腺病毒介导人内皮抑素基因瘤内注射抑制鼻咽癌生长

Induction of cell cycle arrest and apoptosis in human nasopharyngeal carcinoma cells by ZD6474,an inhibitor of VEGFR tyrosine kinase with additional activity against EGFR tyrosine kinaseXia Xiao1,Jiangxue Wu1*,Xiaofeng Zhu1,Peng Zhao1,Jinlin Zhou1,Quentin Qiang Liu1,Limin Zheng1,Musheng Zeng1, Ranyi Liu1and Wenlin Huang1,2,3*1State Key Laboratory of Oncology in South China,Cancer Center,Sun Yat-Sen University,Guangzhou510060,People’s Republic of China2Institute of Microbiology,Chinese Academy of Science,Beijing100080,People’s Republic of China3Guangzhou Doublle Bio-product Inc.,Guangzhou516003,People’s Republic of ChinaZD6474is a vascular endothelial growth factor receptor(VEGFR) and epidermal growth factor receptor(EGFR)tyrosine kinase in-hibitor.The present study was undertaken to investigate the direct antiproliferative effect of ZD6474on human nasopharyngeal car-cinoma(NPC)in vitro and the antitumor activity on NPC xeno-grafts in vivo.Results indicated that ZD6474treatment inhibited EGFR phosphorylation and led to a dose-and time-dependent decrease in NPC cell(CNE-1,CNE-2and C666-1)proliferation. Further investigation demonstrated G0/G1cell cycle arrest in all 3cell lines,which was associated with an upregulation of p21and/ or p27,and downregulation of CDK4,CDK6and CDK2.ZD6474 treatment also induced apoptosis in CNE-1and CNE-2cells.The apoptosis mechanisms involved reduction of Bcl-2and/or Bcl-X L, induction of Bak and/or Bax,and activation of caspases-3,-9and/ or-8.The in vivo antitumor activity was evaluated in CNE-2and C666-1xenografted nude mice.Administration of ZD6474(25–100mg/kg/day,once-daily,p.o.)produced a dose-dependent inhi-bition of tumor growth and prolonged survival in both models. This study suggests that ZD6474exerts direct antiproliferative effects on NPC cell lines in vitro by inducing G0/G1arrest and ap-optosis,and potent antitumor effects on NPC xenografts in vivo.It indicates that ZD6474may offer a new and effective treatment for human NPC.'2007Wiley-Liss,Inc.Key words:vascular endothelial growth factor receptor;epidermal growth factor receptor;tyrosine kinase inhibitor;ZD6474;nasopharyn-geal carcinoma;cell cycle;apoptosisReceptor tyrosine kinases(RTKs)are crucial elements in many cell regulatory processes and are altered or overexpressed in human carcinogenesis.A variety of therapeutic agents have been developed to inhibit the activities of RTKs.A recent highlight is the development of quinazoline-derived agents that are selective ATP competitors of RTKs.ZD6474is an orally bioavailable, small molecule,anilinoquinazoline derivative that is selective for VEGFR-2(KDR)and is currently in Phase III clinical develop-ment.ZD6474has an IC50of 0.04l M against the isolated KDR enzyme and blocks VEGF-induced signaling in endothelial cells.1 Consistent with inhibition of VEGF signaling and angiogenesis, once-daily oral dosing of ZD6474produced significant broad-spectrum antitumor activity in a panel of histologically diverse human tumor xenografts in nude mice.2In addition to its antian-giogenic properties,ZD6474inhibits,to a less extent,the activity of epidermal growth factor receptor(EGFR)tyrosine kinase(IC50 50.5l M).3Overexpression of EGFR or its ligands has been reported in many human tumors.4The aberrant EGFR tyrosine ki-nase activity leads to increased tumor cell proliferation,survival, invasiveness and overexpression of proangiogenic factors such as VEGF.5–8Therefore,ZD6474has the potential to inhibit2pivotal pathways in tumor growth by targeting tumor growth indirectly, via inhibition of VEGF-dependent tumor angiogenesis and endo-thelial cell survival,and targeting tumor growth directly,via inhi-bition of EGFR-dependent tumor cell proliferation and survival.9 The ability of ZD6474to inhibit tumor cell proliferation was examined directly in several cancer cell lines(ovarian,colon, gastric and breast)in vitro with IC50values ranging from0.09to 17.7l M.10It seems that the direct antiproliferative effect of ZD6474is highly tissue-and cell-type specific.Thus,elucidation of the mechanisms underlying the direct antitumor effects of ZD6474on particular cancer cells may provide an opportunity to target specific patient subsets who may obtain further benefit from this pharmacological activity.Nasopharyngeal carcinoma(NPC)is a major malignant disease of the head/neck region that is endemic within the young popula-tion of South China and Southeast Asia.The current treatment reg-imens of ionizing radiation combined with cisplatin chemotherapy yields a5-year survival rate of 70%,11,12indicating that new therapies to combat this disease are urgently needed.The purpose of this study is to examine the direct antiprolifera-tive effects of ZD6474,with its additional activity against EGFR tyrosine kinase,on NPC cell growth in vitro and the underlying molecular basis.In vivo,the antitumor effect of ZD6474on NPC cell xenograft models is also evaluated.Material and methodsReagentsZD6474was synthesized according to a patent(number:wo 2,003,039,551)procedure.ZD6474:1H NMR(DMSO d6):8.64(s, 1H),8.07(s,1H),7.74(d,1H),7.53(m,2H),7.32(s,1H),4.09(d, 2H),3.99(s,3H),3.50(d,2H),3.02(t,2H),2.78(s,3H),2.16(m, 1H),2.05(d,2H),1.65(m,2H).ESI-MS m/z475(M11);Anal. Calcd for C22H24N4O2BrF0.5H2O1.8HCl:C,48.2;H,5.0;N, 10.1;Found C,47.8;H,5.1N10.0.Cell linesThree human NPC cell lines were studied:CNE-1(well-differ-entiated NPC cell line,EBV negative),CNE-2(poorly differenti-ated NPC cell line,EBV negative)and C666-1(undifferentiated NPC cell line,EBV positive).13–16C666-1was a kind gift from Dr.Saiwah Tsao(University of Hong Kong,Hong Kong,People’s Republic of China).Cells were cultured in RPMI1640supple-mented with10%fetal bovine serum(Gibco,Paisley,UK),100 units/ml penicillin and100l g/ml streptomycin at37°C in a5%This article contains supplementary material available via the Internet at /jpages/0020-7136/suppmat.Grant sponsor:National Basic Research Program of China(973Pro-gram);Grant number:2004CB518801;Grant sponsor:China Postdoctoral Science Foundation;Grant number:2004046156;Grant sponsor:Hi-tech Research and Development Program of China(863Program);Grant num-ber:2006AA02Z489;Grant sponsor:Guangzhou Science Foundation; Grant number:2004Z3-E4011.Thefirst two authors contributed equally to this work.*Correspondence to:Cancer Center,Sun Yat-Sen University,Guang-zhou510060,PRC.Fax:186-20-8734-3146.E-mail:wl_huang@Received23January2007;Accepted after revision22May2007DOI10.1002/ijc.22955Published online13July2007in Wiley InterScience(www.interscience. ).Int.J.Cancer:121,2095–2104(2007)'2007Wiley-Liss,Inc.Publication of the International Union AgainstCancerCO2humidified atmosphere.Experiments were carried out in the log phase of growth.For experiments on synchronized cultures, cells were synchronized in G0/G1phase by culturing in RPMI 1640without serum for24hr.Cells were then released to reenter the cell cycle by the addition of10%fetal bovine serum,either in the presence or in the absence of ZD6474,and were harvested at the indicated time points.MTT assayCells were seeded in96-well plate at a density of23103–23 104cells/well,allowed to recover for16–24hr and then treated using complete media with or without ZD6474at different con-centrations(0.1–25.6l M).After the indicated incubation time, cell viability was measured by MTT assay.Briefly,20l l of MTT stock solution(5mg/ml in PBS)was added into each well and incubated for4hr in an incubator(37°C,5%CO2).The plate was centrifuged at1,800rpm for5min at4°C and the medium was removed.One hundred microliter of DMSO per well was added to dissolve the formazan crystal,and the absorbance was recorded on a microplate reader at of570/630nm wave length.Cell cycle analysisCell cycle analysis was performed as described before.17 Briefly,bothfloating and adherent cells were harvested after ZD6474treatment for the indicated time courses,washed with ice-cold PBS andfixed overnight with70%ethanol at4°C,fol-lowed by resuspension in500l l of PBS.After addition of10l l RNase(10mg/ml),cells were left for30min at37°C and stained with10l l propidium iodide(1mg/ml).Then,cells were analyzed by a Coulter Epics Eliteflow cytometer(Beckman-Coulter, Miami,FL).Annexin-V/7-AAD binding assayFor Annexin-V/7-AAD binding assay,bothfloating and adher-ent cells were collected after ZD6474treatment for48hr and washed with ice-cold PBS.Cells were resuspended in100l l of binding buffer,and stained with10l l of FITC-Annexin V and 20l l of7-AAD.After incubation on ice in the dark for15min, every sample was diluted with385l l13binding buffer and im-mediately analyzed by a Coulter Epics Altraflow cytometer (Beckman-Coulter).Caspase activity assayCells were plated in175-cm2flasks.After incubation with ZD6474for the desired time,floating and adherent cells were har-vested and combined.Preparation of cell lysates and measurement of caspase-8,caspase-9and caspase-3activity were performed with Caspase-8Assay Kit,Caspase-9Assay Kit and Caspase-3 Cellular Activity Assay Kit(Calbiochem,La Jolla,CA)according to the manufacturer’s instruction.AntibodiesAll of the antibodies,excluding EGFR(Cell Signaling Technol-ogy,Beverly,MA),Bcl-X L and p-BAD(Ser136)(SAT,CA)and GAPDH(Kangcheng,Shanghai,China)were obtained from Santa Cruz(Berkeley,CA).Western blotting analysis and immunoprecipitationCell lysates were prepared by extracting proteins with RIPA lysis buffer containing Protease Inhibitor Cocktail II(state,Lake Placid,NY).Protein estimation was performed using Bradford method.Twenty to40l g total cell protein extracts were resolved by electrophoresis using NuPAGE14–12%Bis-Tris Gel(Invi-trogen,Carlsbad,CA)and transferred to PVDF membrane. SeeBlue1prestained standard(Invitrogen)was used to determine product size.The membrane was blocked with either5%BSA or 5%nonfat milk in Tris-buffered saline and then incubated with primary antibodies for4hr at room temperature followed by washing3times for5min each with TBST.Blots were incubated with horseradish peroxidase-conjugated secondary antibody for1 hr at room temperature.Bands were detected using enhanced chemiluminescence system(Cell Signaling,Danvers,MA)and quantified by densitometry using Bio-rad Quantity One software (Bio-rad,Hercules,CA).For immunoprecipitations,500l g of total protein from cell lysates was incubated with1l g primary antibody for2hr at4°C.Twenty microliter of appropriate protein G Plus-Agarose suspension(Santa Cruz)was added and then incu-bated at4°C on a rocker platform overnight.The precipitates were washed4times with ice-cold PBS buffer,resuspended in43LDS sample buffer(Invitrogen)and resolved using NuPAGE14–12% Bis-Tris Gel followed by immunoblot analysis.Tumor xenograft modelsFemale BALB/c nude mice(6-to8-week-old,weighing>18g) were obtained from Shanghai Slike experimental animals Co. (Shanghai,China;animal experimental license No.SCXKhu2003-0008).Mice were housed in air-filtered laminarflow cabinets with 12-hr light/dark cycles,provided with sterilized food and water ad libitum.After1-week adaptation,mice were injected s.c.in the scapular region with23106CNE-2cells or13107C666-1cells in100l l of serum-free media.When tumors reached a volume of 30–50mm3,mice were randomly allocated into4groups(n56) and received(p.o.)either ZD6474(25–100mg/kg/day)or vehicle (sterilized water),administered once daily at0.1ml/10g body weight.Tumor volume was assessed twice weekly by calipers measurement of tumor diameters and calculated according to the formula:V5L3W2/2(L,length;W,width).After3.5weeks treatment,mice were killed and tumors were resected and weighed.For survival studies(n510),mice were dosed(p.o., once-daily)with either ZD6474(25–100mg/kg/day)or vehicle (sterilized water)for25days,and then the tumors were allowed to grow.Mice either were found dead or were killed when tumors were estimated by palpation to reach10%body weight or individ-ual mice appeared stressed by weight loss,ruffled fur and/or leth-argy.All the animal experiments were conducted in accordance with‘‘Guidelines for the Welfare of Animals in Experimental Neoplasia’’.Statistical analysisResults were evaluated using2-tailed Student’s t test with SPSS 13.0software(SSPS,Chicago,IL),unless otherwise specified. Results of survival were evaluated using Kaplan Meier survival analysis.A p value<0.05was considered statistically significant.ResultsZD6474treatment inhibits the tyrosine phosphorylation of EGFR VEGFR1,VEGFR2and EGFR expression was evaluated in3 NPC cell lines at protein levels.Results showed that both VEGFR1and VEGFR2proteins were undetectable in all the NPC cell lines(data not shown).The relative expression of EGFR in all 3cell lines was as shown in Figure1a.Densitometric analysis showed that the relative abundance of EGFR was1.1,1.23and 0.66in CNE-1,CNE-2and C666-1,respectively,after normaliz-ing with GAPDH.Figure1b showed that ZD6474treatment inhib-ited the tyrosine phosphorylation of EGFR within15min in CNE-1cells.These results were obtained after immunoprecipitation with an anti-EGFR antibody and subsequent Western blotting with an anti-phosphotyrosine antibody of the cell extracts treated with 3l M ZD6474.Similar results were also observed in CNE-2and C666-1(data not shown).Effect of ZD6474on EGFR downstream signaling pathwaysTo further identify the effect of ZD6474on EGFR downstream signaling that might contribute to the observed direct growth inhi-bition,we examined the phosphorylation of several key regulators involved.Figure1c exhibited that the phosphorylation of Akt was significantly inhibited by ZD6474treatment in all3cell lines.2096XIAO ET AL.Consistent with the inhibition of Akt activity,phosphorylation of GSK-3b ,a target of the Akt kinase,was reduced (Fig.1c ).Figure 1d showed that the phosphorylation of ERK1/2was significantly inhibited by ZD6474treatment in CNE-2and C666-1cells.Simi-lar result was also observed in CNE-1(data not shown).Antiproliferative effects of ZD6474on human NPC cell linesWe evaluated the effect of ZD6474on the growth of human NPC cell lines,CNE-1,CNE-2and C666-1using MTT assay.ZD6474treatment caused a significant growth inhibition in NPC cell lines in a dose-and time-dependent manner.After 24hr of incubation,ZD6474had minor effects on cell growth (data not shown).After 48hr of incubation,the IC 50values of CNE-1,CNE-2and C666-1were 3.6,6.2and 23.4l M,respectively (data not shown).Moreover,the corresponding IC 50values,examined after 72hr of incubation,were 2.3,3.6and 4.86l M,respectively (Fig.2).In addition,the result indicated that among the 3NPC cell lines tested,CNE-1is the most sensitive cell line to the antipro-liferative effect of ZD6474.For example,after exposure to 3.2l M ZD6474for 48hr,the proliferation of CNE-1,CNE-2and C666-1decreased to 55.3,69.4and 77.5%,respectively,compared withuntreated cells (data not shown).The corresponding relative growth rates were further reduced to 43.3,52.4and 68.2%after the same concentration of ZD6474treatment for 72hr (Fig.2).ZD6474induces cell cycle arrest in NPC cell linesBased on the results of MTT assay,ZD6474concentrations at 1.5–6l M for CNE-1and CNE-2and at 3–9l M for C666-1,which inhibited the growth of corresponding cell lines by 25–75%over a time period of 72hr,were adopted for further studies.To examine whether the antiproliferative effect of ZD6474on NPC cell lines was partly mediated via specific cell cycle arrest,we investigated the cell cycle phase distribution of ZD6474-treated cells by flow cytometric analysis.The results revealed that ZD6474treatment caused an accumulation of cells in the G0/G1fraction in all the 3cell lines,and significant alterations were observed by 24hr of treatment (Fig.3a ).For example,after expo-sure to ZD6474(6l M in CNE-1and CNE-2;9l M in C666-1)for 24hr,the percentage of G0/G1phase cells increased from (52.366)%to (79.466.9)%,(45.2610.2)%to (59.967.6)%and (54.961.3)%to (58.761.5)%in CNE-1,CNE-2and C666-1cells (Fig.3a ,upper panel;p <0.05),respectively,with aproportionalF IGURE 1–Effect of ZD6474on EGFR signaling pathways.(a )Western blotting analysis of EGFR expression in CNE-1,CNE-2and C666-1.The GAPDH serves as a loading control.(b )ZD6474inhib-its the phosphorylation of EGFR in CNE-1cells.(c )ZD6474inhibits the phosphorylation of Akt and its downstream signaling in all 3cell lines.(d )ZD6474inhibits the phosphorylation of ERK1/2in CNE-2and C666-1.Cells were treated with ZD6474for the indi-cated time periods.Following har-vesting,cells were lysed and proc-essed for immunoprecipitation or Western blotting analysis using antibodies directed against EGFR,p-Try-20,p-Akt1/2/3,p-GSK3b ,p-Bad (ser136),ERK1/2and p-ERK1/2as described in Materials and Methods.2097ANTIPROLIFERATIVE AND ANTITUMORAL EFFECTS OF ZD6474reduction in the S and G2-M phase fractions.Moreover,the G0/G1cell cycle arrest induced by ZD6474exhibited in a dose-de-pendent manner in CNE-1(Fig.3a ,lower panel;p <0.05),CNE-2and C666-1(data not shown).To further dissect the G0/G1cell cycle arrest of ZD6474,C666-1cells were synchronized in G0/G1phase by serum starvation.Then cells reentered into cell cycle by addition of serum,and the cell cycle progression was monitored in the presence or absence of ZD6474.The results showed that untreated cells rapidly exited G0/G1phase and proceeded through S and G2-M phase (Fig.3b ,top).ZD6474-treated cells,however,progressed more slowly (Fig.3b ,bottom).At 16hr,70.7%of the ZD6474-treated cells remained in G0/G1fraction compared with 60.3%of the untreated cells.By 24hr,in contrast to only 53.5%of the untreated cells in G0/G1phase,69%of the ZD6474-treated cells were still delayed in G0/G1phase.Similar results were obtained in CNE-1and CNE-2cells (data not shown).Effect of ZD6474on proteins regulating the G1to S transition in the cell cycleTo investigate the molecular mechanisms involved in the G0/G1cell cycle arrest caused by ZD6474treatment,a number of key molecules regulating cells from the G1to the S phase of the cell cycle were examined.These include cyclins and its catalytic part-ners,the CDKs and inhibitors of CDKs.The results demonstrated that,in CNE-1cells,ZD6474treatment resulted in a significant reduction in CDK4,CDK6and CDK2levels after 24–72hr of incubation (Fig.3c ,upper panel).In CNE-2cells,the same treat-ment resulted in a moderate decrease in CDK6and CDK2levels,with little or no effect on CDK4expression (Fig.3c ,upper panel).In C666-1cells,a detectable decrease in CDK4and CDK6expres-sion was observed (Fig.3c ,upper panel).In addition to the altera-tions in CDKs expression,the inhibitors of CDKs,including the Cip/Kip and INK4family of proteins,were also regulated by ZD6474treatment.As shown in Figure 3c lower panel,treatment with ZD6474resulted in a marked increase in p21and p27levels in CNE-1and CNE-2cells,and these results could be detected as early as 6hr and became apparent after 16hr.However,in C666-1cells,a significant decrease in p21levels and a slight decrease in p27levels were observed (Fig.3c ,lower panel).No effect on p57was detected in all 3cell lines.Furthermore,the results also revealed that,in the 3NPC cell lines we tested,the protein of p16was undetectable and the other INK4family members,p15,p18,p19,were unaltered (data not shown).ZD6474-treated NPC cell lines did not show any detectable changes in the levels of cyclin D1and cyclin E (data not shown).Determination of ZD6474-induced apoptosis in human NPC cell linesTo investigate the fate of ZD6474-treated NPC cell lines,an Annexin-V/7-AAD binding assay was performed (Fig.4a ).The results exhibited an increased percentage of early apoptotic cells (Annexin-V-positive)after ZD6474treatment for 48hr in CNE-2cells (p <0.05).In the same way,ZD6474treatment of CNE-2cells also induced a significant increase in the fraction of late apo-ptotic/necrosis cells (Annexin-V/7-AAD double-positive,p <0.05).For example,after treatment with 6l M ZD6474for 48hr,the percentage of early apoptotic CNE-2cells was (12.261.1)%when compared with (2.561.2)%of the control group (Fig.4a ;p <0.05),and the percentage of late apoptotic/necrosis CNE-2cells was (12.761.8)%in contrast to (0.560.2)%of the control group (Fig.4a ;p <0.05).Similar result was obtained in CNE-1cells (data not shown).No data indicative of apoptosis induced by ZD6474in C666-1cells were obtained.Furthermore,we evaluated the involvement of various caspases including caspase 3(the executioner caspase),caspase 8(the ini-tiator of the extrinsic pathway)and caspase 9(the initiator of the intrinsic pathway)during ZD6474induced apoptosis in NPC cells.As shown in Figure 4b ,ZD6474treatment significantly increased caspase-3and -9activities in CNE-1and CNE-2cells (p <0.05).Additionally,ZD6474treatment also led to caspase-8activation in CNE-1(Fig.4b ;p <0.05).Effect of ZD6474on proteins regulating apoptosisTo elucidate the molecular basis responsible for ZD6474-induced apoptosis,cells were treated with ZD6474from 24to 72hr,and then the expression of Bcl-2family proteins were exam-ined by Western blotting analysis.As shown in Figure 4c ,in CNE-1cells,a marked decrease in Bcl-2and Bcl-X l levels and a moderate increase in the expression of Bak and Bax which are known to heterodimer with Bcl-2or Bcl-X l to favor apoptosis were detected.In CNE-2cells,ZD6474treatment resulted in a moderate increase in Bak and Bax levels,with no effect on Bcl-2and Bcl-X l expressions (Fig.4c ).In addition,Bid,the BH3domain-only protein,which can be cleaved by caspase-8and also translocate to mitochondria to induce cytochrome c release and mitochondrial damage,was found reduced in full length inCNE-1F IGURE 2–Effect of ZD6474on the proliferation of CNE-1,CNE-2and C666-1cells.Cells were treated with ZD6474at doses ranging from 0.1to 25.6l M for 72hr and the cell viability was measured by MTT assay.Data are given as relative growth rates compared with untreated control group.Each point represents mean of 3independent experiments conducted in triplicate;bars,SE.*p <0.05,compared with cells treated with medium alone.2098XIAO ET AL.cells and unaltered in CNE-2cells (Fig.4c ).Furthermore as a result of blocking,the phosphorylation of Akt by ZD6474treat-ment,the levels of phospho-Bad (ser 136)decreased in all 3cell lines (Fig.1c ).ZD6474inhibits the growth of human NPC xenograftsSince the majority of NPC biopsies belong to undifferentiated cell type,CNE-2and C666-1were focused on for in vivo antitu-mor studies.The experiments were performed twice,and the results of representative experiments were presented.As shown in Figure 5a ,once-daily oral administration of ZD6474produced a significant dose-dependent inhibition of tumor growth in both models,compared with the control (p <0.05).The inhibition rates of ZD6474-treated groups were assessed by tumor weight (Fig.5b ;p <0.05).The growth of tumors after ZD6474treatment was significantly slower than that of the control groups (p <0.05).In the CNE-2-exnograft model,the inhibition rates of 100,50and 25mg/kg/day group were 86.4,47.3and 33.5%,respectively,and in the C666-1-exnograft model,the corresponding inhibitionratesF IGURE 3–ZD6474delays G0/G1cell cycle progression in NPC cell lines.(a )FACS analysis of the cell cycle distribution of asynchronized NPC cell lines.Upper panel,NPC cells were treated with the indicated concen-trations of ZD6474for 24hr.Lower panel,CNE-1cells were treated with increasing concentra-tions of ZD6474(1.5–6l M)for 24hr.The total population of cells in each phase of the cell cycle is shown.Column,mean of 3inde-pendent experiments;Bars,SD.*p <0.05,the proportion of cells in G0/G1phase after ZD6474treat-ment versus that after vehicle treat-ment.(b )representative FACS analysis of the cell cycle distribu-tion of synchronized C666-1cells,which were released to reenter the cell cycle either in the absence (top)or in the presence (bottom)of 9l M ZD6474.The experiments were repeated 3times.(c )Western blotting analysis of proteins involved in G0/G1cell cycle arrest.Synchronized NPC cells were treated with ZD6474(4.5l M in CNE-1and CNE-2,and 6l M in C666-1)for the indicated time periods.Following harvesting,cells were lysed and processed for Western blotting analysis using antibodies directed against CDK2,CDK4,CDK6,p21,p27and p57as described in Materials and Methods.[Color figure can be viewed in the online issue,which is available at .]2099ANTIPROLIFERATIVE AND ANTITUMORAL EFFECTS OF ZD6474were 91.5,57.1and 43.6%,respectively.The in vivo antitumor studies also showed that ZD6474treatment was well tolerated with only slight effects on body weight (particularly at dose of <50mg/kg/day)and no adverse effects on clinical condition (even at 100mg/kg/day for 25days)(data not shown).ZD6474treatment prolongs the survival of mice bearing human NPC xenograftsThe long-term outcome of ZD6474treatment in human NPC-xenografted mice was determined by survival rates.The experi-ment was conducted twice,and the results of representative experiment were shown (Fig.5c ).Our data suggested that,in each exnograft model,survival durations significantly prolonged after ZD6474treatment when compared with that of control group (p <0.05,Kaplan-Meier).For CNE-2cell-xenografted mice,the me-dian survival of 100,50,25mg/kg/day and vehicle-treated groups were 6063.2,4662.4,3661.6and 3261.9days,respec-tively.For C666-1cell-xenografted mice,the corresponding median survival was 6865.5,5564.7,4362.4and 3861.6days,respectively (Fig.5c ).DiscussionTumor cell proliferation and angiogenesis are 2crucial pro-cesses for solid tumor growth.18Activation of VEGFR-2is neces-sary and sufficient to VEGF-induced pathological angiogenesis and vascular permeability.Abnormal EGF/EGFR signaling path-way is closely related with tumor cell proliferation and survival.ZD6474is a small molecule tyrosine kinase inhibitor with activityagainst both VEGFR-2and EGFR.1,3By inhibition of EGFR tyro-sine kinase activity,ZD6474could impart a direct inhibitory effect on tumor cell growth and survival.However,this direct in-hibitory action of ZD6474may be highly cell-type specific,and need further investigation to elucidate which subset of tumors may be sensitive to this agent and the involved mechanisms.In this study,we report for the first time that ZD6474exerts direct antiproliferative effect on NPC cells via induction of G0/G1phase arrest and apoptosis in vitro ,and potent antineoplastic effects on CNE-2and C666-1xenograft models in vivo .More importantly,the molecular mechanisms controlling the direct anti-proliferative effect are well defined.There are some papers that have reported the sensitivities of several cancer cell lines to the direct antiproliferative activity of ZD6474in vitro .However,in different papers,different methods were used to measure the sensitivity.To date,a human lung tumor cell line,PC-9,was confirmed to be hypersensitive to the direct antiproliferative action of ZD6474(IC 5050.09l M)because of harboring a 15-bp deletion in the gene encoding the EGFR.Although compared with PC-9,NPC cell lines did not appear to be hypersensitive to the direct antiproliferative effect of ZD6474,we have showed that the proliferation of the 3NPC cell lines with overexpression of EGFR were directly and significantly inhibited in response to low doses of ZD6474.19,20Furthermore,we have found that G0/G1cell cycle arrest con-tributed to the antiproliferative effect of ZD6474.Investigation of the cki-cyclin-cdk machinery exhibited that ZD6474treatment led to a marked increase in both p21and p27expression in CNE-1and CNE-2cells (Fig.3c ,lower panel).Conversely,amoderateF IGURE 3–(C ONTINUED )2100XIAO ET AL.。