this study.We established the WEHI-3 cells leukemia mice through the injection of murineWEHI-3 cells into BALB/c mice,and they were then treated with As2O3(0.9 and 4.5 mg kg21;p.o.)and/or com-bined with all-trans-retinoic acid(ATRA),(30 mg kg21;i.p.).The results indicated that(1)As2O3alone or As2O3combined with ATRA promoted the total survival rate of leukemia mice and these effects are dose-dependent;(2)As2O3did not affect the body weight but decreased the spleen weight;however,it did not affect liver weight;(3)As2O3alone or As2O3combined with ATRA increased the levels of CD3 and CD19,indicating that the differen-tiation of Tand B cells were promoted;and(4)As2O3alone or As2O3combined with ATRA did not change thelevels of Mac-3 and CD11b markers,indicating that the differentiation of the precursor of macrophage were notinhibited.Based on these observations,As2O3alone or As2O3combined with ATRA have efficacious antileuke-mia activity in WEHI-3 cells leukemia in vivo.#2010 Wiley Periodicals,Inc.Environ Toxicol 27:364371,2012.Keywords:As2O3;WEHI-3 cells;leukemia murine model;in vivoCorrespondence to:J.-G.Chung;e-mail:jgchungmail.cmu.edu.twand J.-S.Yang;e-mail:jaisingmail.cmu.edu.tw*These authors contributed equally to this work.Contract grant sponsor:Department of Health,Executive Yuan,R.O.C(Taiwan).Contract grant number:DOH99-TD-C-111-005.Published online 30 September 2010 in Wiley Online Library().DOI 10.1002/tox.20650?C2010 Wiley Periodicals,Inc.364INTRODUCTIONLeukemia is one of the major causes of death for patientswith cancer.The best treatments for patients with leukemiaare still unsatisfactory.Arsenic trioxide(As2O3),a naturallyoccurring substance,has been adopted from traditional Chi-nese medicine and it has shown to exhibit clinical efficacy inacute promyelocytic leukemia(APL),(Novick and Warrell,2000)and also induce apoptosis in various cancers such asAPL,other myeloid leukemia cells,esophageal,prostate,andovarian carcinomas(Shen et al.,2000;Uslu et al.,2000;Maeda et al.,2001).The in vitro study showed that APL cellsunderwent differentiation with low concentrations of As2O3,but they induced apoptosis at high concentrations(Chen et al.,1997a).Moreover,it was found that As2O3is a potent alterna-tive in treatment of all-trans retinoic acid(ATRA)-resistantAPL(Chen et al.1997b;Kitamura et al.1997).It was reported that As2O3inhibited growth and inducedapoptosis in the HCE 16/3 immortalized cells through adecrease of HPV E6/E7(Zheng et al.,1999)and it inducedapoptosis in cervical cancer cells through down-regulation ofHPV E6/E7(Yu et al.,2007b).These effects are obtainedwith 12 lM As2O3that can be tolerably reached(Shenet al.,1997).Recently,it was also reported that As2O3is aninhibitor of cervical cancer invasion both in vitro and in vivo(Yu et al.,2007a).It was also reported that the differentiatedeffects of As2O3are observed at subapoptotic concentrationsas is the case with other apoptosis-and differentiation induc-ing agents such as ATRA(Baj et al.,2000).Although many reports have shown that As2O3affectedleukemia cells in vitro and in vivo,the effects of As2O3onleukemia mice have seldom been reported.As2O3could bea satisfying therapeutic agent for leukemia mice,and it isour hypothesis.Therefore,the aim of this work was toinvestigate the activity of As2O3against WEHI-3-inducedleukemia mice in vivo.The results indicated that As2O3promoted the survival rate in WEHI-3 leukemia mice.MATERIALS AND METHODSMaterials and ReagentsAs2O3and ATRA were obtained from Sigma Chemical(St.Louis,MO).RPMI 1640,fetal bovine serum(FBS),penicil-lin-streptomycin,and glutamine were obtained from Invi-trogen(Carlsbad,CA).The antibodies anti-Mac-3-PE,CD11b-FITC,CD3-PE,and CD19-FITC were obtainedfrom BD PharMingen(San Diego,CA).Male BALB/c MiceThere was a total of 140 male BALB/c mice,and eachmouse about 2228 g in weight at the age of 8 weeks,pur-chased from the Laboratory Animal Center,College ofMedicine,National Taiwan University(Taipei,Taiwan).Murine WEHI-3 Leukemia CellsThe WEHI-3 cell line(murine myelomonocytic leukemiacells)was purchased from the Food Industry Research andDevelopment Institute(Hsinchu,Taiwan).The WEHI-3cells were immediately cultured into 75 cm2tissue cultureflasks in RPMI 1640 medium supplemented with 10%FBS,2 mML-glutamine,100 U mL21penicillin and 100 lgmL21streptomycin and grown at 378C under a humidified5%CO2.All cells were observed for several generations(Tsou et al.,2009;Wen et al.,2010;Yu et al.,2010).In vivo StudiesThe experiments were divided into two parts:Part I.For primary studies for examining the dose affectsof As2O3on the survival rate of BALB/c normal mice.As2O3TreatmentFifty male BLAB/c mice were divided into five groups.Eachgroupcontained10normalanimals.GroupIwas treated with PBS by oral administration.Group II wastreated with As2O3(5 mg kg21,QD,oral).Group III wastreated with As2O3(10 mg kg21,QD,oral).Group IVwas treated with As2O3(50 mg kg21,QD,oral).Group Vwas treated with As2O3(100 mg kg21,QD,oral).All ani-mals were monitored for 2 weeks,and then counted for thesurvival rate and 50%lethal dose(LD50).Thirty male BLAB/c mice were divided into three groups.Each group contains 10 normal mice.Group I was treatedwith PBS.Group II was treated with ten LD50 doses of As2O3(1/10 LD50:0.9 mg kg21,QD,oral).Group III was treatedwith half LD50 dose of As2O3(1/2 LD50:4.5 mg kg21,QD,oral).All mice were monitored for 4 weeks,and then animalswere measured for the body weight,spleen.and liver weight.Part II.As2O3and ATRA affected the survival rate inWEHI-3 cells leukemia mice.As2O3and ATRATreatmentOverall,the experimental design is shown in Figure 2(A).Each group contains 10 normal animals and all animals werei.p.injected with WEHI-3 cells(1 3 105/mice)in PBS.After14 days,60 BLAB/c mice were divided into six groups.Group I was control(PBS.QD,oral);group II wastreated with As2O3(0.9 mg kg21,QD,oral);group III wastreated with As2O3(4.5 mg kg21,QD,oral);group IVwas treated with ATRA(30 mg kg21,QD,i.p.);group Vwas treated with ATRA(30 mg kg21,QD,i.p.)combined withAs2O3(0.9 mg kg21,QD,oral);group VI was treated with365AS2O3INHIBITS WEHI-3 CELLS LEUKEMIA IN VIVOEnvironmental ToxicologyDOI 10.1002/toxATRA(30 mg kg21,QD,i.p.)combined with As2O3(0.45 mgkg21,QD,oral).All animals were treated for up to 28 daysbefore being weighed(Yang et al.,2006;Tsou et al.,2009).Blood Samples and ImmunofluorescenceStaining for Cell MarkersAt the end of treatment,about 1 mL blood was collectedfrom each animal of all experimental groups and wasindividually added into the lysing buffer(4.15 g ammo-nium chloride and 50 mL 0.1 M Tris-HCl and make up to500 mL with DD water)for lysing of the red blood cells,followed by centrifugation for 15 min at 1500 rpm at48C.The isolated white blood cells were obtained byheart puncture from each mouse for collecting theirblood and then each collected blood was measured usingcell markers including Mac-3,CD11b,CD3,and CD19from the staining with anti-Mac-3-PE,CD11b-FITC,CD3-PE,and CD19-FITC antibodies(BD PharMingen).After being washed with PBS,the cell marker levelswere determined by flow cytometry(FACSCaliburTM,Becton Dickinson,Franklin Lakes,NJ)as described else-where(Tan et al.,2009;Yu et al.,2009).Body and Tissues Samples(Liver and Spleen)WeightsEach animal from each group was weighed before bloodwas sampled.The liver and spleen samples were obtainedfrom each animal and were weighed individually and thespleen tissues were further used for histopathology(Linet al.,2009).Histopathology of Spleen TissueTissue samples from spleen were fixed in 4%formaldehydeand embedded in paraffin.Sections of 5 lm were stainedwith hematoxylin and eosin according to standard proce-dures(Yang et al.,2006;Lu et al.,2007).Fig.1.As2O3affected the survival rate and LD50 in BALB/c mice.The BALB/c micetreated PBS,or As2O3(5,10,50,and 100 mg kg21,QD,oral)for 14 days,then whole sur-vival rate(A)was counted.The BALB/c mice treated by PBS,or As2O3(0.9 and 4.5 mgkg21,QD,oral)for 28 days,then body weight(B),spleen weight(C),and liver weight(D)were measured as described in Materials and Methods.*P0.05 is a significant differencebetween control and experimental groups.366LAI ET AL.Environmental ToxicologyDOI 10.1002/toxStatistics AnalysisThe results were expressed as mean 6 SD and the differ-ence between control(only PBS treatment)and experimen-tal groups(As2O3alone or As2O3combined with ATRA)were analyzed by Students t test.*P 0.05 and*P 0.001 were taken as significant.RESULTSAs2O3Affected the Survival RateBody Weight of BALB/c MiceGroups of BALB/c mice were treated with PBS,As2O3(5,10,50,and 100 mg kg21body weight)of mice,and the rep-resentative survival rates are shown in Figure 1(A).Theresults indicated that after animals were treated with As2O3(5,10,50,and 100 mg kg21body weight)for 14 days,therepresentative survival rates were 100,90,40,30,and 0%in PBS treatment,As2O3(5 mg kg21of mice),As2O3(10mg kg21of mice),As2O3(50 mg kg21of mice)and As2O3(100 mg kg21of mice),respectively.The 50%lethal dose(LD50)of As2O3was 9 mg kg21.The BALB/c mice wastreated with 10 LD50 doses of As2O3(1/10 LD50:0.9 mgkg21)and half LD50 doses of As2O3(1/2 LD50:4.5 mgkg21).All treatments were monitored for 28 days,and thenall animals were measured for their body weight,spleen,and liver weight.The body weight Fig.1(B),spleenweight Fig.1(C),and liver weight Fig.1(D)did notFig.2.As2O3and ATRA affected the survival rate in WEHI-3 cells leukemia mice.The experimental design of As2O3and ATRA affect WEHI-3 cells leukemia animal model(A).The animal will be i.p.injected with WEHI-3 cells(1 3 105cells/100 lL)in PBS then treated with or without As2O3(0.9and 4.5 mg kg21,QD,oral)and combined with ATRA(30 mgkg21,QD,i.p.)for 28 days,then whole survival rate wascounted as treated with As2O3only or As2O3combined withATRA(B)as described in Materials and Methods.Fig.3.As2O3and ATRA affected the weights of body,spleen and liver tissues of animal.BLAB/c mice after injec-tion with WEHI-3 cells(1 3 105cells/100 lL)in PBS andtreated with or without As2O3(0.9 and 4.5 mg kg21,QD,oral)and combined with ATRA(30 mg kg21,QD,i.p.)for 28days.Body weights(A),spleens(B),and livers(C)wereweighed individually.*P 0.05;*P 0.001 is a significantdifference between control and experimental groups.367AS2O3INHIBITS WEHI-3 CELLS LEUKEMIA IN VIVOEnvironmental ToxicologyDOI 10.1002/toxshow any significant difference among PBS treatment andAs2O3(0.9 mg kg21of mice)and As2O3(4.5 mg kg21ofmice)Fig.1(B).As2O3and ATRA Affected the Survival Rateof WEHI-3 Cells Leukemia MiceThe experimental designs of As2O3and ATRA affectedWEHI-3 cells leukemia animal model Fig.2(A).Groups ofleukemia mice were treated with 10 LD50 doses and halfLD50 doses of As2O3(0.9 and 4.5 mg kg21body weight)onlyFig.2(B)and combines with ATRA(30 mg kg21bodyweight)Fig.2(C)of mice;the representative survival ratesare shown in Figure 2(B).Animals were treated with As2O3for28 days and the survival rate was 40,60,and 70%in PBS treat-ment,0.9 mg kg21of mice,4.5 mg kg21of mice with As2O3treatment,respectively.Animals were treated with As2O3and/or combined with ATRA for 28 days and the survival rate was40,70,80,and 90%in PBS treatment,4.5 mg kg21As2O3treatment,0.9 mg kg21As2O3with 30 mg kg21body ofATRA and 4.5 mg kg21As2O3with 30 mg kg21body of ATRtreatment,respectively.Apparently As2O3with 30 mg kg21body of ATRA increased the survival rate of leukemia mice.As2O3with ATRA affected theWEHI-3-induced leukemia mice body,spleen,and liver weights in vivo.Groups of mice were treated with As2O3(0.9 and 4.5 mgkg21body weight)and combined with ATRA(30 mgkg21bodyweight),the representative animalbodyweight,spleen,and liver weights are shown in Figure3(AC).The treatments of both doses of As2O3and/orATRA did not increase the body weight significantlywhen compared to PBS-treated group Fig.3(A).Bothdoses of As2O3and/or ATRA treatment decreased signifi-cantly the spleen weight compared to PBS group Fig.3(B)but they did not significantly affect the liver weightcompared to PBS Fig.3(C)except As2O3(4.5 mg kg21body weight)and it combines with ATRA(30 mg kg21body weight)treatment.As2O3Combined with ATRA Affectedthe Histopathology of SpleenSpleen tissues were isolated from each BLAB/c mice afterinjection with WEHI-3 cells and treated with or withoutFig.4.As2O3and ATRA affected the histopathology of mice spleen.BALB/c mice wereinjected with WEHI-3 cells(1 3 105cells/100 lL)in PBS for 28 days then treated with or with-out As2O3(0.9 and 4.5 mg kg21,QD,oral)and combined with ATRA(30 mg kg21,QD,i.p.)for28 days.Spleens were photographed,weighed and histopathologically examined.Colorfigure can be viewed in the online issue,which is available at .368LAI ET AL.Environmental ToxicologyDOI 10.1002/toxAs2O3(0.9 and 4.5 mg kg21body weight)and combineswith ATRA(30 mg kg21body weight)for 28 days andwere histopathologically examined.Representative picturesare presented in Figure 4.The histopathological examina-tion showed that the spleens either disclosed markedlydecreased numbers of neoplastic cells or the cells were hardto find in the red pulp from animal after exposed to As2O3and/or ATRA.Also,the number of megakaryocytesincreased.The results indicated that As2O3and/or ATRAdecreased the number of leukemia cells in spleen tissues.As2O3and ATRA Affected the Whole BloodCell Surface Marke