Role of autophagy in insulin-promoting cisplatin-induced apoptosis in EC9706 cells

Abstract

Objective To investigate the role of autophagy in apoptosis induced by cisplatin and insulin in human esophageal squamous cells.Methods EC9706 cells were treated with 140 mg/L cisplatin,and cell viability was measured by using methyl thiazol tetrazolium (MTT) assay.The time T1 when the number of living cells reached a plateau was 24 h,then the time T2 was found when cell viability reached a new plateau with the addition of 10 mU/ml insulin after 6 h.At T1 and T2 time points,the cells were stained by Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit and monodansylcadaverine (MDC),respectively.Cell fluorescence was observed qualitatively under a fluorescence microscope,and apoptosis and the fluorescence intensity of MDC were detected quantitatively by using flow cytometry.Western blotting was used to investigate autophagy-related Beclin-1 changes that occurred in the course of treatment.Results (1) Apoptosis ratio of T1 plateau cells [(32.6 ± 4.3) ％] was lower than that of T2 plateau cells [(47.5 ± 5.6) ％] (P ＜ 0.05),and the MDC fluorescence intensity at T1 plateau [(104.9 ± 13.2)] was higher than that at T2 plateau [(82.6 ± 10.3)] significantly (P ＜ 0.05).(2) After co-treatment with insulin,the expression of Beclin-1 was significantly down-regulated (P ＜ 0.0l).Conclusion Inhibition of autophagy by insulin enhances the effect of cisplatin-induced apoptosis in esophageal cancer cells.Insulin is likely to be in this way to realize its chemotherapy sensitization effect. Key words: Esophageal squamous cell carcinoma; Autophagy; Apoptosis; Insulin