Agarwal Chapla, Singh Rana P, Dhanalakshmi Sivanandhan, Tyagi Anil K, Tecklenburg Marianne, Sclafani Robert A, Agarwal Rajesh
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Oncogene. 2003 Nov 13;22(51):8271-82. doi: 10.1038/sj.onc.1207158.
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 microg/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 silibinin 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.
水飞蓟素是一种天然黄酮类化合物的特定混合物，最近的研究表明，它在大鼠模型中对结肠癌发生具有强大的癌症化学预防功效；然而，这种功效的机制尚未阐明。在此，我们使用水飞蓟素中的纯活性剂水飞蓟宾，展示了其对人结肠癌HT - 29细胞的抗增殖和凋亡作用以及相关的分子改变。以50 - 100微克/毫升的剂量用水飞蓟宾处理细胞，会导致中度至非常强烈的生长抑制，且呈剂量和时间依赖性，这主要是由于细胞周期进程停滞在G0/G1期；更高剂量和更长的处理时间也会导致G2/M期停滞。在关于其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蛋白及其mRNA水平上调，同时CDK2、CDK4、细胞周期蛋白E和细胞周期蛋白D1蛋白水平降低，以及CDK2和CDK4激酶活性受到抑制。在其他研究中，我们观察到水飞蓟宾引起的G2/M期停滞与cdc25C、cdc2/p34和细胞周期蛋白B1蛋白水平以及cdc2/p34激酶活性降低有关。在评估水飞蓟宾处理细胞的生物学命运的研究中，水飞蓟宾诱导的细胞周期停滞和生长抑制与细胞分化无关，而是导致凋亡死亡。定量凋亡分析显示，水飞蓟宾处理48小时后，凋亡细胞死亡高达15%。有趣的是，水飞蓟宾诱导HT - 29细胞凋亡与半胱天冬酶激活无关，因为所有半胱天冬酶抑制剂都不能逆转水飞蓟宾诱导的凋亡。水飞蓟宾处理HT - 29细胞后，半胱天冬酶活性增加、半胱天冬酶和PARP裂解缺乏以及细胞溶质中细胞色素c释放缺乏的结果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细胞中发现有效的水飞蓟宾剂量在血浆中是可以达到的，这增加了本研究结果的重要性以及它们在水飞蓟宾对结肠癌的体内抗癌功效方面可能的转化价值。总之，这些结果确定了水飞蓟宾作为人结肠癌HT - 29细胞中的细胞周期调节剂和凋亡诱导剂的功效的分子机制，并证明了进一步研究以调查这种无毒剂在结肠癌预防和干预中的潜在用途的合理性。
