Role Of RIP1 Ubiquitination In Sodium Selenite - Induced Apoptosis Of Colorectal Cancer Cells And Its Regulatory Mechanism

Colorectal cancer is one of the most prevalent cancers in both males and females with increasing number of patients diagnosed with this malignancy each year in China. However, the five-year survival rate of colorectal caner remains low. Thus, developing novel chemotherapy drug is of vital importance. Sodium selenite impedes cell proliferation and induces apoptosis of multiple cancer cell lines including prostate cancer, liver cancer, breast cancer as well as lung cancer. Moreover, sodium selenite can significantly attenuate xenograft tumor mice models as well as tumor patients, but the molecular mechanism remains unclear. We investigated the molecular mechanism of selenite-induced apoptosis of colorectal cancer and provide IAP as potential target for combinational therapy. Moreover, our results identified Lys63 polyubiquitination of RIP 1 as an important regulator in selenite-induced apoptosis. Importantly, we built the HCT116 colorectal cancer xenograft model, and assessed the toxicity as well as efficiency of sodium selenite.We found that the Lys63 polyubiquitination of RIP1 was removed and RIP1/Caspase8/FADD complex was formed upon sodium selenite treatment by immunoprecipitation and immunofluorescence experiments. Moreover, Western blot and immunofluorescence experiments showed that removal of Lys63 polyubiquitination of RIP1 led to inactivation of IKK/NFκB signaling pathway and downregulation of Flip, which bound and inactivated Caspase8. In addition, we found that activated Caspase8 cleaved Bid to a truncated form, which translocated to mitochondria and initiated intrinsic apoptosis pathway. Importantly, to rule out detecting Lys48 polyubiquitin chains, we performed immunoprecipitation with RIP1 antibody and incubated Lys63 polyubiquitination-specific antibody in subsequent Western blot analysis. Unexpectedly, our results revealed that RIP1 was highly modified by ubiquitin proteins in cytoplasm without receptor stimulation, and showed that RIP1 had a preference for the number of ubiquitin proteins conjugated to it. Moreover, our results showed that selenite-induced apoptosis shared some of the signaling proteins with extrinsic apoptosis pathway, and provided novel insights for tumor treatment.We investigated the proteins participated in removal of Lys63 polyubiquitination from RIP1. Western blot results showed that selenite downregulated ubiquinase cIAP while upregulated deubiquinase CYLD. Overexpression of cIAP1 showed that Lys63 polyubiquitination of RIP1 was significantly upregulated, which led to resistant to formation of RIP1/Caspase8/FADD complex. Moreover, Western blot and FACS results showed that overexpression of cIAPl significantly attenuated selenite-induced apoptosis. On the other hand, overexpression of CYLD or silencing cIAP significantly downregulated Lys63 polyubiquitination chain modification of RIP1, thus facilitated formation of RIP1/Caspase8/FADD complex and promoted selenite-induced apoptosis. Finally, we found that the inhibition of LEF1 over CYLD transcription was abolished upon selenite treatment via ChIP experiment.Importantly, we collaborated our results in HCT116 xenograft mice model. Our results showed that 1mg/kg/d or 2mg/kg/d sodium selenite didn’t not exert toxicity in HCT116 xenograft mice, while induced apoptosis in the HCT116 xenograft tumor. In addition, immunohistochemistry and Western blot results showed activated cIAP, CYLD and Caspase8 in the HCT116 xenograft tumor upon selenite treatment, which is consistent with our findings in colorectal cancer cells.In conclusion, our research results showed that removal of Lys63 polyubiquitination from RIP1 initiated selenite-induced apoptosis in colorectal cancer cells and investigated the detail mechanism. Our results provided new insights for clinical application of sodium selenite.