Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells
Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells
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DOI：
10.1038/sj.onc.1207158
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发表时间：
2003-11-13
期刊：
影响因子：
8
通讯作者：
Agarwal, R
中科院分区：
文献类型：
--
作者：
Agarwal, C;Singh, RP;Agarwal, R
水飞蓟素是一种确定的天然类黄酮混合物，最近在大鼠模型中显示出对结肠癌发生具有强效的癌症化学预防作用；然而，这种功效的机制尚未阐明。本研究使用水飞蓟素中的纯活性物质水飞蓟宾，研究了水飞蓟宾在人结肠癌HT-29细胞中的抗增殖和凋亡作用，以及相关的分子改变。水飞蓟宾在50-100马克杯/毫升剂量下处理细胞导致中等至非常强的生长抑制，其剂量和时间依赖的方式，这主要是由于细胞周期进程的G0/G1阻滞；较高的剂量和较长的治疗时间也会引起G2/M骤停。在有关水飞蓟宾对细胞周期进程影响的机制研究中，水飞蓟宾处理导致Kip1/p27和Cip1/p21蛋白和mRNA水平上调，降低CDK2、CDK4、cyclin E和cyclin D1蛋白水平，抑制CDK2和CDK4激酶活性。在其他研究中，我们观察到水飞蓟宾阻滞G2/M与cdc25C、cdc2/p34和cyclin B1蛋白水平以及cdc2/p34激酶活性的降低有关。在评估水飞蓟宾处理细胞生物学命运的研究中，水飞蓟宾诱导的细胞周期阻滞和生长抑制与细胞分化无关，但导致凋亡死亡。定量凋亡分析显示，水飞蓟宾作用48 h后，凋亡细胞死亡率高达15%。有趣的是，水飞蓟宾诱导的HT-29细胞凋亡与caspases的激活无关，因为所有的caspases抑制剂都不能逆转水飞蓟宾诱导的细胞凋亡。水飞蓟宾处理HT-29细胞后，发现半胱天冬酶活性增加，半胱天冬酶和PARP切割缺失，胞浆中细胞色素c释放缺失，进一步证实了这一观察结果。在小鼠中进行的其他研究表明，在HT-29细胞中有效的剂量可以在血浆中实现，这增加了本研究结果的重要性，并可能转化为水飞蓟宾对结肠癌的体内抗癌功效。总之，这些结果确定了水飞蓟宾在人结肠癌HT-29细胞中作为细胞周期调节剂和细胞凋亡诱导剂功效的分子机制，并为进一步研究这种无毒药物在结肠癌预防和干预中的潜在用途提供了依据。
Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50-100 mug/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.