Studies On The Mechanisms For Cell Death And Generation Of Reactive Oxygen Species/Reactive Nitrogen Species Induced By Silibinin In Hela Cells

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing10%fetal bovine serum or in serum free media (SFM) with an IC50of approximately80-100and40-60μM at24h, respectively. Silibinin induced autophagy at12h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at24h, detected by observation of apoptotic bodies and activation of caspase-3.3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin’s inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at12h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and·OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at12h. High concentration of N omega-nitro-1-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiratory functions of mitochondria, leading to ROS production and oxidative damage.Then 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 NAC attenuated silibinin-induced up-regulation of p-p53expression, suggesting that p53might 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 SP600125decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53was suppressed by SP600125and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-a reduced the expression of PUMA and Bax. These results showed that p53could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53in silibinin induced ROS generation, we have chosen the A431cells (human epithelial carcinoma) because they Iack p53activity (p53His273mutation). Interestingly, silibinin did not up-regulate the ROS level in A431cells but lower the ROS level. PFT-α had no influence on ROS level in A431cells. p53activation plays a crucial role in silibinin induced ROS generation.In order to clarify the relationship between RNS and nitric oxide (NO) in HeLa cells, we chose SNP as a NO donor to inhibit the cell viability. We found that silibinin treatment did not reduce the cytotoxicity of NO by reducing the ROS-induced RNS levels; conversely, silibinin treatment enhanced the cytotoxicity of NO. Pre-treatment with the NO scavenger PTIO preserved the viability of SNP-or silibinin-treated cells. Buthionine sulfoximine (BSO) treatment was also used to deplete the level of glutathione (GSH) and subsequently enhance the cytotoxicity of NO. Pre-treatment with BSO enhanced the SNP-induced reduction of cell viability but had no such effects in the silibinin-treated cells. These results led us to investigate whether silibinin treatment could induce the depletion of GSH. To elucidate the role of p53in this process, A431cells were used. To our surprise, silibinin treatment did not lower the GSH level in A431cells but rather elevated the GSH level. Unlike the ROS level, the NO level was still up-regulated by silibinin treatment in A431cells. Cumulatively, these findings support the idea that the silibinin-induced GSH depletion, which is mediated by p53, enhances the cytotoxicity of NO in HeLa cells.