Research On How Irradiation-induced Necroptosis In Tumor Cell Repopulaiton

Purpose:Tumor cell repopulation after radiotherapy is a major cause for the tumor radioresistence and recurrence.This study aims to investigate the underlying mechanism of the tumor repopulation after radiotherapy especially focuses on whether and how necroptosis associated with the process.Methods:The occurrence of necroptosis in irradiated human colorectal cancer derived tumor cells were examined by different methods as flow cytometry and western blot for cultured tumor cells,and immunohistochemical staining(IHC)for xenograft tumor in nude mice.The growth-promoting effect of necroptotic cells after irradiation were investigated by in vitro and in vivo tumor repopulation models,further confirmed by inhibitor or shRNA of necroptosis associated protein or genes.The downstream relevance genes and factors were identified by western blot and Elisa.Finally,the immunohistochemistry staining was conducted in tissue microarray(TMA)from 71 colorectal cancer patients to verify clinical relationship with results from this study.Results:Radiation-induced necroptosis depended on RIP1/RIP3/MLKL pathway,and the evidence in vitro and in vivo demonstrated that the inhibition of necroptosis attenuated growth-stimulating effects on living tumor reporter cells.The JNK/IL8 and COX2/PGE2 were identified as downstream signaling of MLKL during necroptosis,and inhibition of JNK,IL8,IL8 receptor or COX2 significantly reduced tumor repopulation after radiotherapy.Moreover,the high expression of IL8 was associated with poor clinical prognosis in human colorectal cancer.Conclusions:Necroptosis contributed to tumor repopulation after radiotherapy depended on RIP1/RIP3/MLKL pathway.And MLKL/JNK/IL8 or MLKL/COX2/PGE2 could be developed as potential targets for blocking tumor repopulation and enhancing the efficacy in colorectal cancer radiotherapy.