青蒿琥酯对人卵巢癌细胞株增殖及p38通路的影响

目的:探讨青蒿琥酯(Art)抑制卵巢癌细胞株(CAOV3)增殖及p38通路的影响及机制。方法:培养卵巢癌细胞株(CAOV3),以四甲基偶氮唑盐(MTT)比色法及克隆形成实验测定Art抑制卵巢癌细胞株CAOV3增殖的作用。免疫印记法测定p-p38、Cyclin D1及p21waf/cip1表达。免疫沉淀法纯化蛋白并p38试剂盒测定p38活性。结果:MTT实验及克隆分析法均显示Art明显抑制CAOV3细胞增殖,Art抑制p-p38表达及活性、下调Cyclin D1表达,同时上调p21waf/cip1表达。结论:青蒿琥酯可以抑制卵巢癌细胞株(CAOV3)增殖,其机制与影响p38通路及下游Cyclin D1、p21waf/cip1表达有关。     

新型组蛋白去乙酰化酶抑制剂致U251细胞周期阻滞、凋亡及其机制的研究

目的探讨新型组蛋白去乙酰化酶抑制剂(HDACi)2,2,3,3-四甲基环丙酰硫脲(TCCT)诱导人脑胶质瘤U251细胞周期阻滞、凋亡及其作用机制。方法以不同药物浓度与U251细胞共同培养48 h后,采用MTT法检测药物作用48 h后肿瘤细胞的增殖。药物作用24 h后,采用RT-PCR检测肿瘤细胞的p21WAF1/CIP1与Cyclin D1 mRNA的表达,Western blot检测HDAC3、HDAC4、Cyclin D1与p21WAF1/CIP1蛋白的表达,PI单染法分析细胞周期,Annexin V-PI双染法检测肿瘤细胞凋亡。结果 TCCT对U251细胞增殖具有明显抑制作用,药物干预48 h时的IC50为(0.461±0.108)mmol·L-1,并呈现剂量依赖性。TCCT药物干预24 h后U251细胞p21WAF1/CIP1mRNA表达上调,Cyclin D1 mRNA下调;组蛋白去乙酰化酶3(HDAC3)和组蛋白去乙酰化酶4(HDAC4)表达下调,Cyc-lin D1蛋白表达弱下调,p21WAF1/CIP1蛋白表达上调;S期细胞比例明显提高(P<0.05);细胞凋亡率明显增高(P<0.01)。结论 TCCT对U251细胞增殖有明显的抑制作用,引起肿瘤细胞S期阻滞和凋亡。其作用机制可能与其下调HDAC3、HDAC4表达,促进组蛋白乙酰化,而影响p21WAF1/CIP1和Cyclin D1的基因、蛋白的表达有关。     

丙戊酸钠及其代谢产物的体外肝毒性研究

目的 研究丙戊酸钠及3个代谢产物（2-丙基-4-五烯酸、3-羟基丙戊酸、5-羟基丙戊酸）对体外人正常肝细胞L02增殖活性及对肝细胞损伤相关指标的影响。方法 实验分为对照组和实验组,对照组细胞常规培养,实验组加入丙戊酸钠及3个代谢产物,采用CCK-8法检测细胞增殖活性,PCR法检测CYP1A1、CYP1A2、PCNA、Bax及Bcl-2的mRNA相对含量,Western Blotting法检测蛋白表达,同时检测细胞上清液中谷草转氨酶（AST）、谷丙转氨酶（ALT）、乳酸脱氢酶（LDH）的含量。结果 与对照组相比,随着丙戊酸钠及3个代谢产物浓度和时间的增加,对 L02细胞增殖活性的抑制逐渐增强,CYP1A1、CYP1A2及Bax的mRNA相对含量和蛋白表达量升高,PCNA及Bcl-2的 mRNA相对含量和蛋白表达量均有下降,AST、ALT、LDH含量升高。结论 丙戊酸钠及3个代谢产物与肝毒性有关。     

蜂毒素对人胚肺成纤维细胞生长的抑制作用及其机制探讨

目的研究蜂毒素(melittin)对人胚肺成纤维细胞(MRC-5)生长的抑制作用及其机制。方法采用MTT法检测蜂毒素对MRC-5细胞的增殖抑制作用;流式细胞仪检测细胞周期;RT-PCR法检测细胞中Cyclin D1、CDK4及E2F-1mRNA的表达变化;Western blot法检测Cyclin D1、CDK4、p21及E2F-1蛋白表达。结果与对照组比较,蜂毒素处理组细胞增殖明显受到抑制,并呈剂量和时间依赖性(P<0.05);流式细胞仪检测结果发现,随着浓度的增大,G0/G1期细胞百分比逐渐上升,S期细胞百分比逐渐下降;同时Western blot结果提示蜂毒素(1,2,4 mg.L-1)可以明显降低Cyclin D1、CDK4及E2F-1表达,而上调p21表达。结论蜂毒素能抑制MRC-5细胞增殖,其机制可能与干扰该细胞G0/G1期相关基因的转录相关且抑制Cyclin D1/E2F信号转导。     

4-氨基-2-三氟甲基苯基维甲酸酯对K562细胞分化和细胞周期的影响

目的本研究探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯(4-amino-2-trifluoromethyl-phenyl retinate,ATPR)对K562细胞株的抑制增殖和诱导分化活性并对其机制进行研究。方法ATPR作用于K562细胞3d后,通过MTT法检测细胞的增殖,NBT还原实验法分析细胞的分化指标,瑞氏染色法在油镜下观察加药前后细胞形态学变化,FCM检测分析细胞周期,RT-PCR法检测cyclinE、cyclinD1、CDK2、CDK4、CDK6、p21cip1、p27kip1、p57kip2和PCNA mRNA的变化情况。Western blot法检测cyclin D1和CDK4蛋白表达的改变。结果ATPR呈浓度依赖性抑制K562细胞增殖的作用。ATPR诱导分化活性表现为NBT阳性细胞率增加,油镜下观察K562细胞有分化成熟的改变,G0/G1期细胞表达量增加,S期细胞表达量减少,呈G1期阻滞。RT-PCR检测发现cyclin E、cyclin D1、CDK2、CDK4、CDK6表达减少,PC-NA、P21cip1、P27kip1改变不明显,P57kip2表达增加。Western blot检测cyclin D1和CDK4蛋白表达减少。结论ATPR有较强的抑制K562细胞增殖并诱导其分化的活性,并通过上调P57kip2的表达,抑制Cyclin-CDK激酶复合物,发挥细胞周期阻滞的作用。     

熊果酸对脑胶质瘤U251细胞株增殖的影响

目的 观察熊果酸(ursolic acid, UA)对脑胶质瘤U251细胞株增殖及细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)、细胞周期蛋白(cyclin)D1/p21waf/cip1信号通路的影响.方法 培养脑胶质瘤细胞株(U251),四甲基偶氮唑蓝(MTT)比色法、克隆形成实验及流式细胞术测定不同浓度UA对细胞增殖的影响;Western-blot测定p-ERK1/2及下游的cyclin D1、p21waf/cip1表达.结果 MTT、克隆形成实验及流式细胞术均显示UA可明显抑制U251细胞增殖,免疫印记法显示UA可明显抑制p-ERK1/2、cyclin D1表达及增强p21waf/cip1表达.结论 UA可抑制脑胶质瘤U251细胞株增殖,其途径与下调p-ERK1/2表达、增强p21waf/cip1表达进而抑制cyclin D1表达有关.     

山奈酚通过调控miR21抑制非小细胞肺癌A549细胞增殖的研究

摘要 目的：研究山奈酚(Kaempferol, KA)对非小细胞肺癌A549细胞增殖的抑制作用及其作用机制。方法：运用CCK-8细胞活力检测法研究不同浓度的山奈酚对非小细胞肺癌A549细胞存活率的影响,筛选给药浓度;将A549细胞分为对照组及药物组,考察不同浓度山奈酚对A549细胞中miR21表达的影响;运用miR21 precursor转染A549细胞使miR21在A549细胞中高表达,不同浓度山奈酚给药作用后运用实时荧光定量PCR(RT-qPCR)检测miR21、c-Myc、Cyclin D1 mRNA的表达水平,Western Blot法检测PTEN、p-AKT(Thr308)、c-Myc、Cyclin D1等蛋白的表达。结果：不同浓度山奈酚对非小细胞肺癌A549细胞增殖均具有一定的抑制作用;30 、60 μmol/L的山奈酚给药作用24h均能够降低A549细胞中miR21的表达,结果具有统计学意义;运用miR21 precursor转染A549细胞后,c-Myc、Cyclin D1 mRNA的表达水平显著升高,miR21、p-AKT(Thr308)等蛋白的表达升高,PTEN蛋白表达降低,不同浓度的山奈酚给药作用均能降低miR21, c-Myc、Cyclin D1 mRNA的表达及p-AKT(Thr308)、 c-Myc、Cyclin D1等蛋白的表达,升高PTEN蛋白表达。结论：山奈酚能够通过调控miR21/PTEN/AKT信号通路抑制非小细胞肺癌A549细胞增殖。     

双丙戊酸钠和丙戊酸钠对HepG2细胞的毒性作用及机制

目的探讨双丙戊酸钠和丙戊酸钠对肝细胞的毒性作用及其可能的作用机制。方法肝癌细胞株HepG2加入双丙戊酸钠和丙戊酸钠0.1,0.3,1和3mmo.lL-1,培养24h后,MTT法测定HepG2的细胞存活;双丙戊酸钠和丙戊酸钠0.3,0.5和1.0mmol.L-1作用HepG2细胞24h,丙酮酸法测定培养液中乳酸脱氢酶(LDH)活性,赖氏法测定培养液中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性;双丙戊酸钠和丙戊酸钠62.5,125,250,500和1000μmol.L-1作用24h,实时定量逆转录聚合酶链反应RT-PCR测定细胞色素P450家族中CYP1A1mRNA和CYP1A2mRNA表达的变化。结果与溶剂对照组比较,双丙戊酸钠和丙戊酸钠0.1,0.3,1和3mmo.lL-1均显著抑制细胞的存活(P<0.05,P<0.01),且存在浓度依赖关系。双丙戊酸钠与丙戊酸钠0.3,0.5和1mmol.L-1使HepG2细胞培养液中GPT,GOT和LDH的活性明显升高(P<0.05,P<0.01),且随浓度升高,肝酶活性进一步升高。双丙戊酸钠与丙戊酸钠62.5,125,250,500和1000μmol.L-1使HepG2细胞中CYP1A1mRNA和CYP1A2mRNA的表达水平亦逐渐升高。结论双丙戊酸钠和丙戊酸钠对HepG2细胞都有明显的毒性作用,CYP1A1mRNA和CYP1A2mRNA表达水平的升高可能是丙戊酸类药物诱发肝毒性的机制之一。     

降钙素基因相关肽对大鼠血管平滑肌细胞CDK2和Cyclin E的影响

目的:研究降钙素基因相关肽(CGRP)对大鼠血管平滑肌细胞中细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)和细胞周期蛋白E(Cyclin E)的影响,为阐明CGRP抑制血管平滑肌细胞增殖的细胞周期机制提供实验依据。方法:培养大鼠胸主动脉血管平滑肌细胞,分别用CGRP或/和10%FBS处理细胞。噻唑蓝比色法(MTT)观察CGRP对10%FBS诱导大鼠血管平滑肌细胞增殖的影响;流式细胞术分析细胞周期;Western blot检测CDK2和Cyclin E的表达。结果:CGRP能抑制10%FBS诱导的血管平滑肌细胞增殖,升高G0/G1期细胞百分比,降低S+G2+M期细胞百分比,抑制细胞内CDK2和Cyclin E表达。结论:CGRP能通过阻滞细胞周期由G0/G1期进入S期而抑制血管平滑肌细胞增殖,其作用机制可能与降低CDK2和Cyclin E表达有关。     

Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞增殖及细胞周期的影响

目的观察Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞(VSMC)增殖及细胞周期的影响。方法组织贴块法培养大鼠VSMC细胞,采用TNF-α诱导其增殖,加入不同浓度的Roscovitine预处理15 h,将细胞分为:对照组、TNF-α组、Roscovitine 5、10、15、30μmol·L~(-1)组。MTT比色法检测细胞增殖活性;Western blot检测增殖细胞核抗原(PCNA);流式细胞仪检测细胞周期;荧光定量RT-PCR及Western blot检测细胞周期蛋白(Cyclin A、Cyclin B、Cyclin D、Cyclin E)、细胞周期蛋白依赖激酶(CDK4、CDK5)、细胞周期抑制蛋白(p53、p21、p27)的表达。结果 Roscovitine能抑制VSMC增殖;抑制细胞周期从G_0/G_1期向S期转化。与TNF-α组比较,Roscovitine 5、10、15、30μmol·L~(-1)组能降低细胞周期蛋白Cyclin A、Cyclin B、Cyclin D、Cyclin E蛋白表达,降低细胞周期蛋白依赖激酶CDK4、CDK5蛋白表达,升高细胞周期抑制蛋白p53、p21、p27蛋白表达(P<0.05)。结论 Roscovitine可抑制大鼠VSMC细胞周期进程及增殖活性。     

小檗胺对人肝癌SMMC-7721细胞增殖作用及其机制的初步研究

目的：研究小檗胺对人肝癌SMMC-7721细胞增殖作用的影响及可能的机制。方法：经不同浓度小檗胺处理体外培养的肝癌SMMC-7721细胞,采用MTT法检测细胞增殖的变化;采用PI染色法检测肿瘤细胞周期分布的变化;采用RT-PCR和Western blot法检测肿瘤细胞中Cyclin B1、Cyclin D1、p21和p27的表达水平。结果：与溶剂对照组比较,不同浓度的小檗胺均可明显抑制人肝癌SMMC-7721细胞增殖。PI染色结果显示,小檗胺可明显地诱导肝癌细胞周期阻滞于G₀/G₁期（P<0.05）。RT-PCR和Western blot结果均显示,应用小檗胺处理肿瘤细胞后,p21和p27的表达明显升高,而Cyclin B1和Cyclin D1的表达水平明显下降（P<0.05）。结论：小檗胺能抑制人肝癌SMMC-7721细胞增殖,并使其细胞周期阻滞于G₀/G₁期,其机制可能与其上调p21和p27的表达和下调Cyclin B1和Cyclin D1的表达有关。     

Genistein-induced neuronal apoptosis and G2/M cell cycle arrest is associated with MDC1 up-regulation and PLK1 down-regulation

The aim of the present study is to investigate the effect of genistein on human neuroblastoma SK-N-MC cells. MTT proliferation assay, LDH cytotoxicity assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting were used to investigate the effect of genistein on cell survival, cellular toxicity, cell cycle progression, and mRNA and protein alterations of selected DNA damage-, cell cycle- and apoptosis-related genes in SK-N-MC cells. Genistein suppressed cell proliferation, increased LDH release and modulated cell cycle distribution through accumulation of cells at G2/M- and S-phase and sub-G0 (cell death) with a concurrent decrease of cells at G0/G1 phase. Genistein increased the MDC1 (Mediator of DNA damage Checkpoint protein 1), p53, p21(waf1/cip1), Cdc2 and Bax mRNA levels in a dose-dependent manner. However, PLK1 (Polo-Like Kinase 1) and Cyclin B1 mRNAs were down-regulated after genistein treatment. Furthermore, Genistein did not alter Chk2 (Checkpoint Kinase 2), Bcl-2 and Cdc25C mRNA levels. On western blotting analyses; genistein increased the protein level of MDC1, p53, p21(waf1/cip1), and Bax in a dose-dependent manner. Genistein also increased the phosphorylation of Chk2 and Cdc25C at Thr-68 and Ser-216, respectively. In addition, consistently with PLK1 down-regulation, the phosphorylation of Cdc25C at Ser-198 was markedly decreased after genistein treatment. Additionally, Chk2, Cdc25C, Cyclin B1, p-Cyclin B1 (Ser-147), and Cdc2 as well as Bcl-2 proteins were down-regulated after genistein treatment. Altogether, these results suggest for the first time the involvement of MDC1 up-regulation after genistein treatment in DNA damage-induced Chk2 activation- and PLK1 down-regulation-mediated apoptosis and cell cycle checkpoint pathways.     

抑制组蛋白去乙酰化酶与DNA甲基转移酶活性对胶质瘤恶性表型影响的研究

【摘要】 目的：探讨阻断组蛋白去乙酰化酶与DNA甲基转移酶的活性后对胶质瘤恶性表型的抑制作用。方法：选择丙戊酸（VPA）为组蛋白去乙酰化酶抑制剂,5-氮杂-2′-脱氧胞苷（Aza）为DNA甲基转移酶抑制剂。将U251胶质瘤细胞分为对照组、VPA治疗组、Aza治疗组和VPA+Aza联合治疗组。采用四唑盐(MTT)比色法分析肿瘤细胞增殖活性,流式细胞术分析细胞周期,Annexin V-FITC染色检测细胞凋亡,2D Matrigel、3D Matrigel、Transwell法检测胶质瘤细胞侵袭能力,并建立U251细胞裸鼠皮下移植瘤模型,进行肿瘤局部多点注射上述药物治疗,测量肿瘤体积。结果：与对照组比较,各治疗组肿瘤细胞的增殖在治疗2 d后均出现明显抑制,S期和G2M期细胞比例减少,诱导细胞周期阻滞于G0/G1期,治疗组细胞凋亡率明显下降,侵袭和运动迁移能力均明显下降,各治疗组鼠移植瘤体积增长较对照组明显减缓,与对照组相比差异均有统计学意义（P<0.05）。以上结果均以VPA+Aza联合治疗组最为显著。结论：联合阻断DNA甲基转移酶和组蛋白去乙酰化酶活性可抑制胶质瘤表观遗传学性征,抑制胶质瘤恶性表型。     

Cell cycle- and protein kinase C-specific effects of resiniferatoxin and resiniferonol 9,13,14-ortho-phenylacetate in intestinal epithelial cells

We have previously reported that protein kinase C (PKC) signaling can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells, including downregulation of cyclin D1, induction of p21(Waf1/Cip1), and activation of the growth suppressor function of pocket proteins. In the current study, we compared the cell cycle- and PKC-specific effects of the vanilloid resiniferatoxin (RTX), its parent diterpene resiniferonol 9,13,14-ortho-phenylacetate (ROPA), and the PKC agonist PMA in the IEC-18 non-transformed intestinal crypt cell line. ROPA and PMA were found to produce strikingly similar alterations in cell cycle progression and PKC activity in IEC-18 cells, although PMA was approximately 1000-fold more potent in producing these effects. Both agents induced a transient PKC-dependent blockade in G1---> S progression associated with transient downregulation of cyclin D1 and induction of p21(Waf1/Cip1). In contrast, RTX produced a prolonged PKC-independent cell cycle arrest in G(0)/G(1) phase which was maintained for longer than 24h. This arrest was vanilloid receptor-independent and associated with prolonged downregulation of cyclin D1 mRNA and protein, with little effect on levels of p21(Waf1/Cip1). Combined exposure to RTX and ROPA produced a sustained and complete cell cycle blockade in IEC-18 cells, associated with depletion of cyclin D1 and sustained enhancement of p21(Waf1/Cip1) levels. PMA, ROPA, RTX and the RTX/ROPA combination were capable of activating ERK1/2 signaling in IEC-18 cells, albeit with different kinetics. In contrast, only PMA and ROPA activated JNK1/2 and p38 in this system. Notably, some preparations of commercially obtained RTX produced effects indistinguishable from those of the RTX/ROPA combination, suggesting that certain batches of the compound may contain significant amounts of ROPA (or another PKC agonist activity). Together, these data demonstrate that structurally related compounds can produce similar cell cycle-specific effects but through distinct mechanisms. In addition, they add to a growing body of evidence that vanilloids can have antiproliferative effects in a variety of cell types.     

曲古抑菌素A对甲状腺鳞癌细胞中Cyclin D1、CDK4、pRB表达的影响

目的:观察曲古抑菌素A(TSA)对甲状腺鳞癌(SW579)细胞中细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性激酶4(CDK4)、视网膜母细胞瘤基因(RB)的蛋白产物(pRB)表达的影响。方法:体外培养SW579细胞,以二甲基亚砜(DMSO)为溶剂对照组,另设空白对照(未作任何处理)组,观察50、100、200、400nmol·L-1TSA对SW579细胞生长抑制率的影响,及细胞中Cy-clin D1、CDK4、RB基因和蛋白的表达情况。结果:与溶剂对照组和空白对照组比较,各剂量TSA作用后SW579细胞的细胞生长抑制率明显升高(P<0.01),且呈剂量依赖性。与空白对照组比较,各剂量TSA作用后SW579细胞中Cyclin D1 mRNA表达明显降低(P<0.01),且随剂量增加表达降低,但CDK4、RB mRNA表达和溶剂对照组中这2个基因的表达均无明显变化(P>0.05);与空白对照组比较,各剂量TSA作用后细胞中Cyclin D1和pRB蛋白表达明显降低(P<0.01),且随剂量增加表达降低,但CDK4蛋白表达无明显变化(P>0.05)。结论:TSA能明显抑制SW579细胞的生长,且呈剂量依赖性;其机制可能与Cyclin D1基因和蛋白、pRB蛋白的表达有关。     

Physalis angulata induced G2/M phase arrest in human breast cancer cells.

Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.     

曲古抑菌素A对结肠癌细胞细胞周期影响的机制研究

目的 研究组蛋白去乙酰化酶（HDAC）的抑制剂曲古抑菌素A（TSA）对结肠癌细胞株SW480细胞周期、凋亡的影响,初步探讨TSA作用细胞周期的可能机制,为HDAC抑制剂用于结肠癌治疗提供理论依据.方法 培养人结肠癌细胞系SW480,采用HDAC抑制刺TSA干预细胞,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用Western blot对细胞周期相关的基因进行检测.结果 TSA处理细胞后,流式细胞计数分析显示,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyelinE、cyclinA聚集,而对凋亡无明显的影响.Western blot显示,TSA能够上调p21wafl/Cipl、p27Kipl的表达,下调CDK2、cyclinE以及cyclinA的表达.结论 在结肠癌细胞中,TSA能够通过上调p21Wafl/Cip1、p27Kip1的表达以及下调CDK2、cyclinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长.     

HDAC inhibition delays cell cycle progression of human bladder cancer cells in vitro

Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.