冬凌草乙素对白血病NB4细胞的体外诱导凋亡作用研究

Investigation of apoptosis inducing effect of ponicidin on leukemia NB4 cells in vitro

目的:探讨冬凌草乙素(PON)对急性白血病NB4细胞的增殖抑制和诱导凋亡作用及其作用机制。方法:以不同浓度的PON(0～50μmol/L)作用于体外培养的NB4细胞0、24、48及72h,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,Hoechst33258染色法检测细胞凋亡时的形态学变化。应用免疫印迹法检测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Bax、Bcl-2、Bak、Bad及Survivin的表达水平进行检测。结果:>20μmol/L的PON可显著抑制细胞的生长,并呈现出明显的量-效与时-效关系,FCM检测结果表明,>20μmol/L的PON作用48h后,凋亡细胞逐渐增多,在Hoechst染色后可见典型的细胞凋亡现象。蛋白质印迹法检测结果表明,药物作用48h后Caspase-3被活化出现17×103的亚单位,同时PARP被裂解出现89×103的亚单位片段。蛋白质印迹法检测结果显示,凋亡抑制蛋白Survivin的表达水平明显降低,促凋亡蛋白Bax的表达水平显著升高,而其他凋亡调节基因Bcl-2、Bak、Bad则无明显变化。结论:PON可以通过诱导白血病NB4细胞凋亡而发挥体外抗白血病作用,降低Survivin以及升高Bax的表达水平可能是PON诱导白血病细胞发生凋亡的重要作用机制之一。

OBJECTIVE：To investigate the anti-proliferation and apoptosis inducing effects of ponicidin （PON） on leukemic NB4 cells and its mechanisms of action.METHODS：NB4 cells in culture medium in vitro were given with different concentrations of PON （0-50 μmol/L） for 0,24,48 and 72 h.The inhibitory rate of the cells was measured by MTT assay and 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,Bad and Survivin.RESULTS：PON （over 20 μmol/L） inhibited the growth of NB4 cells in both time-and dose-dependent manners.The cell apoptosis was demonstrated obviously by FCM and marked morphological changes of cell apoptosis such as the condensation of chromatin was clearly observed by Hoechst staining after treatment for 48 h.Western blotting showed cleavage of the Caspase-3 zymogen protein,with the appearance of its 17×103 subunit,and a cleaved 89×103 fragment of PARP was also found.Furthermore,Western blotting showed that anti-apoptotic protein Survivin decreased dramatically while pro-apoptotic protein Bax increased significantly after the cells were treated by PON for 48 h.CONCLUSIONS：PON exhibits in vitro anti-leukemia effect by induction of apoptosis in NB4 cells,and downregulation of Survivin as well as upregulation of Bax expression may be one of its significant apoptosis inducing mechanism.