自噬抑制剂3-MA对顺铂诱导的人胶质瘤U251细胞凋亡的影响  

Effect of 3-methyladenine on cisplatin induced apoptosis in human glioma U251 cells

作　　者：张瑞剑[1] 陈谦学[1] 

机构地区：[1]武汉大学人民医院神经外科,430060

出　　处：《中华神经外科杂志》2015年第4期406-411,共6页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金（81372683）

摘　　要：目的 用自噬抑制剂3-甲基腺嘌呤（3-methyladenine,3-MA）抑制自噬,观察其对顺铂诱导的U251细胞凋亡的影响及其作用机制.方法 MTT法检测3-MA对顺铂诱导的U251细胞生长抑制的影响;Hoechst33342染色,观察3-MA对顺铂诱导细胞凋亡的影响;蛋白免疫印迹法检测内质网应激（endoplasmic reticulum stress,ERS）相关蛋白,泛素化蛋白（ubquitinated proteins Ub1）,蛋白二硫键异构酶（protein disulfide isomerase,PDI）,葡萄糖调节蛋白78（glucose-regulated protein78,Grp78）的表达,并检测了C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白（C/EBP homologous protein,CHOP）,活化的Caspase-4和活化的Caspase-3的表达水平;免疫荧光染色后共聚焦显微镜观察3-MA与顺铂联合作用于U251细胞,微管相关蛋白轻链3（microtubule-associated protein 1 light chain 3,LC3）、PDI的表达.结果 MTT结果显示,3-MA可以促进顺铂对U251生长抑制（顺铂组与对照组相比,P=0.00324;联合组与顺铂组相比P=0.02249）,Hoechst33342染色结果显示3-MA可以促进顺铂对U251凋亡诱导作用;免疫印迹法结果显示,3-MA增加了顺铂诱导的细胞内泛素化蛋白、ERS标志蛋白PDI、Grp78的表达（顺铂组与对照组相比,P值分别为0.00294,0.0012,0.00179;联合组与顺铂组相比,P值分别为0.01896,0.01515,0.00734）;促进顺铂诱导的CHOP表达,增强了Caspase-4和Caspase-3的活化（顺铂组与对照组相比,P值分别为0.00081,0.00017,0.00018;联合组与顺铂组相比,P值分别为0.00671,0.00934,0.01124）,3-MA可以有效抑制顺铂诱导的U251细胞LC3II的表达（顺铂组与对照组相比,P=0.00447;联合组与顺铂组相比,P=0.01588）;免疫荧光染色结果显示,3-MA与顺铂联合作用于U251细胞,抑制了LC3的聚集、促进了PDI的表达.结论 3-MA通过抑制自噬,增强了顺铂诱导的ERS,从而促进顺铂诱导的U251细胞凋亡。Objective 3-methyl adenine （3-methyladenine,3-MA） was used to test the effect on cisplatin induced U251 cell apoptosis.Methods MTT assay was used to test the effect of 3-MA on cisplatin cytotoxicity in U251 cells.Hoechst33342 staining was used to test the effect of 3-MA on cisplatin induced apoptosis.Western bolt was used to detect the expression of ER stress associated proteins,ubiquitinated protein,protein disulfide isomerase （PDI）,glucose-regulated protein78 （Grp78）,C/EBP homologous protein （CHOP）,and activation of caspase-4 and caspase-3.The expression of microtubule-associated protein 1 light chain 3 （LC3） and PDI were observed by immunofluorescence staining and confocal microscopy.Results MTT assay showed that 3-MA could enhance cisplatin induced growth inhibition rate in human glioma U251 cells （P =0.00324 cisplatin troup vs.control group;P =0.02249 3-MA ＋ cisplatin group vs.cisplatin group）.Hoechst33342 staining showed that 3-MA could increase cisplatin induced apoptotic effect in U251 cells.Western blot showed that 3-MA increases the expressions of ubiquitinated proteins,ERS marker proteins PDI,Grp78 （P =0.00294,0.0012,0.00179 cisplatin group vs.control group;P =0.01896,0.01515,0.00734 3-MA ＋ cisplatin group vs cisplatin group）,CHOP,and active Caspase-4 and active Caspase-3 （P =0.00081,0.00017,0.00018 cisplatin group vs.control group;P =0.00671,0.00934,0.01124 3-MA ＋ cisplatin group vs.cisplatin group）.3-MA could inhibit the expression of LC3II induced by cisplatin in U251 cell （P =0.00447 cisplatin group vs.control group;P =0.01588 3-MA ＋ cisplatin group vs.cisplatin group）.Indirect immunofluorence staining showed that 3-MA inhibited the aggregation of LC3 and increased the expression of PDI.Conclusion 3-MA combined with cisplatin enhances cisplatin induced apoptosis by increasing ER stress.

关 键 词：顺铂 自噬 内质网应激 凋亡 神经胶质瘤 

分 类 号：R739.41[医药卫生—肿瘤]