Sodium Selenite In Colon Cancer Cells Induced By Redox-dependent Bax Activation And Apoptosis Research

It is well supported that selenium has chemopreventive effects, and emerging evidence suggests that selenium has chemotherapeutic potential by inducing cancer cell apoptosis with minimal side effects to normal cells within a proper dose range. Colorectal cancer is a common neoplasm and the incidence in China is increasing. We proved that supranutritional doses of selenite could induce apoptosis both in cultured colon cancer cells and xenograft tumor models. However, the mechanism by which selenium induces apoptosis is not well understood. We have investigated the role of Bcl-2 family proteins in selenite-induced apoptosis and the molecular mechanism leading to Bax activation.We found that 5-10μM selenite could induce typical apoptosis in SW480, HCT116 and HT29 colon cancer cell-lines with multiple methods, and selenite induces cytochrome c release from mitochondria into cytosol, indicating activation of the mitochondrial apoptotic pathway. Bcl-2 family proteins are central regulators in apoptosis, especially in the mitochondrial apoptotic pathway, we focused on the role of Bax in selenite-induced apoptosis. Selenite induces Bax translocation from cytosol to mitochondria, we stably expressed GFP tagged Bax in SW480 cells and constructed a real-time cellular model to observe the functional changes in Bax. We found that selenite induces the sequential events including Bax conformational change, translocation, oligomerization and cytochrome c release in this model; and clarified the causal relationships among them. Overexpression of Bcl-xL, an antiapoptotic member of Bcl-2 family, inhibited significantly the sequential events above. We further evaluated the importance of Bax in selenite-induced apoptosis using Bax/Bak deficient cells. Bax/Bak double deficient MEF cells are strongly resistant to selenite-induced cell death compared with their wildtype counterparts, and Bax deficient HCT116 cells are less sensitive to selenite-induced cell death compared with wildtype HCT116 cells, these indicate that Bax is an important mediator in selenite-induced cell death. We further investigated the molecular mechanism leading to Bax activation, and found that selenite induced ROS accumulation in SW480 cells. In SW480 cells stably expressing GFP-Bax, a MnSOD mimic MnTMPyP inhibits selenite-induced Bax activation; NAC and BSO potentiate selenite-induced ROS generation and Bax activation. Among the possible clues responsible for Bax activation induced by selenite, we found Bim upregulation caused by oxidative stress might be the one. Besides, we found that selenite induced Bax oxidation using biotin conjugated thiol-reactive probes, and MnTMPyP could abrogate Bax oxidation. These suggest selenite could change the redox status of Bax by inducing endogenous ROS generation, and free thiols on cysteine residues might be the targets. To validate this hypothesis, we constructed a Bax expressing vector with the two conserved cysteine residues mutated using site-directed mutation methods (C62/126S GFP-Bax) and stably expressed it in SW480 cells, and compared the sensitivity to selenite-induced Bax activation with the wildtype GFP-Bax transfectant. Cysteine 62/126 mutation inhibited Bax conformational change, translocation, oligomerization and cytochrome c release induced by selenite and H₂O₂, but had no effects on Bax activation and cytochrome c release induced by DNA toposiomerose inhibitors including etoposide and camptothecin. These results indicate that selenite induces alteration of redox status of cysteine 62/126 of Bax by endogenous ROS generation, and hence leads to Bax conformational change and activation.Although 5-10μM selenite could induce apoptosis of colon cancer cells in vitro, but it has minimal effects in primary cultured human embryo intestinal epithelial cells when administrated at the same dose. We established a xenograft colon cancer model by implanting SW480 cells in nude mice, and found that 2 mg/kg/d selenite could inhibit the growth of tumors without obvious adverse effects. TUNNEL staining showed elevated apoptosis rate in tumor tissues after selenite treatment, these results suggest that supranutritional doses of selenite could selectively kill colon cancer cells. In summary, apoptosis is not only a good explanation to the chemopreventive effect of selenite, but also provide chances for using this compound in conlon cancer treatment.