摘要
目的 观察自噬在蛋白酶体抑制剂MG132诱导的食管鳞癌细胞死亡中的作用.方法 用20 μmol/L MG132和（或）5 mmol/L 3-甲基腺嘌呤（3-MA）处理人食管鳞癌EC9706细胞,膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）/碘化丙锭（PI）双染色流式细胞术检测细胞凋亡率；吖啶橙（AO）染色荧光显微镜观察细胞酸性自噬泡的变化；Western blot法检测自噬相关蛋白Beclin-1及凋亡相关蛋白半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-9的表达.结果 流式细胞仪检测示MG132、3-MA及MG132＋ 3-MA组细胞凋亡率为（57.37±0.81）％、（9.43±0.15）％、（79.40±0.92）％.MG132组经吖啶橙染色后,细胞内酸性自噬泡明显增加,呈明亮的红色荧光,而加入3-MA联合作用后,则上述现象被抑制.Western blot结果示对照组、MG132、3-MA及MG132＋ 3-MA组Beclin-1和Caspase-9相对灰度值分别为0.57 ±0.01、0.90 ±0.03、0.49 ±0.14、0.65±0.01和0.61±0.05、0.88±0.02、0.70 ±0.01、0.98±0.02.结论 MG132可以诱导食管鳞癌细胞凋亡,并可激活其自噬；3-MA可能通过上调Caspase-9的表达增强MG132对食管癌细胞的杀伤作用.
Objective To investigate the role of autophagy on proteasome inhibitor MG132 induced-death of esophageal squamous cancer cells.Methods The esophageal squamous cancer EC9706 cells were treated with 20 μmol/L MG132 and/or 5 mmol/L 3-Methyladenine （3-MA）.Flow cytometry was used to examine the apoptosis rate of EC9706 cells treated with Annexin V-FITC/propidium iodide （PI） double staining.The change of acidic vesicle organelles （AVO） was detected by using acridine orange （AO） staining under fluorescence microscope.The expression of autophagy-associated protein Beclin-1 and apoptosis-associated protein cysteinyl aspartate-specific protease （Caspase）-9 was detected by using Western blotting.Results Flow cytometry showed that the apoptosis rate of MG132 group,3-MA group and MG132 ＋3-MA group was （57.37 ± 0.81） %,（9.43 ± 0.15） % and （79.40 ± 0.92） % respectively.The autophagic vacuoles were obviously increased in MG132 group,but the phenomenon was inhibited after combined use of 3-MA.The protein average values of Beclin-1 and Caspase-9 in control group,MG132 group,3-MA group and MG132 ＋3-MA group were 0.57 ±0.01,0.90 ±0.03,0.49 ±0.14 and 0.65 ±0.01,and 0.61 ±0.05,0.88 ±0.02,0.70 ±0.01 and 0.98 ±0.02,respectively.Conclusion MG132 can induce autophagy and apoptosis in esophageal squamous cancer cells,while 3-MA can promote MG132-induced cell killing effect possibly by upregulating the Caspase 9 expression.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2013年第11期2339-2341,共3页
Chinese Journal of Experimental Surgery
基金
河南省高校科教创新团队
吴阶平医学基金资助项目（320.6750.1319）
郑州大学第一附属医院青年创新基金资助项目
关键词
MG132
3-甲基腺嘌呤
自噬
食管鳞癌
Keywords：MG132
3-methyladenine
Autophagy
Esophageal squamous carcinoma
