China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, China.
Free Radic Res. 2012 Mar;46(3):310-9. doi: 10.3109/10715762.2012.655244. Epub 2012 Feb 2.
Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.
水飞蓟宾是从奶蓟草（水飞蓟）中提取的一种有效成分。我们之前的研究表明，水飞蓟宾通过生成活性氧（ROS）诱导 HeLa 细胞发生自噬和凋亡。在这项研究中，我们研究了自噬和凋亡相关分子是否也参与了 ROS 的生成。水飞蓟宾以剂量依赖的方式促进磷酸化 p53（p-p53）的表达。p53 的特异性抑制剂 Pifithrin-α（PFT-α）降低了 ROS 的产生并逆转了水飞蓟宾的生长抑制作用。ROS 清除剂 N-乙酰半胱氨酸（NAC）减弱了水飞蓟宾诱导的 p-p53 表达上调，表明 p53 可能受到 ROS 的调节，并与 ROS 形成正反馈环。另一方面，水飞蓟宾剂量依赖性地促进磷酸化 c-Jun N 末端激酶（p-JNK）的表达。用 SP600125 抑制 JNK 降低了 ROS 的产生。NAC 下调了 p-JNK 的表达，表明 JNK 可以被 ROS 激活。SP600125 抑制了 p53 的激活，PFT-α 抑制了 p-JNK 的表达，因此水飞蓟宾可能激活了 ROS-JNK-p53 循环以诱导细胞死亡。水飞蓟宾上调了 PUMA 和 Bax 的表达并降低了线粒体膜电位（MMP）水平。PFT-α 降低了 PUMA 和 Bax 的表达。这些结果表明，p53 可以通过 PUMA 途径干扰 MMP 等线粒体功能，从而导致 ROS 的产生。为了阐明 p53 在水飞蓟宾诱导的 ROS 生成中的作用，我们选择了 A431 细胞（人上皮癌细胞），因为它们缺乏 p53 活性（p53His273 突变）。有趣的是，水飞蓟宾并没有上调 A431 细胞中的 ROS 水平，反而降低了 ROS 水平。PFT-α 对 A431 细胞中的 ROS 水平没有影响。p53 的激活在水飞蓟宾诱导的 ROS 生成中起着关键作用。
