Study On Role Of Cav-1in The Resistance To Radiation Of Triple Negative Breast Cancer Cells

Part1:Downregulate Cav-1abrogates radiation-induced EGFR nuclear accumulation and increases radiosensitivity of basal-like TNBC cellsObjective Radiation-induced Cav-1-mediated EGFR nuclear transportation is an important mechanism of radioresistance. The aim of this study is to determine the specific expression and function of Cav-1in TNBC cell lines and investigate whether downregulation of Cav-1would diminish EGFR nuclear trafficking following irradiation and eventually enhance the radiosensitivity of basal-like TNBC cell lines.Methods WB assay was used to compare protein expression in a panal of breast cancer cell lines. Colony formation assay was performed to determine the inherent radiosensitivity among breast cancer cell lines with varied expression of Cav-1. In two Cav-1highly-expressed TNBC cell lines, nuclear accumulation of EGFR at multiple time points following irradiation with or without Cav-1siRNA pretreatment was investigated using WB and confocal assay. The radiosensitising effect of Cav-1siRNA was evaluated using colony formation assay.Results We found that Cav-1is over-expressed in basal-like TNBC cell lines while do not express in HER-2positive cells. We observed that Cav-1positive MDA-MB-231and Hs578T cells were more radioresistant than BT474cells in which there is no expression of Cav-1. Radiation-induced EGFR nuclear translocation in basal-like TNBC cell lines could be impaired by knockdown of Cav-1. Colony formation assay verified that basal-like TNBC cells had been radiosensitized after knockdown of Cav-1.Conclusion These results support the hypothesis that knockdown of Cav-1by siRNA abrogates radiation-induced EGFR nuclear translocation and resultes in increased radiosensitivity of basal-like TNBC cells. Part2:Exploration on the mechanism of radiosensitizing effect of Cav-1siRNA in basal-like TNBC cellsObjective Our previous research found out that Cav-1siRNA could sensitize basal-like TNBC cells to irradiation.In this study, we aimed to explore the involved possible mechanisms.Methods DNA repair and DNA damage at multiple time points following irradiation with or without Cav-1siRNA pretreatment were investigated using immunofluorescence assay in two basal-like TNBC cell lines. Flowcytometry were performed to analyze cell apoptosis induction and cell cycle alteration.Results Radiation-induced phosphorylation of DNA repair protein DNA-PKcs had been hampered when pretreated with cav-1siRNA before RT in both basal-like TNBC cell lines. Silencing of Cav-1also promoted and prolonged DNA damage as indicated by γ-H2AX expression lasting to24h post-RT. Furthermore, Cav-1siRNA alone could induce cell apoptosis and G2/M cell-cycle arrest. When combined with RT, it led to a greater extent of alteration.Conclusion Reduced DNA repair and enhanced DNA damage, G2/M cell-cycle arrest and apoptosis induction are important involved mechanisms of radiosensitizing effect of Cav-1siRNA in basal-like TNBC cells.