Goel Ajay, Fuerst Florentine, Hotchkiss Erin, Boland C Richard
Division of Gastroenterology, Department of Internal Medicine, Baylor University Medical Center and Baylor Research Institute, Dallas, Texas 75246, USA.
Cancer Biol Ther. 2006 May;5(5):529-35. doi: 10.4161/cbt.5.5.2654. Epub 2006 May 5.
While there is an increasing interest in selenium chemoprevention against human colon polyp recurrence and other cancers, the mechanism(s) by which these agents inhibit carcinogenesis are uncertain. Some of the proposed mechanisms include the inhibition of cytosine methyltransferases, carcinogen bioactivation, and inhibition of cyclooxygenase (COX). More recently, it has been suggested that selenium may exert growth inhibitory effects by activating p53. However, the molecular mechanisms of action of selenomethionine, an organoselenium compound present in selenized yeast and currently being investigated in human clinical trials for colon polyp prevention, are unclear. In the present study we tested the hypothesis that selenomethionine might affect colon cancer cell growth by p53 mediated apoptosis and/or cell cycle regulation. Four human colon cancer cell lines including HCT116 and RKO (wild type p53), HCT116-p53KO (isogenic control of HCT116 cells with p53 knocked out) and Caco-2 (mutant p53) were treated with 0-100 microM of selenomethionine for 24, 48 and 72 h. Cell viability rates were determined by the MTT assay. Cell cycle analysis was performed by flow cytometry and apoptosis measured by Annexin V-Cy5 staining. Expression of p53 protein was determined by Western blotting and immunofluorescence assays. All cell lines showed concentration and time dependent growth inhibition with selenomethionine, although HCT116 and RKO cells were the most sensitive to such treatments. Interestingly, although HCT116 and HCT116-p53KO are isogenic cell lines, selenomethionine caused a G2/M cell cycle arrest in HCT116 and RKO cells, but not in HCT116-p53KO cells. Similarly, both HCT116 and RKO demonstrated a significant increase in apoptosis (100-170%; p < 0.01) with 50-100 microM selenomethionine. Cell cycle arrest and apoptosis observed in HCT116 and RKO cell lines were accompanied by a marked increase in p53 protein expression following selenium treatment. These results clearly suggest that selenomethionine exerts p53 dependent growth inhibitory effects in colon cancer cells by inducing G2/M cell cycle arrest as well as apoptosis.
尽管人们对硒在预防人类结肠息肉复发及其他癌症方面的化学预防作用越来越感兴趣，但这些物质抑制致癌作用的机制尚不清楚。一些提出的机制包括抑制胞嘧啶甲基转移酶、致癌物生物活化以及抑制环氧化酶（COX）。最近，有人提出硒可能通过激活p53发挥生长抑制作用。然而，硒代蛋氨酸（一种存在于富硒酵母中的有机硒化合物，目前正在进行预防结肠息肉的人体临床试验）的分子作用机制尚不清楚。在本研究中，我们检验了以下假设：硒代蛋氨酸可能通过p53介导的细胞凋亡和/或细胞周期调控影响结肠癌细胞的生长。用0至100微摩尔的硒代蛋氨酸处理包括HCT116和RKO（野生型p53）、HCT116 - p53KO（p53基因敲除的HCT116细胞的同基因对照）和Caco - 2（突变型p53）在内的四种人类结肠癌细胞系24、48和72小时。通过MTT法测定细胞活力率。通过流式细胞术进行细胞周期分析，通过膜联蛋白V - Cy5染色测定细胞凋亡。通过蛋白质免疫印迹和免疫荧光测定法测定p53蛋白的表达。所有细胞系均显示出硒代蛋氨酸浓度和时间依赖性的生长抑制，尽管HCT116和RKO细胞对这种处理最为敏感。有趣的是，尽管HCT116和HCT116 - p53KO是同基因细胞系，但硒代蛋氨酸在HCT116和RKO细胞中引起G2/M期细胞周期阻滞，而在HCT116 - p53KO细胞中则没有。同样，50至100微摩尔的硒代蛋氨酸使HCT116和RKO的细胞凋亡均显著增加（100 - 170%；p < 0.01）。在HCT116和RKO细胞系中观察到的细胞周期阻滞和细胞凋亡伴随着硒处理后p53蛋白表达的显著增加。这些结果清楚地表明，硒代蛋氨酸通过诱导G2/M期细胞周期阻滞以及细胞凋亡在结肠癌细胞中发挥p53依赖性的生长抑制作用。
