Role Of Autophagy In Esophageal Squamous Cancer Cells Induced By Proteasome Inhibitor

BackgroudThere are mainly two protein degradation ways in eukaryotic cells, one is theubiquitin-proteasome pathway,the other is autophagic-lysosomal pathway. Theubiquitin-proteasome pathway (UPP) is composed of ubiquitin、proteasome and aseries of related enzymes.The premise of Proteasome hydrolyzing protein is theubiquitination of a target protein. MG132is a common, effective aldehyde peptideprotease inhibitor.Studies have demonstrated that inhibiting proteasome activity cannot only induce apoptosis in glioma cells but also activate cell autophagy.Currently, there are surely two regulation pathways of autophagy,one is mTORsignaling pathway and the other is phosphoinositide-3-kinasepathway(PI3K)/Akt(PKB) pathway.3-MA is a classic autophagy inhibitor,whichinhibits autophagy by inhibiting the activity of Class Ⅲ PI3K（Vps34）.In China, the most common pathological type of esophageal cancer isesophageal squamous cell carcinoma, which accounting for90%of all esophagealcancer. Although the comprehensive treatment which is mainly by surgery includingradiotherapy and chemotherapy has been more mature, but the prognosis is still notideal. The causes of death are tumor metastasis or recurrence.Currently, theesophageal cancer studies mainly focus on apoptosis,while more and more researcherspay much attention to autophagy which has not only become the popular areas of lifesciences,but also of great significance in the field of tumor research.The role of autophagy on proteasome inhibitor-induced esophageal cancer cell death has not seen in the relevant reports at home and abroad. Accordingly, wepropose the academic hypothesis that inhibiting autophagy can enhance theproteasome inhibitor-induced esophageal cancer cell killing effect.ObjectiveThe aim of the study is to explore the role of3-methyladenine (3-MA) onproteasome inhibitor MG132-induced apoptosis of esophageal squamous cancer cells.In order to provide new ideas and direction for esophageal squamous cell carcinoma.MethodsThe esophageal cancer EC9706cells were treated with MG132and/or3-MA,then the drug optimal concentration and action time were tested with MTT assay;fluorescence microscope was performed to examine the change of autophagicvacuoles and acidic vesicle organelles(AVO);the apoptosis rate of the EC9706cellstreated with AnnexinV-FITC/PI double staining was detected by flow cytometry; Theexpression of apoptosis-associated protein Caspase9and autophagy-associated proteinBeclin1were tested by Western Blot assay.ResultsThe MTT assay showed that the48h IC50of MG132was about20umol/L,the48h IC10of3-MA was about5mmol/L.The FCM analysis showed thatthe EC9706cell inhibitory rate was（57.37±0.81）%(MG132group),（9.43±0.15）%(3-MA group)and（79.4±0.9）%(MG132+3-MA group) respectively. The autophagicvacuoles were obviously increased in MG132group treated with MDC staining,butthe above phenomenon was inhibited after co-treated with3-MA and MG132.Thechang of acidic vesicle organelles(AVO) is the same as the autophagic vacuoles. Theprotein average values of expression of Beclin1and caspase9in control group,MG132group,3-MA group and MG132+3-MA group were0.57±0.01，0.90±0.03，0.49±0.14and0.65±0.01，and0.61±0.05，0.88±0.02，0.70±0.01and0.98±0.02，respectively。ConclusionMG132can induce apoptosis and enhance autophagy in EC9706.However3-MAcan inhibit autophagy, while enhance MG132-induced cell killing effect.