[半胱天冬酶-3在雷帕霉素诱导肝癌BEL-7402细胞凋亡中的作用]

[The role of caspase-3 in rapamycin-induced apoptosis of hepatocellular carcinoma BEL-7402 cells].

作者信息

Zhang Jun-Feng, Lu Min-Qiang, Cai Chang-Jie, Yang Yang, Li Hua, Yi Hui-Min, Chen Gui-Hua

机构信息

Department of Liver Transplantation, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510630, P. R. China.

出版信息

Ai Zheng. 2006 Dec;25(12):1508-11.

PMID:17166376

Abstract

BACKGROUND & OBJECTIVE: Rapamycin, isolated from Streptomyces hygroscopicus, is recently reported to have immunosuppressive and antitumor effects on a large variety of cancers. This study was to investigate the role of Caspase-3 in rapamycin-induced apoptosis of hepatocellular carcinoma BEL-7402 cells.

METHODS

BEL-7402 cells were treated with different concentrations (5, 10, 20, 30, 40, 50 nmol/L) of rapamycin. Cell viability was detected by MTT assay; cell apoptosis was observed by flow cytometry (FCM) and Hoechst 33258 staining. The activity of Caspase-3 was determined by Caspase colorimetric assay kit and Western blot.

RESULTS

Rapamycin inhibited the growth of BEL-7402 cells and induced apoptosis significantly in time- and dose-dependent manners. Marked morphologic changes of cell apoptosis, such as chromatin condensation and nuclear fragmentation, were observed clearly at 48 h after exposion to rapamycin; Caspase-3 was activated by the loss of Caspase-3 proenzyme (32-ku) and its 20-ku subunit appeared at 24 h after incubation. Caspase-3 inhibitor z-DEVD-FMK could block the apoptosis induced by rapamycin.

CONCLUSIONS

Rapamycin can inhibit growth and induce apoptosis of BEL-7402 cells. The activation of Caspase-3 may play an important role in cell apoptosis.

摘要

背景与目的

雷帕霉素是从吸水链霉菌中分离得到的，最近有报道称其对多种癌症具有免疫抑制和抗肿瘤作用。本研究旨在探讨半胱天冬酶 -3（Caspase-3）在雷帕霉素诱导肝癌BEL-7402细胞凋亡中的作用。

方法

用不同浓度（5、10、20、30、40、50 nmol/L）的雷帕霉素处理BEL-7402细胞。采用MTT法检测细胞活力；通过流式细胞术（FCM）和Hoechst 33258染色观察细胞凋亡情况。使用Caspase比色法检测试剂盒和蛋白质免疫印迹法测定Caspase-3的活性。

结果

雷帕霉素抑制BEL-7402细胞的生长，并以时间和剂量依赖性方式显著诱导细胞凋亡。在暴露于雷帕霉素48小时后，明显观察到细胞凋亡的显著形态学变化，如染色质浓缩和核碎裂；在孵育24小时后，Caspase-3原酶（32-kD）消失，其20-kD亚基出现，Caspase-3被激活。Caspase-3抑制剂z-DEVD-FMK可阻断雷帕霉素诱导的细胞凋亡。