目的研究丹参酮ⅡA对人急性白血病K562细胞的诱导凋亡作用及其作用机制。方法以不同浓度的丹参酮ⅡA(10~50μmol/L)作用于体外培养的K562细胞24、48、72 h,应用MTT法检测细胞生长抑制率,流式细胞术(FCM)检测细胞凋亡率,并对50μmol/L的药物作用不同时间后亚G1期细胞进行检测。应用免疫印迹法(Western blotting)检测Caspase-3及其裂解底物多聚ADP-核糖聚合酶(PARP)的表达水平,并对凋亡调节蛋白Fas、Bax、Bcl-2、Bak、Bid的表达水平进行检测。结果 20μmol/L以上的丹参酮ⅡA可显著抑制K562细胞的生长、诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系;FCM检测结果表明50μmol/L丹参酮ⅡA作用不同时间后,亚G1期细胞(凋亡细胞)逐渐增多。20μmol/L以上的丹参酮ⅡA作用48 h后Caspase-3逐渐被活化出现17 000的亚单位,Caspase-3的作用底物PARP被活化裂解出现89 000的亚单位片段,而且Caspase-3的激活以及PARP的裂解可被Caspase-3的特异性抑制剂z-DEVD-FMK所阻断,促凋亡蛋白Fas以及Bax的表达水平明显升高,而凋亡抑制蛋白Bcl-2及其他促凋亡蛋白Bak和Bid的表达水平则无明显变化。结论丹参酮ⅡA可以通过诱导白血病K562细胞凋亡而发挥体外抗白血病作用,上调促凋亡蛋白Fas和Bax的表达水平及激活Caspase-3可能是丹参酮ⅡA诱导白血病K562细胞发生凋亡的重要作用机制之一。

Objective To investigate the apoptosis inducing effect of tanshinone ⅡA(Tan ⅡA) on human acute leukemic K562 cells and its mechanisms.Methods K562 Cells were treated by different concentration of Tan ⅡA(10—50 μmol/L) for 24,48,and 72 h.The inhibitory rate of the cells was measured by MTT assay,cell apoptosis was examined by flow cytometry(FCM).Western blotting was used to detect caspase-3 and poly(ADP-ribose) polymerase(PARP) expression as well as apoptotic modulators,such as Bax,Bcl-2,Bak,Bid,and Fas.Results Tan ⅡA(over 20 μmol/L) could inhibit the growth and induce apoptosis in K562 cells in both time-and dose-dependent manners.FCM Analysis showed that the sub-G1 cells(apoptotic cells) were gradually increased after treatment by 50 μmol/L Tan ⅡA for different hours.Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17 000 subunit,and a 89 000 subunit cleavage product of ADP-ribose PARP after Tan ⅡA(over 20 μmol/L) treatment for 48 h.Moreover,activation of caspase-3 and PARP was dramatically inhibited by caspase-3 specific inhibitor zDEVD-FMK.Western blotting also revealed that the expression of pro-apoptotic protein Fas and Bax was up-regulated significantly while expression of anti-apoptotic protein Bcl-2 as well as other pro-apoptotic protein Bak and Bid remained unchanged.Conclusion Tan ⅡA exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells,and activation of caspase-3 as well as upregulation of the expression of pro-apoptotic protein Fas and Bax may be one of its significant apoptosis inducing mechanisms.

教育部新世纪优秀人才支持计划资助项目(NCET-0721);广东省科技计划资助项目(2010B030700006)