隐丹参酮对K562细胞凋亡的影响及其机制

Effect of cryptotanshinone on apoptosis in K562 cell and its mechanism

目的探讨隐丹参酮诱导人慢性髓系白血病K562细胞凋亡的作用及其线粒体途径相关机制。方法 MTT法检测隐丹参酮对细胞增殖的抑制作用;Annexin V-FITC/PI双标记流式细胞术检测丹参酮对细胞凋亡的影响,DAPI染色法观察细胞形态的变化;Western blotting检测丹参酮对细胞凋亡相关蛋白Caspase-3、Caspase-9、PARP、Bax、Bcl-2和细胞色素C(Cyt C)表达的影响,线粒体膜电位检测试剂盒检测丹参酮对线粒体膜电位的影响;检测线粒体膜通透性转运孔(PTP)抑制剂环孢霉素A和caspase抑制剂z-VAD-fmk对隐丹参酮药效的影响。结果隐丹参酮对K562细胞的生长有显著抑制作用,可引起细胞形态发生明显改变,诱导细胞发生凋亡,提高蛋白Bax/Bcl-2的比例,降低线粒体膜电位,促进Cyt C从线粒体释放到胞浆,增强促凋亡蛋白Caspase-3、Caspase-9和PARP的活性。环孢霉素A和cz-VAD-fmk均能减弱隐丹参酮抑制细胞增殖和诱导细胞凋亡的作用。结论隐丹参酮可显著诱导K562细胞凋亡,其机制与线粒体凋亡途径密切相关。

Objective To investigate the apoptosis inducing effects of cryptotanshinone （CPT） in human chronic myeloid leukemia K562 cells and its molecular mechanisms. Methods K562 cells were firstly treated with CPT at different concentration. Then, the inhibitory effect of CPT was detected by MTT assay; morphological changes of cells were observed by inverted microscope; cell apoptosis was detected by Annexin V-FITC/PI staining. Western blotting was carried out to determine the expression of apoptosis-related proteins, including Caspase-3, Caspase-9, PARP, Bax, Bcl-2 and cytochrome C （Cyt C）. The mitochondrial membrane potential was measured using JC-1 probe. Finally, the effects of CPT on the proliferation and apoptosis were detected in the absence or presence of inhibitors for mitochondrial permeability transition pore （PTP） and caspase-family. Results CPT significantly inhibited the proliferation, induced apoptosis and morphological changes of K562 cells. Furthermore, CPT increased the Bax/Bcl-2 ratio obviously, decreased the mitochondrial membrane potential, and enhanced the Cyt C release. CPT also significantly increased the activities of pro-apoptotic proteins, such as Caspase-3, Caspase-9, and PARP. The inhibitors of PTP and caspase-family reduced the pharmacodynamics of CPT obviously. Conclusion These results show that CPT could significantly induce the apoptosis of human chronic myeloid leukemia cells, in which its function is related with the mitochondrial pathway.