Combined Inhibition Of Autophagy And Ionizing Radiation Increase The Radiosensitivity Of Esophageal Cancer

Part I The impact of autophagy inhibitors on autophagy expression in Radiation-induced Eca-109 cellsObjective To investigate the impact of autophagy inhibitors on autophagy expression in Radiation-induced Eca-109 cells, monitor the changes of autophagosome in Radiation-induced Eca-109 cells.Methods Esophageal carcinoma cell line Eca-109 were divided intocontrol grou p,5mmol/L treatment group, 10mmol/L treatment group, 6GY group,6GY+5mmol/L group,6GY+10mmol/L group, a total ofsix different treatment groups. Western blot was used to detect the expression of autophagy marker LC3B in different treatment groupsand monitor ed the changes of autophagosome in esophageal squamous cell carcinoma Eca-109 cells transfected by GFP-LC3.Results Autophagy level increased after radiation, however LC3BII expression and LC3BII/LC3BI ratio significantly decreased after autophagy inhibition (F=25.64, P<0.05); we found that autophagosome fluorescent spots significantly increased in the transfected cells after radiation, autophagosome significantly decreased after autophagy inhibition(F=127.16,P<0.05).Conclusion 3-MA effectively inhibited autophagy induced by radiation, and autophagy decreased significantly in 6GY+10mmol/L group than in 6GY+5mmol/L group, it was in a dose-dependent manner.Part II The impact of autophagy inhibitors on cell cycle and apoptosis of Radiation-induced Eca-109 cellsObjective To investigate the impact of autophagy inhibitors on cell cycle and apoptosis of Radiation-induced Eca-109 cellsMethods Esophageal carcinoma cell line Eca-109 were divided intocontrol group,5mmol/L treatment group, 10mmol/L treatment group,6GY group,6GY+5mmol/L group,6GY+10mmol/L group, a total of six different treatment groups. MTT method was used to detect cell viability of different treatment groups; Morphological characteristics of apoptosis cells in different treatment groups were observed by fluorescein stain in Hoechst33342; The percentage of apoptotic cells and cell cycle analysis in different treatment groups were assessed by flow cytometry.Results We found that compared with 6GY group alone,6GY+10mmol/L group significantly decreased cell viability (F=129.54, P<0.05);Inhibition of autophagy increased radiation-induced apoptosis and the percentage of G2/M-phase cells.Conclusion 3-MA effectively blocked the G2/M phase of esophageal cancer cells induced by radiotherapy, and 3-MA effectively increased the apoptosis of esophageal cancer cells induced by radiotherapy which promotes cell death.Part ? The impact of autophagy inhibitors on radiosensitivity of Radiation-induced Eca-109 cellsObjective To investigate the impact of autophagy inhibitors on radiosensitivity of Radiation-induced Eca-109 cellsMethods Esophageal carcinoma cell line Eca-109 were divided intocontrol grou p,5mmol/L treatment group, lOmmol/L treatment group, 6GY group,6GY+5mmol/L group,6GY+10mmol/L group, a total of six different treatment groups. The radiosensitivity of different treatment groups of esophageal carcinoma cell line Eca-109 was detected by clone formation assay, and the cell survival curve was fitted by the multiple target model.Results DO values of 6GY group,6GY+5mmol/L group and 6GY+10mmol/L group were (2.75�0.26) GY, (2.19�0.18) GY and (1.6�0.05) GY respectively.Conclusion 3-MA can enhance the radiosensitivity of radiation-induced Eca-109 cells.Part ? The role of LKB1 protein in Radiation-induced Eca-109 cellsObjective To investigate the role of LKB1 in Radiation-induced Eca-109 cellsMethods Esophageal carcinoma cell line Eca-109 were divided intocontrol grou p,2GY group,4GY group,6GY group, a total of six different treatment groups. Western blot was used to detect the expression of LKB1 protein, P-LKB1 protein, AMPK protein and P-AMPK pro tein in the different treatment group; Esophageal carcinoma cell line Eca-109 w ere divided into control group and siRNA silencing LKB1 group, a total of tw o different treatment groups. After 6GY radiation exposure, Western blot was us ed to detect the expression of P-LKB1 protein, P-AMPK protein, LC3B? prote in and LC3B? protein in the different treatment group.Results After radiation exposure, both LKB1 and AMPK were activated (phos phorylation) in Esophageal carcinoma cell line Eca-109 cells. In particular, the expression of P-LKB1 and P-AMPK increased in a dose-dependent manner. Fur thermore, siRNA-mediatedknockdown of LKB1 in Eca-109 cells blocked the ra diation-mediated LC3B? to LC3B? conversion after 6GY radiation exposure, an d LKB1 siRNA reduced LKB1 and AMPK phosphorylation.Conclusion LKB1 plays an important role on autophagy expression in radiation-induced Eca-109 cells.Part V The antitumor effect of autophagy inhibitor combined with radiotherapy in animal model of esophageal cancerObjective To investigate the antitumor effect of autophagy inhibitor CQ combined with radiotherapy in animal model of esophageal cancerMethods After established esophageal cancer xenograft model with esophageal carcinoma cell line Eca-109, the nude mice were divided into PBS group, CQ group, 6GY group, CQ+6GY group, a total of four groups. After treated by the different conditions, tumors were measured with calipers twice weekly and were removed as specimens on day 27; Western blot and IOD was used to detect the expression of LC3B and P-LKB1; TUNEL staining to was used to detect apoptosis.Results Tumor tissues were measured on day 27 in the different groups. The tumor volumes were 2089 � 68mm3 in PBS group,2033 � 72 mm3 in CQ group,1405�81 mm3 in 6GY group,239�54 mm3 in CQ+6GY group. The tumor volumes were smaller in CQ+6GY group compared with other groups (P<0.05); LC3BII expression which were detected by IOD were 63.36 � 187 in PBS group,538.64 � 96 in CQ group,751.8�311 in 6GY group,2622.98�443 in CQ+6GY group. The LC3BII expression were higher in CQ+6GY group compared with other groups (P<0.05). P-LKB1 protein expression were 204.5�87 in PBS group,584.9�65 in CQ group, 2630.48�327 in 6GY group,861.41�150 in CQ+6GY group. The P-LKB1 protein expression were higher in 6GY group compared with other groups (P<0.05). Apoptosis which were detected by Tunel were 11�4 in PBS group,19�2 in CQ group,37�6 in 6GY group,49�15 in CQ+6GY group. The apoptosis were stronger in CQ+6GY group compared with other groups (P<0.05).Conclusion Autophagy inhibitor combined with radiotherapy can effectively treat esophageal cancer, and increase radiosensitivity.