Mechanisms Of The Effect Of Radiotherapy On Bortezomib On Esophageal Squamous Cancer Cell Lines ECA-109 And TE13 Cells In Hypoxia

Objective Esophageal cancer is the Sixth most common cancer and the fourth most common cause of cancer deaths in China,Radiation therapy is a important treatment for esophageal squamous cell carcinoma(ESCC), but radiosensitivity is low and the effect of treatment effect is not ideal. Bortezomib is a specific and reversible boronate inhibitor. Here we investigate the radiosensitization of bortezomib on Eca-109 and TE13 cells in hypoxia and its potential radioprotective mechanisms.Methods ESCC cell lines ECA109 and TE13 cells were used for the study.(1)The CCK-8 assay was used to detect the inhibition of proliferation of bortezomib for TE13 cells, and got the Suitable concentration.(2) The apoptosis and cell cycle of bortezomib combination with irradiation on ESCC cell lines were evaluated by Flow cytometry.(3) Radiosensitization of bortezomib were investigated by colony formation assay.(4) Immunfluorescence detected the number of phospho- H2 AX foci and the percentage of the positive cells that expressed phospho- H2 AX to investigate DSB and DNA injury repairing.(5) The expression of HIF-1α 、 VEGF and apoptosis-related proteins on ECA109 and TE13 cells in hypoxia were inhibited due to the treatment of bortezomib by Western blot.Results(1)Bortezomib inhibited growth of ECA109 and TE13 cells in a dose-time-dependent manner. After 24 h treatment with bortezomib, the IC50 for ECA109 and TE13 was 34.98 n M and 30.78 n M, respectively, and then at 48 h, the IC50was15.67 n M and 13.52 n M.(2) The apoptosis rate of the cells treated with bortezomib inereased obiviously than the cells without bortezomib and the cells were arrested at G2 / M phase.(3) Clonogenic assay showed that bortezomib could enhance the hypoxic ECA109 and TE13 cells radiosensitivity and the sensitizing enhancement ratio(SER) of 5n M、10n M bortezomib were 1.21、1.61 and 1.42、171 respectively.(4)The percentage of the positive cells significantly increased in the combination group.(5) With increasing concentrations of bortezomib, the protein expression of HIF-1α and VEGF on ESCC cells in hypoxia were significantly inhibited.(6) ESCC cells treated with 8 Gy radiation plus 10 n M bortezomib compared with bortezmob or radiation alone induced lower levels of Cleaved-Caspase-9、 Cleaved-Caspase-8、Cleaved-Caspase-3 and cleaved-PARP1 in both ECA-109 and TE-13 cells.Conclusions(1)Bortezomib enhances radiosensitivity of ESCC under hypoxia condition and the mechanisms involved were revealed;(2)decreasing the expression of HIF- 1α and VEGF;inducing apoptosis by activating Caspase-9、 Caspase-8、Caspase-3; arresting at G2 / M phase of cell cycle; delaying DNA damage repair after radiation.