Caspase 3 gene expression and [Ca²⁺]_i homeostasis underlying desipramine-induced C6 glioma cell apoptosis  

在地昔帕明诱导胶质瘤C6细胞凋亡过程中caspase 3基因表达和[Ca2+]i稳态（英文）

作　　者：祁红[1] 陈红专[1] 金正均[1] 

机构地区：[1]上海第二医科大学药理学教研室

出　　处：《Acta Pharmacologica Sinica》2002年第9期37-41,共页中国药理学报（英文版）

摘　　要：AIM: To study desipramine (Des)-induced apoptosis and regulation of caspase 3 gene expression and [Ca2+]i homeostasis in rat glioma C6 cells. METHODS: Apoptotic DNA breaks were quantified by propidium iodide (PI) incorporation using flow cytometry (FCM) and were detected by DNA agarose gel electrophoresis. Expression of apoptotic effector gene caspase 3 was assessed by reverse transcription polymerase chain reaction (RT-PCR). Single cell [Ca2+]i was measured using fluorescence indicator Fura-3/AM with confocal laser scanning microscopy. RESULTS: Des induced apoptotic DNA breaks in a concentration-dependent manner evidenced by hypodiploid peak on FCM histogram and the apoptotic cell percentage induced by Des 10, 20, and 40 μmol/L for 24 h was 5.2 %, 21.9 % , and 41.9 %, respectively. Apoptotic DNA breaks were further confirmed by a typical "DNA ladder" on agarose gel electrophoresis after exposure to Des 40 μmol/L for 24 h. Meanwhile, expression of caspase 3 gene was observed following Des 20 μmol/L treatment. Des 40 μmol/L resulted in an early sustained increase in [Ca2+]i over 28 min and the elevation magnitude was greatly decreased by removal of extracellular free [Ca2+]i with calcium-chelator egtazic acid, suggesting that Des elicited [Ca2+]i influx rather than intracellular calcium mobilization. CONCLUSION: Up-regulation of caspase 3 gene expression and disturbance of homeostasis in calcium signaling system might play pivotal roles in Des-induced apoptotic DNA breaks of C6 cells.目的:研究抗抑郁药地昔帕明（Des）对胶质瘤C6细胞的凋亡诱导作用以及对凋亡关键效应分子caspase3和凋亡早期信号[Ca²⁺]_i的调控作用.方法:采用流式细胞术（FCM）和凝胶电泳观察Des对C6细胞凋亡的DNA裂解作用,RT-PCR分析。caspase 3基因的表达以及激光扫描共聚焦显微镜测量单个活细胞[Ca²⁺]_i浓度.结果:Des（10,20,40μmol/L）处理C6细胞24 h后,FCM图的G₁峰左侧出现凋亡特征性亚二倍体细胞峰,凋亡细胞百分率分别为5.2％,21.9％和 41.9％.同时,凝胶电泳显示典型的DNA“梯带”.DeS 20 μmol/L处理C6细胞24 h可明显增强caspase 3基因的表达,而未经Des处理的C6细胞则检测不到caspase 3基因的表达.此外,Des 40 μmol/L可使 C6细胞[Ca²⁺]_i迅速升高并维持超过28 min,而钙螯合剂依他酸可显著降低C6细胞[Ca²⁺]_i增高幅度,提示Des致C6细胞[Ca²⁺]_i增高主要与细胞外钙内流有关.结论:Des诱导C6胶质瘤细胞凋亡可能与caspase 3基因表达的上调以及细胞内钙稳态的失衡有关.

关 键 词：DESIPRAMINE GLIOMA apoptosis CASPASES calcium 

分 类 号：R73-3[医药卫生—肿瘤]