An Experimental Study On The Mechanism Of EC9706 Cell-line Chemotherapy Sensitivity By Autophagy Expression Modulation In ESCC

PART I:Expression and Clinical Significance of Autophagy-related Proteins in Esophageal Squamous Cell CarcinomaObjective:Many studies have indicated that the change of autophagy might be related with the growth and development of tumor. This study was to investigate the expression and implication of atuophagy-related gene Beclinl and microtubule-associated protein 1 light chain 3 (LC3) in esophageal squamous cell carcinoma (ESCC) and their relationships with clinical pathological features of ESCC.Methods:Immunohistochemistry was used to detect the expression of Beclin 1 and LC3 proteins in 51 ESCC tissues and 30 normal para-cancerous tissues. And the correlations of their expression to the clinicopathologic characterisics were analyzed by SPSS 13.0.Results:The positive rates of Beclinl and LC3 were 62.7%(32/51) and 66.7%(34/51) respectively. The expression of Beclinl and LC3 protein was detected in a significantly greater proportion in ESCC carcinoma tissues than that in para-cancerous tissues (15.7% and 9.80%, P<0.05). The positive rates of Beclinl and LC3 were correlated with lymph node metastasis and TNM stage. There was no relationship was between the two factors and age, sex, pathological subtypes and histological grades. Beclinl expression was associated with LC3 expression (P<0.05). Beclin 1 and LC3 were over-expressed in ESCC.Conclusion:LC3 and Beclinl expresson are up-regulated in ESCC. The increase of autophagic capacity may be involved in the carcinogenesis of ESCC, and play an important role in invasion and metastasis of ESCC. Partâ…¡:Role of Autophagy in Esophageal Carcinoma Cell Line induced by CisplatinObjective:To explore the expression of autophagy in EC9706 cell line with treatment of Cisplatin, and study whether the expression level of autophagy is related to the death induced by Cisplatin.Methods:CCK-8 assays were used to detect the growth inhibition rate, and MDC staining was used to examine the autophagosome. Western bolt was used to detect the protein expression of Beclinl and LC3. RT-PCR analysis was used to dectect the expression of Beclin 1 mRNA.Results:DDP can evidently inhibit the growth of EC9706 cell line. After DDP treatment for 24h, MDC staining indicated that autophagosomes were increased in DDP group with respect to the control group, which showed that autophagy was induced by DDP in EC9706 cells. From the expression of LC3 and Beclinl by Western blot and the Beclin 1mRNA expression by RT-PCR, the perk of autphagy indcued by DDP is 6 hours and the expression of autophagy can continue for 24 hours. There was no obvious change the control group.Conclusion:Cisplatin can strongly inhibit proliferation of EC9706. DDP can induce autophagy in EC9706 cells. The expression of autophagy induced by DDP in EC9706 cells may be related with Beclinl signal transduction pathway. Part III:Role of Autophagy in esophageal carcinoma cell line induced by cisplatinObjective:To explore the role of autophagy in cisplatin-induced death of EC9706 cell line.Methods:To explore the role of autophagy in cisplatin-induced death of EC9706 cell line, the autophagy specific inhibitor 3-MA was added with cisplatin. The apoptosis rate and cell death rate were examined by flow cytometry and CCK-8 assays. Western bolt was used to detect the protein expression of PI3K III and cyt-c induced by cisplatin in EC9706 cell line.Results:After treatment of EC9706 cells with 3-MA and cisplatin for 24h, MDC staining indicated that autophagy induced by cisplatin was attenuated by 3-MA. course of treatment. Moreover, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclinl and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy,3-MA.Conclusion:3-MA could attenuate cisplatin-induced autphagy of EC9706 cells and enhace the inhibitory effect on EC9706 cells. Cisplatin induced apoptosis and activated cyt-c in EC9706 cells. In a word, the inhibition of autophagy could be a novel strategy for the adjuvant chemotherapy of esophageal cancer...