Akt/beta - Catenin/foxo3a Signaling Pathway In Colorectal Cancer Cell Apoptosis Induction Of Sodium Selenite Mechanism Research

Selenium is an essential trace element for human health. Mounting epidemiological and clinical evidence indicate that it possesses chemopreventive and chemotherapeutic efficacy against malignant cancers. Exploring the molecular mechanisms underlying the anti-cancer effect of selenium is a hot spot of current research. A growing number of people are diagnosed with colorectal cancer in China and the cure rate is very low. Exploiting novel chemotherapeutic drugs treating colorectal cancer is of great significance.Our previous data show that supranutritional doses of selenite could induce apoptosis in colorectal cancer cell lines and xenograft colon tumor in nude mice. But the detailed mechanism is unclear.In this study, we focused on the role of AKT/β-catenin in selenite-treated colorectal cancer cells, β-catenin is aberrantly accumulated in most colorectal cancer cells and is pivotal for development of colorectal cancer. From western blot, immunofluorescence and luciferase assays, we discovered that selenite inhibited β-catenin accumulation in the nucleus and caused downregulation of its targets cyclin D1and survivin. AKT, a pro-survival factor, is frequently upregulated in tumor cells. From western blot and AKT kinase activity assay results, we detected that selenite treatment inhibited activation of AKT. Furthermore, suppression of AKT activity resulted in less nuclear accumulation of β-catenin and inhibition of its targets cyclin D1and survivin. Both these changes were found to be correlated with selenite-induced apoptosis in colorectal cancer cells.In addition, we found that selenite could regulate PTEN/PI3K/PDK1upstream of AKT. On one hand, selenite suppressed PI3K/PDK1signaling axis to block activation of AKT; On the other hand, PTEN was activated by selenite to negatively regulate PI3K/AKT signaling. In the context of AKT inhibition, FoxO family proteins were activated, which could be reflected from the decrease of inhibitory phosphorylation of them by selenite treatment. Of note, FoxO3a was discovered to accumulate in the nucleus after selenite treatment. Inhibiting or activating AKT indicated that the change of FoxO3a was under the control of AKT and was associated with apoptosis.Accumulated FoxO3a in the nucleus was found to bind more intensely with promoters of Bim, enhancing its transcription and expression. Correspondingly, upregulated Bim translocates from cytoplasm to mitochondria to promote apoptosis. JC-1assay demonstrated that the mitochondria outer membrane potential decreased in response to selenite treatment Specific knockdown of Bim attenuated the apoptotic rate of cells treated by selenite, indicating the participation of Bim in selenite-induced apoptosis. It is worth noting that FoxO3a was also found to bind more intensely with promoters of PTEN by ChIP experiments. Enhanced expression of PTEN was also involved in regulating AKT/FoxO3a signaling, further amplifying the pro-apoptotic response of selenite.Reactive oxygen species (ROS) was demonstrated to be a vital factor in initiating apoptosis of tumor cells. We probed the ROS level using DCFH-DA probe and discovered that selenite induced ROS increase in colorectal cancer cells. Depletion of ROS by MnTMPyP reversed the change of AKT/β-catenin/FoxO3a signaling axis and apoptosis by selenite. These data indicate that ROS is an important upstream factor in modulating AKT/β-catenin/FoxO3a and inducing apoptosis of colorectal cancer cells.In vivo, we constructed a colon xenograft nude mice model and analyzed the alteration of AKT/β-catenin/FoxO3a in tumor tissues using western blot and immunohistochemical assays. These results indicate that selenite induce the similar change pattern of this signaling axis with that in vitro.Finally, we explored the effect of selenite on autophagy in HCT116and SW480colorectal cancer cells. By carrying out western blot, electron microscopy and GFP-LC3assays, we detected that selenite treatment enhanced autophagy in cells. When autophagy was inhibited by3-MA or Bafiloymycin A1, selenite-induced apoptosis increased. While autophagy was enhanced by rapamycin treatment, the apoptosis was blocked to some degree. These results demonstrate that selenite-induced autophagy acts as a pro-survival factor to antagonize apoptosis, but the detailed mechanisms remain to be discovered.Our findings provide novel insights into screening novel molecular targets in killing tumor cells by selenite. These results help to elucidate the chemopreventive and chemotherapeutic efficacy of selenium in treating tumor. Also clinical translation of selenium would be fastened based on these findings.