Molecular Mechanism Of PKD1 And Its Related Signaling Pathway In The Apoptosis Of Colorectal Cancer Cells Induced By Sodium Selenite In Vivo And In

The essential trace element selenium (Se) is involved in several physiological processes of the body and has been applied to cancer prevention and treatment. Supranutritional selenite induces apoptosis, an important cellular event that accounts for the anti-cancer effects of selenite.Evidence from several cancer cell lines has demonstrated that sodium selenite treatment possesses chemo-preventive and chemotherapeutic efficacy against malignant cancer; however, the detailed mechanisms underlying these effects are not clearly understood. Further studies would broaden our understanding of the anti-cancer effects of this compound and provide a theoretical basis for its clinical application.In this study, we primarily found that selenite exposure inhibited phosphorylation of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) in HCT116 and SW480 colorectal cancer (CRC) cells. From western blot, ChIP experiments and luciferase assays, we discovered that the inhibition of CREB transcriptional activity further resulted in enhanced selenite-induced inhibition of Bcl-2 expression and increased PARP and caspase-9 cleavage. The results also showed CREB is a PKD1 substrate and selenite treatment greatly decreased the association between p-PKD1 and p-CREB in CRC cells.Furthermore, our western blot assays and immunofluorescence results indicated that selenite inhibited CREB/Bcl-2 survival signalling via PKD1 activity in CRC cells. We then determined which upstream signal causes PKDI inhibition. After screening several proteins, we found that p38 MAPK inhibits PKD1 activation in selenite-treated CRC cells. Further experiments strongly suggested that p38 MAPK inhibited the PKD1/CREB/Bcl-2 survival signalling pathway in selenite-treated CRC cells.BCL-2 antagonist of cell death (Bad) plays important in triggering mitochondrial apoptosis and 14-3-3 protein could phospho-Bad sequestered in the cytoplasm. In this study, we found selenite treatment caused Bad dephosphorylation and released from 14-3-3 protein, which up-regulated Bad translocated from cytoplasm to mitochondria to promote apoptosis. CoIP, Immunofluorescence and western blot assays also suggested that selenite induced Bad-mediated mitochondrial apoptosis by depressing AKT phosphorylation. Further experiments indicated that selenite treatment induced Bad accumulation to mitochondria by inhibiting the PKD1/AKT signalling pathway in selenite-treated CRC cells.Reactive oxygen species (ROS) was demonstrated to play an important role in initiating apoptosis of cancer cells. By probing the ROS level using DCFH-DA probe we discovered that sodium selenite treatment increased the level of ROS in CRC cells. To explore the possible link between ROS and the PKD1-mediated pathway, we depleted ROS in selenite-treated cells with the ROS scavenger MnTMPyP and found that depletion of ROS almost completely inhibited selenite-induced PKD1 dephosphorylation. Further experiments indicated that ROS is an important upstream factor in modulating PKD1/CREB/Bcl-2 and PKD/AKT/Bad pathway and inducing CRC cells apoptosis.To investigate the effects of selenite in vivo, we constructed colon xenograft nude mice model of both HT116 and SW480 CRC cells to analyze the alteration of p38 MAPK/PKD1/CREB/Bcl-2 and PKD/AKT/Bad pathway in tumor tissues using western blot and immunohistocheical assays. These results indicate that selenite treatment of tumour-bearing mice markedly attenuated tumour growth and induce the similar change pattern of p38 MAPK/PKD1/CREB/Bcl-2 and PKD/AKT/Bad pathway with that in vitro.Our study provided more evidence to understand the detailed mechanisms of apoptosis induced by supranutritional selenite in CRC cells. These findings provide a theoretical basis for the use of sodium selenite in clinical applications as a treatment for malignant tumours.