苯丁酸钠对人舌鳞癌Tca8113细胞株p21和survivin基因表达的影响

目的研究苯丁酸钠对人舌鳞癌Tca8113细胞株的生长、凋亡及其对p21和survivin分子表达的影响。方法应用MTT法检测苯丁酸钠对人舌鳞癌Tca8113细胞株的抑制作用,采用流式细胞仪研究苯丁酸钠对人舌鳞癌Tca8113细胞株的细胞周期阻滞和诱导凋亡作用,应用Western blot和RT-PCR检测p21和survivin基因的转录和表达。结果实验表明苯丁酸钠对Tca8113细胞株增殖有明显的抑制作用;流式细胞仪检测显示苯丁酸钠诱导Tca8113细胞株G1期阻滞和细胞凋亡;苯丁酸钠能使p21的mRNA及蛋白质表达升高,同时survivin的mRNA及蛋白质表达下降。结论苯丁酸钠通过刺激p21表达增加和抑制survivin的表达,从而抑制Tca8113细胞株增殖,诱导细胞G1期阻滞和细胞凋亡;并且p21mRNA水平的上升和survivin mRNA水平的下降变化呈负相关(rs=-0.548,P<0.01),二者蛋白表达的变化亦如此(rs=-0.514,P<0.01)。     

苯丁酸钠对人肝癌细胞分化及P~(21WAF1/CIP1)表达的调控

目的:探讨苯丁酸钠诱导人肝癌细胞Bel7402,HepG2生长抑制、分化和细胞周期阻滞,以及对抑癌基因P21WAF1 /CIP1表达的影响。方法:培养Bel7402,HepG2,应用MTT比色法观察苯丁酸钠对Bel7402,HepG2的生长抑制作用,倒置相差显微镜观察细胞形态改变,流式细胞术分析细胞周期,以RTPCR检测Bel 7402,HepG2细胞中P21WAF1 /CIP1基因表达水平,WESTERN印记法检测P21WAF1 /CIP1蛋白的表达。结果:苯丁酸钠 2, 4, 8mmol·L-1处理 72h后Bel 7402, HepG2 的抑制率分别为 28. 43 %,57. 61 %, 71. 32 %和 27. 42 %, 57. 11 %, 70. 31 %处理后的细胞成纤维母细胞样改变,细胞阻滞于G期,P21WAF1 /CIP1基因和蛋白表达水平均增强。结论:苯丁酸钠抑制 2种人肝癌细胞株的生长,诱导部分人肝癌细胞分化,使细胞阻滞于G1期,苯丁酸钠能诱导P21WAF1 /CIP1基因的表达,增加P21WAF1 /CIP1蛋白的表达水平。     

2β(3羟丙氧基)骨化三醇对人肝癌细胞周期阻滞及P27kip1蛋白的诱导表达

目的 探讨2β(3羟丙氧基)骨化三醇(ED-71)诱导人肝癌细胞HepG2生长抑制和细胞周期G1阻滞,及对抑癌基因P27kipl表达的影响.方法 培养HepG2,应用四甲基偶氮唑盐(MTT)比色法观察ED-71对HepG2的生长抑制作用,流式细胞术分析细胞周期,以Western-blot检测HepG2细胞中P27kipl蛋白的表达水平.结果 ED-71处理后HepG2的生长缓慢,细胞生长受到明显抑制.细胞阻滞于G1期(80.6±2.6,48.7±3.0,P＜0.05),P27kipl蛋白表达水平增强(0.11±0.06,0.67±0.08,P＜0.05).结论 ED-71抑制人肝癌细胞株HepG2的生长,诱导人肝癌细胞分化,使细胞阻滞于G1期,可能与ED-71诱导人肝癌细胞中抑癌基因P27kipl蛋白的表达有关.     

2β(3羟丙氧基)骨化三醇对人肝癌细胞周期阻滞及P~(27kip1)蛋白的诱导表达

目的探讨2β(3羟丙氧基)骨化三醇(ED-71)诱导人肝癌细胞HepG2生长抑制和细胞周期G1阻滞,及对抑癌基因P27kip1表达的影响。方法培养HepG2,应用四甲基偶氮唑盐(MTT)比色法观察ED-71对HepG2的生长抑制作用,流式细胞术分析细胞周期,以Western-blot检测HepG2细胞中P27kip1蛋白的表达水平。结果ED-71处理后HepG2的生长缓慢,细胞生长受到明显抑制。细胞阻滞于G1期(80.6±2.6,48.7±3.0,P<0.05),P27kip1蛋白表达水平增强(0.11±0.06,0.67±0.08,P<0.05)。结论ED-71抑制人肝癌细胞株HepG2的生长,诱导人肝癌细胞分化,使细胞阻滞于G1期,可能与ED-71诱导人肝癌细胞中抑癌基因P27kip1蛋白的表达有关。     

表观遗传学药物FK228治疗肝癌的实验研究

目的观察表观遗传学药物FK228对人肝癌细胞HepG2的体内外抑制作用,并初步探讨其机制。方法采用不同浓度的FK228和氟尿嘧啶(5-FU)处理HepG2细胞48 h,采用CCK-8法观察比较FK228对人肝癌细胞HepG2的生长抑制作用。将5μg/L FK228和25μg/ml 5-FU分别单独或联合作用于2种肝癌细胞48 h,使用流式细胞仪检测细胞凋亡率。体内实验构建BALB/c裸鼠皮下移植瘤模型,通过腹腔注射FK228 (1 mg/kg)和5-FU (20 mg/kg),比较FK228对HepG2裸鼠移植瘤生长抑制作用。结果 FK228对人肝癌细胞HepG2具有明显的生长抑制作用,且呈剂量依赖性,其48 h的半数抑制浓度(IC50)为(4.20±0.24)μg/L,而5-FU的IC50值为(117.50±8.40)μg/ml;FK228联合5-FU处理组与相应浓度的单药组相比,联合用药组的细胞存活率明显降低(P<0.05)。流式细胞仪检测结果显示,FK228可以显著诱导肝癌细胞HepG2凋亡,且联合用药后细胞凋亡率明显增加(P<0.05)。给药20 d后,FK228组、5-FU组、联合给药组及对照组的肿瘤体积分别为(344.71±118.87)、(351.97±73.46)、(220.36±72.12)、(639.76±241.47)mm3。FK228组、5-FU组、联合给药组的裸鼠肿瘤体积均小于对照组(P<0.05)。免疫组化结果显示,与对照组相比,FK228对肝癌细胞的体内促凋亡作用并不明显(P>0.05),但与5-FU联合应用后,促凋亡作用明显增强(P<0.05);与对照组和5-FU组相比,FK228可以明显抑制肿瘤内的血管生成(P<0.05)。结论 FK228对人肝癌细胞HepG2具有明显的体内外生长抑制作用,且与5-FU联合应用后,抑制作用更加明显。FK228与5-FU联合应用,可以明显促进HepG2肝癌细胞的凋亡和抑制肿瘤的血管生成。     

白藜芦醇烟酸酯抑制人肝癌细胞HepG2增殖并诱导细胞的凋亡

目的：观察白藜芦醇的修饰物白藜芦醇烟酸酯（ResT）对人肝癌细胞HepG2生长增殖的影响及诱导凋亡的作用。方法：用不同浓度的ResT处理HepG2细胞，MTT法检测ResT对HepG2细胞生长增殖的抑制作用；应用Hochest荧光染色法观察凋亡细胞的发生；流式细胞术（FCM）检测分析细胞周期和细胞凋亡率；比色法测定Caspase-3酶活性。结果：ResT抑制HepG2细胞的增殖并呈现一定的量效和时效关系，HepG2细胞与ResT作用后出现典型的凋亡细胞形态改变，FCM分析显示大部分细胞阻滞于G1期，S期细胞比例降低。且药物作用组出现凋亡峰。药物作用12、24、48h后，细胞的凋亡率分别为8．7％、21．1％、和32.7％。显示ResT诱导的细胞凋亡作用随时间的延长而增加，同时Caspase-3酶活性显著增强。结论：ResT可抑制人肝癌细胞HepG2的生长增殖，其作用机制之一可能与阻滞细胞于G1期及诱导细胞凋亡有关。     

人剪切修复基因XPD对p52和p21基因的影响

目的:探讨人剪切修复基因XPD转染人HepG2肝癌细胞后p52和p21基因表达的变化以及对肝癌细胞生长的影响。方法:人肝癌细胞HepG2为靶细胞,XPD基因通过Lipofectamine2000脂质体转染HepG2。将细胞分为重组质粒HepG2-pEGFP-N2-XPD组(XPD组)、空载质粒HepG2-pEGFP-N2组(N2组)和HepG2空白对照组。分别用逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Westernblot)法检测各组细胞中XPD、p52以及p21的mRNA和蛋白质的表达量,并用四甲基偶氮唑盐微量酶反应比色(MTT)法检测各组细胞的增殖能力。结果:XPD组中的p52的mRNA表达较其他2组显著下调,XPD、p21的mRNA表达较其他2组明显上调(均P<0.01)。Westernblot检测结果显示各组细胞中XPD、p52及p21的蛋白表达各组间差异与其mRNA各组间差异一致。MTT检测显示XPD转染入HepG2后细胞增殖能力减弱。结论:XPD基因可以抑制癌细胞的生长和基因p52的表达,促进p21的表达。     

钠钾泵抑制剂通过调节细胞周期相关蛋白的生成介导肝癌HepG2细胞周期S期阻滞与凋亡

目的研究钠泵抑制剂哇巴因(ouabain)和华蟾毒配基(cinobufogenin)对人肝癌HepG2细胞增殖的抑制作用及细胞周期的改变,初步分析其机制。方法以人肝癌细胞HepG2为靶细胞,MTT比色法检测哇巴因和华蟾毒配基对HepG2细胞增殖的影响;Hoechst 33342荧光染色检测细胞形态学变化;流式细胞术检测细胞周期;实时定量PCR和Western blot检测CyclinA1、CDK2、PCNA和p21CIP1表达的变化。结果哇巴因和华蟾毒配基可明显抑制HepG2细胞增殖,抑制作用呈时间-浓度依赖性。荧光染色显示药物处理24h后,细胞呈现典型的凋亡形态特征;细胞周期分析显示,实验组S期细胞比例升高,实时定量PCR和Western blot结果显示:哇巴因和华蟾毒配基可下调CyclinA1、CDK2和PC-NA的表达(P<0.05),上调p21CIP1的表达(P<0.05)。结论钠泵抑制剂可抑制肝癌HepG2细胞的增殖,引起细胞周期S期阻滞,诱导细胞凋亡,这与其调节细胞周期相关蛋白的生成关系密切。     

槲皮素对肝癌HepG2细胞生长影响的体外研究

目的 观察槲皮素对肝癌HepG2细胞生长及hTERT基因表达的影响.方法 以台盼蓝拒染法计数肝癌细胞的生长抑制率,用透射电镜从形态变化方面了解凋亡的发生,流式细胞术检测细胞周期变化,Western-blot检测hTERT基因表达改变,PCR-TRAP法检测端粒酶活性.结果 槲皮素抑制肝癌HepG2细胞增殖的作用明显,且呈浓度和时间依赖性,槲皮素处理48 h后的半数抑药浓度（IC5o）为25.5μmol/L;形态学检测显示出了细胞凋亡的特征变化,经10-20 μm/L的槲皮素处理,肝癌HepG2细胞周期阻滞于G0/G1期,且HepG2细胞hTERT蛋白降低,端粒酶活性被抑制.结论 槲皮素能呈时间、剂量依赖性地抑制肝癌细胞的生长,能诱导HepG2细胞发生凋亡,其抑制增生与诱导凋亡的机制可能与下调hTERT基因表达、抑制端粒酶活性、破坏端粒稳定性有关.     

曲古抑菌素A对肾癌细胞系GRC-1生长的抑制作用

目的 研究组蛋白去乙酰化酶(HDAC)抑制药曲古抑菌素A(TSA)对肾癌GRC-1细胞生长的影响及其作用机制. 方法使用TSA处理GRC-1细胞. 噻唑蓝(MTT)法检测细胞生长;流式细胞仪分析细胞凋亡及细胞周期;Western blot免疫印迹分析p53,p21和bcl-2表达. 结果 TSA能明显抑制GRC-1细胞的增殖,且具有明显的剂量依赖性;TSA处理72 h的GRC-1细胞早期凋亡率明显提高,G0/G1期细胞比例显著升高,S期细胞比例显著降低. TSA能够明显下调bcl-2的表达,上调p21的表达,而对p53的没有显著影响. 结论TSA可以通过诱导肾肿瘤细胞的凋亡和周期阻滞而抑制癌细胞生长;其发生机制可能与下调抗凋亡基因bcl-2和上调肿瘤抑制基因p21的表达有关,TSA可能依赖非p53途径调控p21的表达.     

吡格列酮对HepG2细胞增殖和凋亡的影响及机制研究

目的观察吡格列酮对体外培养的HepG2细胞增殖和凋亡的影响,并探讨其是否通过PPARγ依赖途径发挥上述药理作用。方法将不同浓度的吡格列酮作用于体外培养HepG2细胞,以MTT比色法检测HepG2细胞增殖情况,以3H-TdR参入实验检测细胞DNA合成速率,采用RT-PCR和Western blot检测PPARγmRNA和蛋白的表达,以流式细胞术检测细胞凋亡和细胞周期;同时观察PPARγ特异性拮抗剂GW9662和(或)瞬时转染pSG5-PPARγ真核表达质粒对吡格列酮细胞增殖作用的影响;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒稳定转染HepG2细胞,观察PPARγ沉默后吡格列酮对HepG2细胞增殖作用的影响。结果吡格列酮作用于HepG2细胞后,导致HepG2细胞的增殖受到抑制、DNA合成速率减慢,并诱导细胞凋亡,呈一定的剂量依赖关系;在此过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达没有变化;GW9662部分拮抗吡格列酮的增殖抑制作用,但转染pSG5-PPARγ真核表达质粒可以逆转GW9662的作用;吡格列酮在高浓度(20μmol.L-1)时对pGCsi-PPARγ表达质粒稳定转染的HepG2细胞仍表现出增殖抑制作用。结论吡格列酮能够抑制HepG2细胞的增殖并诱导凋亡,具有潜在的抗瘤作用,这种作用与其诱导细胞G0/G1期的停滞有关,PPARγ依赖和非依赖途径参与上述过程。     

沉默Rock2对肝癌细胞增殖及凋亡作用的研究

目的:探讨沉默Rock2基因对人肝癌细胞Huh-7和HepG2增殖和凋亡作用的影响.方法:实验分为空白对照组、干扰无意义组、转染PBS组及干扰Rock2组.将Rock2干扰质粒shRock2转染到人肝癌细胞Huh-7和HepG2中,通过实时荧光定量PCR检测Rock2 mRNA的表达水平;Western blot检测Rock2蛋白的表达水平;MTT比色法检测沉默Rock2后对Huh-7和HepG2细胞增殖抑制的影响;流式细胞术检测沉默Rock2对Huh-7和HepG2细胞周期及早期凋亡的变化.结果:将shRock2转染肝癌细胞系Huh-7和HepG2后,Rock2 mRNA以及Rock2蛋白的表达水平均明显下降.沉默Rock2表达后,MTT结果显示Huh-7和HepG2细胞较对照组细胞增殖能力明显减弱(P＜0.01);Huh-7和HepG2细胞G0/G1期细胞比例明显升高,而S期和G2/M期细胞比例明显降低,与对照组比较差异有统计学意义(P＜0.01);肝癌细胞的早期凋亡较对照组明显增加(P＜0.01).结论:沉默Rock2能明显抑制肝癌细胞系Huh-7和HepG2的增殖,并诱导其早期凋亡,提示Rock2可作为肝癌基因治疗的一个新的分子靶点.     

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC₅₀ values of 49 μg/ml and 30 μg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21^cip1 and p27^kip1) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature.     

Troglitazone but not rosiglitazone induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines

Rosiglitazone (RSG), an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma), induces minor toxicity in humans relative to another PPARgamma agonist, troglitazone (TRO). In contrast, recent reports suggest that RSG causes growth arrest and apoptosis of normal and cancerous cells. Therefore, in this study, we investigated the relative toxicities of TRO and RSG on three different hepatoma cell lines, and observed that TRO, but not RSG, was cytotoxic. Additionally, we studied the mechanism by which TRO induced damage to HepG2 hepatoma cells. Our results indicated that TRO increased the levels of p53, p27, and p21, while it reduced the levels of cyclin D1 and phospho-Rb in a time-dependent manner. Increased p27 and p21 levels coincided with reduced activities of cell cycle dependent kinases (cdk) such as cdk2- and cyclin A-protein kinases 24 h after TRO treatment. These results demonstrate that TRO, but not RSG, causes G1 arrest of hepatoma cells, most likely through changing the levels of cell cycle regulators. Furthermore, because RSG did not affect the levels of cell cycle regulators, TRO-mediated growth inhibition appears independent of PPARgamma activation.     

Effects of FK228, a novel histone deacetylase inhibitor,on human lymphoma U-937 cells in vitro and in vivo

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.     

Inhibition of G1/S transition potentiates oxaliplatin-induced cell death in colon cancer cell lines

In a series of colorectal cancer cell lines, both necrosis and apoptosis were induced upon exposure to oxaliplatin, and enhanced by co-administration of the Hsp90 inhibitor 17-AAG. We analyzed the effects of these interventions on the cell cycle, and found that oxaliplatin treatment caused G1 and G2 arrest in HCT116 cells, and S-phase accumulation in two p53-deficient cell lines (HT29 and DLD1). Addition of 17-AAG enhanced cell cycle effects of oxaliplatin in HCT116, and induced G1 arrest and decrease in S-phase population in the other cell lines. Analysis of cell cycle proteins revealed that the major difference between the cell lines was that in HCT116, 17-AAG resulted in profound inhibition of expression and phosphorylation of late G1 proteins cyclin E and cdk2, with no effect on p21/WAF1 induction. Consistent with these, an HCT116 p53(-/-) line, lacking p21, showed resistance to oxaliplatin, failure to enter apoptosis, and an accumulation of cells in S-phase. Introduction of p21 in these cells caused reversal of that phenotype, including restoration of the G1 block and re-sensitization to oxaliplatin. Inhibition of G1/S progression using cdk2 inhibitor also enhanced oxaliplatin cytotoxicity. We conclude that in colon cancer cells with impaired p53 function, interventions directed to cycle arrest in G1 may potentiate oxaliplatin activity.     

Ethanol Elicits Inhibitory Effect on the Growth and Proliferation of Tongue Carcinoma Cells by Inducing Cell Cycle Arrest

Cellular effects of ethanol in YD-15 tongue carcinoma cells were assessed by MTT assay, caspase activity assay, Western blotting and flow cytometry. Ethanol inhibited the growth and proliferation of YD-15 cells in a dose- and time-dependent manner in an MTT assay. The effects of ethanol on cell cycle control at low percent range of ethanol concentration (0 to 1.5%), the condition not inducing YD-15 cell death, was investigated after exposing cells to alcohol for a certain period of time. Western blotting on the expression of cell cycle inhibitors showed that p21 and p27 was up-regulated as ethanol concentration increases from 0 to 1.5% whilst the cell cycle regulators, cdk1, cdk2, and cdk4 as well as Cyclin A, Cyclin B1 and Cyclin E1, were gradually down-regulated. Flow cytometric analysis of cell cycle distribution revealed that YD-15 cells exposed to 1.5% ethanol for 24 h was mainly arrested at G2/M phase. However, ethanol induced apoptosis in YD-15 cells exposed to 2.5% or higher percent of ethanol. The cleaved PARP, a marker of caspase-3 mediated apoptosis, and the activation of caspase-3 and -7 were detected by caspase activity assay or Western blotting. Our results suggest that ethanol elicits inhibitory effect on the growth and proliferation of YD-15 tongue carcinoma cells by mediating cell cycle arrest at G2/M at low concentration range and ultimately induces apoptosis under the condition of high concentration.     

FK228对HepG2细胞NF-κB活化及炎症因子转录的影响

目的:研究FK228对TNF-α诱导的人肝癌细胞HepG2核转录因子κB(nuclear factor-κB,NF-κB)活化及炎症因子IL-6、IL-8转录的影响。方法:培养的HepG2细胞分为对照组、TNF-α刺激组和FK228干预组。分别用TNF-α刺激和FK228+TNF-α共同作用,免疫印迹(Western blot)法分析细胞核中NF-κBp65及其细胞浆中抑制因子IκBα的表达;RT-PCR对炎症因子IL-6、IL-8 mRNA作半定量分析。结果:FK228(4～32μg·L~(-1))干预组与TNF-α刺激组比较,细胞核内NF-κB显著减少(P<0.01);FK228(8～32μg·L~(-1))减少胞浆中IκBα的降解且各组之间差异有统计学意义(P<0.01);FK228降低TNF-α诱导的IL-6、IL-8 mRNA表达,FK228干预组与TNF-α刺激组相比,差异具统计学意义(P<0.01)。结论:FK228减少胞浆中IκBα降解、阻碍NF-κB的过度活化可能是降低炎症因子释放、发挥抗炎作用的重要机制。     

Inhibition of cell cycle progression by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G0/gap phase 1 (G1) by (Ac)5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac)5-GP in C6 glioma cells. The inhibitory effect of (Ac)5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G0/G1 phase was associated with a marked decrease in the protein expression of cyclin D1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac)5-GP, arresting cells at the G0/G1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D1/cdk 4 and the phosphorylation of Rb.