Silymarin induces a multi-targeted cell death process in the human colon cancer cell line HT-29

This study investigated the Silymarin (SM) effects on growth of HT-29 human colon cancer cell line and its cellular death mechanism. HT-29 cells were treated by 25μM/ml of SM for 48h. HT-29 cells were also pretreated with 10mmol zVAD (apoptosis inhibitor), 10mmol 3-MA (autophagy inhibitor) and 3mmol Nec (necroptosis inhibitor) for evaluation cell death induced by apoptosis, outophagy and necroptosis. MTT and clonogenicity assays revealed that the SM without inhibitors induced a significant decrease in cell viability and proliferation of HT-29 cells (p<0.05). SM in presence of Nec and 3-MA significantly decreased viability and proliferation of HT-29 cells. Apoptotic indexes were significantly increased compare to other groups. SM in presence of zVAD and 3-MA significantly decreased viability and proliferation of HT-29 cells, and expression of RIPK1 and RIPK3 in compare to absence the inhibitors. Necroptotic index was also increased. zVAD could not completely blocked apoptosis. This indicate that SM may stimulate both caspase-dependent and caspase-independent apoptotic pathways. SM in presence of zVAD and Nec significantly decreased cell viability and proliferation of HT-29 cells in compare to control and other experimental groups. LC3-II positive cells were significantly increased in this group in compare to the control and SM without inhibitors treatment. Our results revealed that the high SM toxicity to HT-29 cells depends on multiple cell death pathways, which involved mainly autophagy.