Piceatannol, a natural analog of resveratrol, inhibits progression through the S phase of the cell cycle in colorectal cancer cell lines

Piceatannol, a naturally occurring analog of resveratrol, was previously identified as the active ingredient in herbal preparations in folk medicine and as an inhibitor of p72(Syk). We studied the effects of piceatannol on growth, proliferation, differentiation and cell cycle distribution profile of the human colon carcinoma cell line Caco-2. Growth of Caco-2 and HCT-116 cells was analyzed by crystal violet assay, which demonstrated dose- and time-dependent decreases in cell numbers. Treatment of Caco-2 cells with piceatannol reduced proliferation rate. No effect on differentiation was observed. Determination of cell cycle distribution by flow cytometry revealed an accumulation of cells in the S phase. Immunoblotting demonstrated that cyclin-dependent kinases (cdk) 2 and 6, as well as cdc2 were expressed at steady-state levels, whereas cyclin D1, cyclin B1 and cdk 4 were downregulated. The abundance of p27(Kip1) was also reduced, whereas the protein level of cyclin E was enhanced. Cyclin A levels were enhanced only at concentrations up to 100 micromol/L. These changes also were observed in studies with HCT-116 cells. On the basis of our findings, piceatannol can be considered to be a promising chemopreventive or anticancer agent.     

Resveratrol enhances the differentiation induced by butyrate in caco-2 colon cancer cells

Butyrate, a short-chain fatty acid produced in the colon by microbial fermentation of fiber, inhibits growth of colonic carcinoma cells while inducing differentiation. Resveratrol, a plant polyphenol found in red wine and peanuts, has been shown to exert chemopreventive properties on colon cancer cells. The aim of this study was to determine whether resveratrol modulates the effects of butyrate on Caco-2, a colonic adenocarcinoma cell line. The growth inhibitory effect of resveratrol (50 micromol/L) was more powerful than that of butyrate (2 mmol/L). Butyrate did not intensify the inhibition of proliferation exerted by resveratrol. Although the polyphenol enhanced the differentiation-inducing effect of butyrate, it did not elevate alkaline phosphatase activity or E-cadherin protein expression, markers of epithelial differentiation, when applied alone. Butyrate-induced transforming growth factor-beta1 secretion was inhibited by resveratrol. Treatment with the combination of resveratrol and butyrate attenuated levels of p27(Kip1), whereas resveratrol enhanced butyrate's effect on the induction of p21(Waf1/Cip1) expression. These data demonstrate a possible combined chemopreventive effect of two substances naturally occurring in the colonic lumen after ingestion of fibers and resveratrol-containing food.     

全反式视黄酸阻断苯并(a)芘致成纤维细胞周期改变的通路

目的 观察全反式视黄酸（ATRA）逆转苯并（a）芘诱导的人胚肺成纤维细胞（HELF）细胞周期紊乱过程中细胞周期素D1（cyclin D1）、细胞周期素依赖性激酶K4（CDK4）、转录因子E2F-1和E2F-4的作用.方法 用0.1 μmol/L ATRA预处理细胞后,再用2 μmol/L苯并（a）芘作用细胞,用Western blotting方法测定其蛋白表达水平;建立稳定转染了反义cyclin D1质粒和反义CDK4质粒的细胞系,应用反义技术证明上下游关系;用流式细胞技术 测定细胞周期.结果 2μmol/L苯并（a）芘作用于HELF24h后,cyclin D1、E2F-1蛋白表达水平明显增高,CDK4和E2F-4蛋白表达水平无明显变化;用0.1 μmol/LATRA预处理24h后,cyclin D1、E2F-1蛋白表达水平明显下降;与相同作用的对照组相比,苯并（a）芘刺激反义cyclin D1或反义CDK4细胞后E2F-1表达增加的幅度明显降低;与相同作用的对照组相比ATRA预处理的反义cyclin D1或反义CDK4细胞中,苯并（a）芘诱导的E2F-1过表达水平降低的幅度明显降低;流式细胞术测定结果显示,苯并（a）芘诱导细胞从G1期进入S期,ATRA通过聚积G1期细胞而阻滞苯并（a）芘诱导的细胞周期进程.结论 ATRA通过cyclin D1/E2F-1途径阻断苯并（a）芘诱导的HELF的细胞周期改变.     

cyclinD1/E2F通路在苯并(a)芘引起人胚肺成纤维细胞周期改变中的作用

目的探讨细胞周期蛋白D1（cyclinD1）／细胞周期蛋白依赖激酶4（CDK4），E2F-1／4通路在苯并（a）芘[B（a）P]引起的人胚肺成纤维细胞（HELF）周期改变中的作用。方法分别用2μmol／L的B（a）P作用HELF24h和100μmol／LB（a）P作用3次，每次24h，细胞具有转化细胞的部分特征（THELF）。用转染反义cyclinD1和CDK4质粒的HELF（A-D1，A-K4）和T-HELF（T-A-D1，T-A-K4）研究cyclinD1／CDK4与E2F-1／4的上下游关系。用流式细胞仪和Western blot法检测细胞周期、cyclin D1、CDK4和E2F-1／4蛋白表达的改变。结果2μmol／LB（a）P作用24h HELF中G1期细胞数明显减少。在A-D1和A-K4的HELF中，cyclinD1和CDK4表达的抑制阻断了由B（a）P引起的细胞周期从G1期进入S期的变化。B（a）P刺激后HEIF中cyclinD1和E2F-1的表达明显增加。在A-D1中，E2F-1的高表达被抑制。B（a）P作用A-K4细胞后，CDK4表达明显升高，E2F-4表达明显降低。T-A-D1和T-A-K4的T-HELF多数停留在G1期。与HELF相比，T-HELF中cyclin D1的表达明显增加；与T-HELF相比，T-A．D1和T-A-K4中E2F-4表达明显增加。结论在2μmol／L B（a）P作用的HELF中，B（a）P通过cyclinD1／CDK4-E2F-1／4信号转导通路引起细胞周期的改变。在T-HELF中，B（a）P通过cyclinD1／CDK4-E2F-4信号转导通路引起细胞周期的改变。     

Dietary isothiocyanates inhibit Caco-2 cell proliferation and induce G2/M phase cell cycle arrest, DNA damage, and G2/M checkpoint activation

Benzyl isothiocyanate and phenethyl isothiocyanate, two aromatic phytochemicals present in substantial concentrations in edible vegetables of the genus Brassica, were investigated for their effects on Caco-2 cell proliferation. Benzyl and phenethyl isothiocyanate inhibited DNA synthesis, with 50% inhibitory concentrations of 5.1 and 2.4 micromol/L, respectively, and significantly increased the doubling times of Caco-2 cells from 32 h to 220 and 120 h, respectively. There was no adverse effect of either chemical on cell viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, but benzyl isothiocyanate and phenethyl isothiocyanate both caused an accumulation of cells in the G(2)/M phase of the cell cycle, which was maintained for at least 48 h in cells synchronized at prometaphase with nocodazole and subsequently treated with 10 micromol/L benzyl isothiocyanate or phenethyl isothiocyanate. Both benzyl and phenethyl isothiocyanate increased DNA strand breakage, increased phosphorylation of the G(2)/M checkpoint enforcer Chk2, and induced p21 expression. These results suggest that the antiproliferative effects of benzyl and phenethyl isothiocyanates toward Caco-2 cells are due at least in part to the activation of the G(2)/M DNA damage checkpoint, and that sustained G(2)/M phase cell cycle arrest in response to benzyl and phenethyl isothiocyanates may be maintained through upregulation of p21. This study indicates that some dietary isothiocyanates may exert an antiproliferative effect through activation of the G(2)/M DNA damage checkpoint.     

Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice

Resveratrol is a naturally occurring phytoalexine found in medicinal plants. We found that resveratrol, at doses of 2.5 and 10 mg/kg, significantly reduced the tumor volume (42%), tumor weight (44%) and metastasis to the lung (56%) in mice bearing highly metastatic Lewis lung carcinoma (LLC) tumors, but not at a dose of 0.6 mg/kg. Resveratrol did not affect the number of CD4(+), CD8(+) and natural killer (NK)1.1.(+) T cells in the spleen. Therefore, the inhibitory effects of resveratrol on tumor growth and lung metastasis could not be explained by natural killer or cytotoxic T-lymphocyte activation. In addition, resveratrol inhibited DNA synthesis most strongly in LLC cells; its 50% inhibitory concentration (IC(50)) was 6.8 micromol/L. Resveratrol at 100 micromol/L increased apoptosis to 20.6 +/- 1.35% from 12.1 +/- 0.36% (P < 0.05) in LLC cells, and decreased the S phase population to 22.1 +/- 1.03% and 29.2 +/- 0.27% from 35.2 +/- 1.72% (P < 0.05) at concentrations of 50 and 100 micromol/L, respectively. Resveratrol inhibited tumor-induced neovascularization at doses of 2.5 and 10 mg/kg in an in vivo model. Moreover, resveratrol significantly inhibited the formation of capillary-like tube formation from human umbilical vein endothelial cells (HUVEC) at concentrations of 10-100 micromol/L; the degree of the inhibition of capillary-like tube formation by resveratrol was 45.5% at 10 micromol/L, 50.2% at 50 micromol/L and 52.6% at 100 micromol/L. Resveratrol inhibited the binding of vascular endothelial growth factor (VEGF) to HUVEC at concentrations of 10-100 micromol/L, but not at concentrations of 1 and 5 micromol/L. The degree of inhibition of VEGF binding to HUVEC by resveratrol was 16.9% at 10 micromol/L, 53.2% at 50 micromol/L and 47.8% at 100 micromol/L. We suggest that the antitumor and antimetastatic activities of resveratrol might be due to the inhibition of DNA synthesis in LLC cells and the inhibition of LLC-induced neovascularization and tube formation (angiogensis) of HUVEC by resveratrol     

Fisetin inhibits the activities of cyclin-dependent kinases leading to cell cycle arrest in HT-29 human colon cancer cells

Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 micromol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 +/- 1% decrease in cell number observed 72 h after the addition of 60 micromol/L fisetin. Perturbed cell cycle progression from the G(1) to S phase was observed at 8 h with 60 micromol/L fisetin treatment, whereas a G(2)/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 micromol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21(CIP1/WAF1) levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities.     

苯并芘诱导人胚肺成纤维细胞cyclinD1、CDK4和E2F-1/4的表达改变

目的建立苯并芘诱导的具有转化细胞部分特征的人胚肺成纤维细胞(T-HELF)模型,并观察T-HELF细胞中cyclin D1、CDK4和E2F-1/4蛋白表达的改变。方法以200、100、50、25、5、1μmol/L的苯并芘处理人胚肺成纤维细胞(HELF)24小时,用噻唑蓝(MTT)比色法测定苯并芘的细胞毒性。根据MTT结果确定以100和200μmol/L苯并芘给HELF细胞染毒3次,染毒结束后培养6周观察细胞的形态变化,培养12周后观察其形态变化及软琼脂中细胞克隆的生长。用流式细胞仪检测T-HELF细胞周期的变化,用Western blot检测T-HELF细胞中cyclin D1、CDK4和E2F-1/4蛋白表达的改变。结果MTT结果显示苯并芘浓度在25~200μmol/L之间时,细胞存活率为78%~80%。苯并芘200μmol/L组染毒4周后细胞死亡。100μmol/L组染毒结束后培养6周,观察到细胞变大,核增大或多核;12周后,在软琼脂中可以生长为细胞克隆,说明HELF细胞具有了转化细胞的部分特征。T-HELF细胞在无血清培养时,细胞周期没有停滞。用Western blot检测发现和正常HELF细胞相比T-HELF细胞的cyclin D1表达明显增加,CDK4、E2F-1和E2F-4的表达没有明显变化。结论建立了苯并芘诱导的具有转化细胞部分特征的人胚肺成纤维细胞模型,可用于苯并芘致癌的分子机制研究。无血清培养时,T-HELF细胞周期没有停滞,和正常HELF细胞相比T-HELF细胞cyclin D1表达明显增加,cyclin D1可能与T-HELF细胞的快速增殖有关。     

活化蛋白-1和细胞周期蛋白在石英诱导的细胞周期改变中的作用

目的 探讨在石英刺激的人胚肺成纤维细胞(HELF)中AP-1/细胞周期蛋白D1 (cyclin D1)通路在细胞周期改变中的作用.方法 建立稳定转染空载体PXJ的HELF系(HELF-PXJ)及空载体PXJ与反义cyclin D1质粒或反义细胞周期蛋白依赖激酶(CDK4)质粒分别共转染的HELF系(简称anti-D1和anti-K4),将HELF-PXJ、anti-D1和anti-K4细胞分为对照组和石英刺激组(共6组),各对照组不加任何处理,石英刺激组用200 μg/ml石英处理细胞24h.检测AP-1对石英诱导的cyclin D1、CDK和E2F-4表达改变的影响.AP-1的化学抑制剂姜黄素(curcumin) 20μmol/L预处理细胞1h后,200 μg/ml石英刺激24 h,将HELF分为4组,分别为HELF对照组、HELF+石英组、HELF+curcumin对照组、HELF+curcumin+石英组.用免疫印迹(Western blot)法和免疫荧光法检测cyclin D1、CDK4和E2F-1/4蛋白表达;运用反义RNA技术证明通路的上下游关系;用流式细胞术检测细胞周期变化.结果 200 μg/ml石英处理HELF细胞,cyclin D1和CDK4蛋白表达升高,E2F-4蛋白表达明显降低,而E2F-1蛋白的表达没有明显改变.HELF-PXJ+石英组G1期细胞所占比例下降,S期细胞所占比例升高,与HELF-PXJ对照组的差异有统计学意义(P＜0.05).抑制cyclin D1或CDK4表达后,与对照组比较,anti-D1+石英组和anti-K4+石英组G1期细胞和S期细胞百分比无明显变化.抑制AP-1活性,与HELF+石英组比较,HELF+curcumin+石英组cyclin D1和CDK表达均减低,E2F-4表达增多.结论200μg/ml石英可诱导cyclin D1和CDK4蛋白表达增高,E2F-4蛋白表达减少,并通过AP-1/cyclin D1通路诱导细胞周期改变.     

绿茶主要活性成分对叙利亚地鼠胚胎癌前细胞生长和凋亡的影响

目的建立叙利亚地鼠胚胎细胞（Syrian hamster embryo cell,SHE细胞）混合培养模型,研究表没食子儿茶素没食子酸酯（epigallocatechin-3-Gallate,EGCG）对SHE癌前细胞生长、增殖和凋亡的作用,探讨EGCG的抑癌机制。方法将SHE癌前细胞和正常细胞分别按1：10000、1：1000、1：100、1：10比例接种于6孔的培养皿中,培养7d,建立SHE细胞混合培养模型。以0μmol／L EGCG为对照组,分别选取浓度为0．5、1、5、10、50μmol／L的EGCG,通过SHE细胞生长实验,原位细胞凋亡实验、原位细胞增殖实验和基因芯片检测EGCG对SHE正常细胞、SHE癌前细胞和混合培养模型中SHE癌前细胞的生长、增殖、凋亡及相关调控基因表达的影响。结果SHE癌前细胞与正常细胞的比例为1：100是合适的混合培养模型。0．5、1、5、10μmol／L的EGCG促进了SHE正常细胞的生长,而浓度为50μmol／L的EGCG抑制了SHE正常细胞的生长。在1：200比例的SHE癌前细胞和正常细胞混合培养模型中,与对照组相比,浓度为5、10μmol／L的EGCG抑制了不同大小的SHE癌前细胞克隆的生长。对照组中SHE癌前细胞的DNA合成指数和凋亡指数分别是39．3％和6．5％,5、10p,mol／LEGCG作用后SHE癌前细胞的DNA合成指数分别降至25．6％和24．8％,细胞凋亡指数分别升至12．65％和14．5％。EGCG抑制了混合培养模型中SHE癌前细胞的生长和增殖,促进了其凋亡。EGCG对于细胞凋亡的调控可能通过p53、NF—KB、bcl-2通道的基因表达来实现;EGCG对于细胞增殖的调控可能通过阻滞细胞周期G1／S期实现。结论EGCG可能是通过抑制癌前细胞的增殖和促进其凋亡进而抑制癌症。     

Cyelin D1和CDK4正性调节苯并(a)芘所致人胚肺成纤维细胞周期改变

目的 研究苯并(a)芘[B(a)P]对人胚肺成纤维细胞(HELF)的细胞周期分布及细胞周期蛋白D1(cyclin D1)和细胞周期蛋白依赖激酶4(CDK4)蛋白表达的影响,并探讨两种蛋白含量改变与细胞周期效应之间的关系.方法 将反义cychn D1质粒和反义CDK4质粒导入HELF细胞内.建立两种质粒稳定转染的细胞模型.用0.1、0.5、2.5和12.5μmol/L的B(a)P处理HELF细胞24h.用蛋白印迹方法检测cvclin D1和CDK4蛋白表达水平;利用流式细胞技术检测B(a)P处理对HELF细胞及两种稳定转染细胞系细胞周期的影响.结果 成功建立了反义cyelin D1和反义CDK4稳定转染的细胞系.不同剂量B(a)P处理可引起cvclin D1蛋白表达的显著增加,但对CDK4蛋白表达无明显影响;2.5/μmol/L的B(a)P处理HELF细胞24h后,引起其细胞周期G1期显著下降,S期显著增加;2.5μmol/L的B(a)P处理反义cyclin D1和反义CDK4稳定转染的HELF细胞24h后,对其细胞周期的分布无显著影响.结论 Cyelin D1和CDK4基因均参与了B(a)P所致细胞周期改变过程,并发挥正性调节作用.     

人生长激素对人结肠癌细胞LS-174-T增殖周期的影响

目的：研究人生长激素（hGH)对体外培养的结肠癌细胞株增殖周期和DNA倍体的影响和意义。方法：取指数生长期的结肠癌细胞株LS-174-T（简称174）,以顺铂和（或）不同浓度的hGH体外培养,24h后以流式细胞仪测定细胞增殖周期等指标。结果：hGH能诱导174细胞S期数率显著上升（P＜0.01),G2/M期参数下调（P＜0.01),DNA指数未见增高。加用顺铂后细胞大量凋亡,细胞存活率显著下降（P＜0.01),各组S期百分数或显著下降,或与对照组无差异。结论：hGH体外作用于174细胞,能促使细胞进入对化疗药敏感的DNA合成期,没有上调DNA倍体数;顺铂能诱导大量凋亡,并且抑制hGH促进细胞增殖的作用。     

他克莫司在体外对HepG2细胞增殖及细胞周期的影响

目的研究他克莫司在体外对肝癌细胞HepG2的细胞增殖、细胞周期及细胞周期蛋白A(cyclin A)表达的生物学影响。方法选择肝癌细胞HepG2进行体外培养,经含有不同浓度的他克莫司(低、中和高浓度组剂量分别为50μg/L、100和500μg/L、1 000和3 000μg/L,对照组0μg/L)的培养基干预后,采用四甲基偶氮唑盐微量酶反应比色法(MTT)及流式细胞技术,分别检测细胞增殖、细胞周期及cyclin A水平。结果①中浓度(500μg/L)和高浓度(1 000和3 000μg/L)的他克莫司对HepG2细胞有增殖抑制作用,且抑制作用随浓度的增加而增加,而低浓度的他克莫司则无抑制作用。②他克莫司对HepG2细胞周期的影响:中浓度(500μg/L)和高浓度(1 000和3 000μg/L)的他克莫司作用时,HepG2细胞停止在G0/G1期,从而对肿瘤细胞的生长有抑制作用,且抑制作用随浓度的增加而增加,而低浓度的他克莫司则无抑制作用。③他克莫司可以降低HepG2细胞中cyclin A的表达,且与浓度相关,他克莫司浓度越高,cyclin A表达越少。结论他克莫司在体外对HepG2增殖有抑制作用,其中cyclin A发挥一定的作用。     

苯并(a)芘通过活化蛋白-1-细胞周期蛋白D1/E2F-1信号通路引起细胞周期的改变

目的 探讨活化蛋白-1(activated protein-1,AP-1)在苯并(a)芘[benzo(a)pyrene,B(a)P]引起的人胚肺成纤维细胞(HELF)周期改变中的作用以及AP-1与细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖激酶4(CDK4)和E2F-1/4的上下游关系.方法 转染AP-1荧光报告质粒的HELF细胞(AP-H)无血清培养48 h后,加入2μmol/L B(a)P作用24 h,用荧光检测法检测AP-1的相对活性.用AP-1的化学抑制剂姜黄素(curcumin)抑制其活性,观察它与cyclin D1/CDK4和E2F-1/4的上下游关系.采用流式细胞仪检测细胞周期的变化,用Western blot检测细胞中cyclin D1、CDK4和E2F-1/4蛋白水平的改变.结果 2 μmol/LB(a)P作用24 h后,G1期细胞比例由(71±2)%减少为(48±3)%,差异有统计学意义(P＜0.05);AP-1的活性增加;cyclin D1/E2F-1的蛋白含量增加;CDK4/E2F-4的蛋白水平没有明显改变.抑制AP-1活性后,B(a)P诱导的细胞周期的改变被逆转,B(a)P诱导的cyclin D1/E2F-1蛋白含量的增加被抑制,CDK4/E2F-4蛋白含量没有明显改变.结论 B(a)P通过AP-1引起HELF周期的改变.AP-1是cyclin D1/E2F-1的上游信号分子,但对CDK4/E2F-4的表达不具有调节作用.     

NS-398对肝癌细胞系HepG2环氧合酶-2表达的影响

目的 研究环氧合酶-2(COX-2)抑制剂氮-2,环己氧-4,硝基苯-甲基磺胺(NS-398)对肝癌细胞株HepG2细胞体外抗肿瘤作用,探讨其抗癌作用的分子机理.方法 分别用100、200、300、400μmol/L NS-398处理HepG2细胞,以四甲基偶氮唑蓝(MTT)法测定肿瘤细胞增殖抑制率,流式细胞仪(FCM)检测细胞周期的改变及凋亡百分比的变化,用Westen blotting检测COX-2蛋白的表达,用放射免疫测定法检测NS-398对HepG2细胞前列腺素E2(PGE2)释放水平.结果 NS-398呈剂量依赖性的方式抑制HepG2细胞增殖,并诱导其凋亡,细胞周期分析表明,随着浓度增大,S期细胞明显减少,有G1期细胞累积现象,24 h时半数有效剂量(IC50)为300μmo/L.NS-398明显抑制COX-2的活性和表达.随着浓度的增加,其COX-2蛋白表达逐渐降低,100、200、300、400μmol/L NS-398组灰度值分别为1515.1±127.2、1023.5±108.4、691±94.3和493.6±86.6,与对照组1822±157.4相比差异有统计学意义(t值分别为3.736、1.623、1.810、2.587,P<0.01).NS-398可明显抑制HepG细胞PGE2释放水平,其吸光度值(A值)分别为0.70±0.02、0.48±0.02、0.29±0.01和0.18±0.01,与对照组比较0.03±0.01差异有统计学意义.结论 NS-398对肝癌细胞增殖有抑制作用并诱导其凋亡,可能与细胞G1期阻滞以及通过抑制COX-2活性,抑制COX-2下游产物PGE2表达有关.     

An inhibitory effect of resveratrol in the mitotic clonal expansion and insulin signaling pathway in the early phase of adipogenesis

Resveratrol is known as a potent antiobesity compound that acts partly through inhibition of adipogenesis. However, the direct targets responsible for its antiadipogenic action are unclear. Our hypothesis is that resveratrol inhibits adipogenesis through modulation of mitotic clonal expansion (MCE) and cell signaling pathways in the early phase of differentiation. To test this, we examined the effects of resveratrol on MCE and insulin signaling pathway in the early phase of adipogenesis in murine preadipocytes. We observed that the antiadipogenic action of resveratrol is largely limited to the early phase of adipogenesis. Specifically, the presence of resveratrol in the first 24 hours of adipogenesis was required for its antiadipogenic effect. During the first 24 hours of adipogenesis, resveratrol impaired the progression of MCE by suppressing the cell cycle entry of preadipocytes to G2/M phase, and expression of cell cycle regulators cyclin A and cyclin-dependent kinase 2. Concomitantly, resveratrol inhibited insulin signaling pathway in the early phase of adipogenesis. Furthermore, we revealed an inhibitory effect of resveratrol on insulin receptor (IR) activity, and this is likely through a direct physical interaction between resveratrol and IR. The antiadipogenic effect of resveratrol is through inhibition of the MCE and IR-dependent insulin signaling pathway in the early phase of adipogenesis.     

trans-10,cis-12 conjugated linoleic acid inhibits the G1-S cell cycle progression in DU145 human prostate carcinoma cells

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid, and has evidenced anti-cancer activities in experimental animal cancer models and in vitro studies. The two predominant isomers of CLA are cis-9,trans-11 CLA (c9t11) and trans-10,cis-12 CLA (t10c12). The present study was performed to study the effect of the individual CLA isomers on DU145 cell growth. The cells were incubated in serum-free medium with different concentrations of the fatty acids. Treatment of cells with t10c12 (at 2.5-10 micromol/L) resulted in a dose-dependent reduction in the numbers of viable cells, whereas c9t11 CLA at a concentration of 5 micromol/L slightly increased viable cell numbers at 3 days (P < .05). DNA flow cytometric analysis revealed that the treatment of DU145 cells with t10c12 for 24 hours induced a small but significant increase in the number of cells in the G1 phase, accompanied by a complementary decrease in cells in the S phase. c9t, however, had no effect on cell cycle progression. To determine the molecular mechanisms underlying t10c12-induced G1 arrest, the levels of cell cycle regulatory proteins were estimated by western blot analyses. t10c12 induced a marked increase in p21(CIP1/WAF1) protein levels in a dose-dependent manner. p27(KIP1) was not affected by t10c12. t10c12 moderately decreased cyclin A and cyclin D1 protein levels (P > .05). However, t10c12 did not affect the expression of cyclin-dependent kinase (CDK) 2, CDK4, or cyclin E. t10c12 increased p21(CIP1/WAF1) bound to CDK2 and attenuated CDK2 activity. These results indicate that t10c12-induced p21(CIP1/WAF1) binds to CDK, and inhibits the activity of this enzyme, which results in the observed decrease in the G1-S progression in DU145 cells.     

Disparate in vitro and in vivo antileukemic effects of resveratrol,a natural polyphenolic compound found in grapes

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenol found in grapes and grape wine, has been reported to exhibit cardioprotective and chemopreventive activity against chemical carcinogenesis. It has also been shown to have growth inhibitory activity toward solid tumors in vivo. However, the antitumor activity of resveratrol against hematologic tumors in vivo has not been examined. In this study, the antileukemic activity of resveratrol in vitro and in vivo was examined using a mouse myeloid leukemia cell line (32Dp210). Treatment of 32Dp210 leukemia cells with resveratrol at micromolar concentrations (25-50 micromol/L) significantly and irreversibly inhibited their clonal growth in vitro. The clonal growth inhibition by resveratrol was associated with extensive cell death and an increase in hypodiploid (sub-G1) cells. Resveratol caused internucleosomal DNA fragmentation, suggesting apoptosis as the mode of cell death in 32Dp210 cells. DNA fragmentation was associated with activation of caspase-3, because cleavage of procaspase-3 was detected in resveratrol-treated cells. Although 32Dp210 cells treated with resveratrol in vitro did not produce leukemia in vivo, only a weak antileukemic effect of resveratrol was observed when administered orally. At doses of 8 mg or 40 mg/kg body daily, five times/wk, resveratrol did not affect the survival of mice injected with leukemia cells. Weak potential antileukemic activity of resveratrol was suggested only at a dose of 80 mg/kg body (2 survivors of 14 mice treated). Thus, despite strong antiproliferative and proapoptotic activities of resveratrol against 32Dp210 cells in vitro, a potential antileukemia effect in vivo, if present, occurs only in a small fraction of mice.     

Soy isoflavones alter expression of genes associated with cancer progression, including interleukin-8, in androgen-independent PC-3 human prostate cancer cells

High consumption of soy isoflavones in Asian diets has been correlated with a lower incidence of clinically important cases of prostate cancer. The chemopreventive properties of these diets may result from an interaction of several types of isoflavones, including genistein and daidzein. The present study investigated the effects of a soy isoflavone concentrate (ISF) on growth and gene expression profiles of PC-3 human prostate cancer cells. Trypan blue exclusion and [3H]-thymidine incorporation assays showed that ISF decreased cell viability and caused a dose-dependent inhibition of DNA synthesis, respectively, with 50% inhibition (IC50) of DNA synthesis at 52 mg/L (P = 0.05). The glucoside conjugates of genistein and daidzein in ISF were converted to bioactive free aglycones in cell culture in association with the inhibition of DNA synthesis. Flow cytometry and Western immunoblot analyses showed that ISF at 200 mg/L caused an accumulation of cells in the G2/M phase of the cell cycle (P < 0.05) and decreased cyclin A by 20% (P < 0.05), respectively. The effect of ISF on the gene expression profile of PC-3 cells was analyzed using Affymetrix oligonucleotide DNA microarrays that interrogate approximately 17,000 human genes. Of the 75 genes altered by ISF, 28 were upregulated and 47 were downregulated (P < 0.05). Further analysis showed that IL-8, matrix metalloproteinase 13, inhibin beta A, follistatin, and fibronectin mRNA levels were significantly reduced, whereas the expression of p21(CIP1), a major cell cycle inhibitory protein, was increased. The effects of ISF on the expression of IL-8 and p21(CIP1) mRNA and protein were validated at high and low ISF concentrations. Our data show that ISF inhibits the growth of PC-3 cells through modulation of cell cycle progression and the expression of genes involved in cell cycle regulation, metastasis, and angiogenesis.