联合磷酸肌醇3激酶／哺乳动物雷帕霉素靶蛋白抑制剂NVP—BEZ235增强奥沙利铂诱导的结肠癌细胞凋亡  

Dual phosphatidylinositol 3 kinase/mammalian target of rapamycin inhibitor NVP -BEZ235 enhances apoptosis of colon cancer cells induced by oxaliplatin

作　　者：王延楠 戚少龙 姜春晓 陈雪松 张佳宇[1] 沈可欣[1] 

机构地区：[1]吉林大学中日联谊医院胃肠结直肠肛门外科,长春130033 [2]吉林省永吉县医院,132200

出　　处：《中华实验外科杂志》2018年第2期222-225,共4页Chinese Journal of Experimental Surgery

摘　　要：目的观察磷酸肌醇3激酶（PI3K）/哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂NVP-BEZ235对奥沙利铂诱导的人结肠癌HCT116细胞凋亡的影响。方法不同浓度的奥沙利铂和（或）PI3K/mTOR抑制剂NVP-BEZ235处理体外培养的结肠癌HCT116细胞，采用噻唑蓝（MTT）法检测奥沙利铂和（或）PI3K/mTOR抑制剂NVP-BEZ235对HCT116细胞生存率的影响。流式细胞术（FCM）检测奥沙利铂和（或）PI3K/mTOR抑制剂NVP-BEZ235对HCT116细胞凋亡率的影响。免疫荧光检测奥沙利铂和（或）PI3K/mTOR抑制剂NVP-BEZ235对HCT116细胞核形态的影响。Western blot检测奥沙利铂和（或）PI3K/mTOR抑制剂NVP-BEZ235对DNA损伤标志基因磷酸化组蛋白H2AX（γ-H2AX）及凋亡相关蛋白表达水平的影响。结果MTT结果显示，2、4、8、16、32、64 μg/ml奥沙利铂以剂量依赖的方式抑制了HCT116细胞增殖，且约16 μg/ml奥沙利铂能够抑制50%的HCT116细胞增殖；流式细胞术的结果显示，4、16、64 μg/ml奥沙利铂作用HCT116细胞24 h后，凋亡率分别是11%、36%和80%（P＝0.026）；免疫荧光结果显示，奥沙利铂和NVP-BEZ235联合应用明显地增加了HCT116细胞凋亡率，观察到HCT116细胞核明显地发生皱缩；Western blot结果显示，凋亡相关蛋白裂解的半胱氨酰天冬氨酸特异性蛋白酶-3（Cleaved Caspase-3）的表达水平明显升高（P＝0.026），同时也检测到DNA损伤标志基因γ-H2AX表达明显上调（P＝0.017）。结论PI3K/mTOR抑制剂NVP-BEZ235明显地增强了奥沙利铂诱导的结肠癌细胞凋亡作用，并增强了奥沙利铂导致的结肠癌细胞DNA损伤效果。Objective To investigate the effect of phosphatidylinositol 3 kinase （PI3K）/mammalian target of rapamycin （mTOR） inhibitor NVP-BEZ235 on apoptosis induced by oxaliplatin in human colon cancer HCT116 cells.Methods Colon cancer HCT116 cells were cultured in vitro with different concentrations of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235. Methyl thiazol tetrazolium （MTT） assay was used to detect the effects of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on cell viability of HCT116 cells. Flow cytometry （FCM） was used to detect the effect of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on HCT116 cells. Immunofluorescence was used to detect the effect of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on the nuclear morphology of HCT116 cells. Western blotting was used to detect the effects of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on DNA damage marker gene gamma histone variant H2A histone family member X （γ-H2AX） and apoptosis-related protein expression.Results The results of MTT showed that 2, 4, 8, 16, 32 and 64 μg/ml oxaliplatin inhibited proliferation of HCT116 cells in a dose-dependent manner, and about 16 μg/ml oxaliplatin platinum could decrease 50% HCT116 cells on the growth. The results of flow cytometry revealed that the apoptosis rate of HCT116 cells treated with 4, 16 and 64 μg/ml oxaliplatin was 11%, 36% and 80% respectively （P=0.026）. The results of immunofluorescence demonstrated that the combination of oxaliplatin and NVP-BEZ235 obviously induced the apoptosis of HCT116 cells, and the nuclear shrinkage of HCT116 cells. Western blotting indicated that the expression of Cleaved cysteinyl aspartate-specific protease （Caspase）-3 was significantly increased （P=0.026）, and the level of γ-H2AX was also up-regulated （P=0.017）.Conclusion Dual PI3K/mTOR inhibitor NVP-BEZ235 significantly enhanced the apoptosis of colon cancer cells induced by oxaliplatin and enhanced the DNA damage of colon cancer cells induced by

关 键 词：结肠癌 磷酸肌醇3激酶/哺乳动物雷帕霉素靶蛋白 奥沙利铂 脱噬作用 DNA损伤 

分 类 号：R735.35[医药卫生—肿瘤]