摘要
目的:探讨维生素E琥珀酸酯(VES)通过酸性神经磷脂酶(ASmase)-神经酰胺(Cer)诱导人胃癌细胞凋亡过程中对死亡受体信号通路以及氧化应激反应的影响.方法:将人胃癌SGC-7901细胞分为对照组、ASMase抑制剂地昔帕明(DES)组(12.5μmol/L)、VES处理组(20μg/mL)以及VES+DES组(12.5μmol/L的DES预处理细胞2 h后加入20μg/mL的VES).在不同时间点用ELISA法测定ASMase活性,免疫荧光法检测Cer表达情况;处理24 h时用DAPI染色检测细胞凋亡率,Western blot法检测死亡受体信号通路蛋白Fas、死亡受体5(DR5)、caspase-8、caspase-9和PARP的蛋白表达水平,流式细胞术检测活性氧簇(ROS)水平.结果:VES处理细胞后ASMase活性在1.5 h时开始增加,同时Cer开始在细胞膜上的聚集增加,两者的表达水平分别在3、6 h时达到高峰,24 h的细胞凋亡率为(41.00±1.00)%;抑制ASMase活性显著降低了VES处理组细胞的凋亡率,Fas、DR5、c-caspase-8、c-caspase-9、c-PARP蛋白表达水平和氧化应激水平(P均<0.01).结论:ASMase/Cer可能是VES通过死亡受体信号通路及氧化应激反应促进胃癌细胞发生凋亡的上游因子.
OBJECTIVE:To investigate the effect of acid sphingomyelinase (ASMase) and ceramide (Cer) on the death receptor pathway and on oxidative stress in vitamin E succinate (VES)-induced apoptosis in gastric cancer cells. METHODS: Human gastric cancer cells in cultures were treated with medium, desipramine (DES,12.5 μmol/L),VES (20 μg/mL) or VES+DES (cells were pretreated with DES at 12.5 μmol/L for 2 h, then with VES at 20 μg/mL). ASMase activity was assessed using the ELISA assay and cellular expression of Cer was detected by immunostaining at different time points. Apoptosis was evaluated using DAPI staining at 24 h. Western blotting was used to detect the expression of proteins in the death receptor pathway, including Fas, death receptor 5 (DR5), caspase-8, caspase-9 and PARP. 2′,7′-dichlorofluorescein diacetate was used to quantitate the levels of reactive oxygen species (ROS). RESULTS:VES increased the activity of ASMase and accumulation of Cer in cellular membrane at 1.5 h and reaching the peak at 3 h. The ratio of apoptosis was (41.00± 1.00)% at 24 h. The inhibition of ASMase activities was significantly associated with blockage of VES-induced apoptosis (20.00±2.00)%,via perturbing the levels of Fas,DR5,caspase-8/9 and PARP cleavage,and oxidative stress (all P<0.01). CONCLUSION:In the VES-induced apoptosis in the SGC- 7901 human gastric cancer cells, our results demonstrate that ASMase and Cer were upstream events which were followed via the death receptor pathway and expression of oxidative stress.
作者
张旭光
杜瑞琥
陈彦平
赵艳
ZHANG Xuguang;DU Ruihu;CHEN Yanping;ZHAO Yan(Department of Child Healthcare, Harbin Children's Hospital, Harbin 150010;Department of Preventive Medicine, School of Public Health, Harbin Medical University, Harbin 150081;Department of Nutrition and Food, Harbin Medical University, Harbin 150081, Heilongjiang, China)
出处
《癌变.畸变.突变》
CAS
2019年第5期379-384,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
哈尔滨市科技局青年后备人才项目基金(2016RAQYJ145)