苞叶香茶菜庚素诱导人白血病细胞系HL-60凋亡的分子机制研究

Melissoidesin G-inducing HL-60 cell apoptosis and its possible anticancer mechanism

目的:观察苞叶香茶菜庚素(melissoidesin G,MOG)对多种肿瘤细胞系的细胞毒活性,并研究MOG诱导白血病细胞系HL-60凋亡的相关分子机制。方法:用MTT法评价化合物对多种肿瘤细胞的增殖抑制活性;通过流式细胞术PI单染法观察化合物对肿瘤细胞内DNA含量的影响,以分析周期变化;Annexin V染色法分析凋亡;荧光底物发光法检测caspase活性;膜电位依赖性结合的荧光探针JC-1标记法检测线粒体膜电位(△Ψm);Western blotting检测蛋白表达。结果:(1)MOG对多种肿瘤细胞系均有一定的增殖抑制作用;(2)MOG诱导HL-60细胞发生剂量依赖性凋亡;(3)在MOG作用下,HL-60细胞出现时间依赖性的△Ψm降低;(4)HL-60细胞在MOG作用6～12h后caspase-3和caspase-7均被明显激活,且在抑制剂Z-VAD-FMK的作用下,MOG诱导的caspase激活和细胞凋亡被完全抑制;(5)抗氧化剂NAC可以抑制MOG诱导的△Ψm降低、细胞凋亡及caspase-3激活。结论:MOG能够明显抑制肿瘤细胞增殖,并通过诱导细胞凋亡发挥其抗肿瘤作用。推测MOG可能通过引起细胞内氧化还原状态失平衡,进一步破坏线粒体功能,并激活caspase通路引起肿瘤细胞凋亡。

Objective： To observe the cytotoxicity of melissoidesin G （MOG） on different tumor cell lines and to investigate the MOG-inducing apoptotic mechanism on HL-60 cells. Methods： MTT assay for proliferation inhibitory activity evaluation of the compound on a variety of tumor cells, flow cytometry assay （P1 staining） detecting DNA content for cycle change of tumor cells, Annexin V staining assay for apoptosis analysis, fluorescent substrate luminescence for caspase activity measurement, fluorescent probe JC-1 for mitochondrial membrane potential （△ψm） detection, Western blotting for protein quantitation were adopted. Results： （1） MOG exerted different antiproliferative activities on different cell lines. （2） MOG induced apoptosis in a dosedependent manner. （3） MOG could decrease △ψm in a time-dependent manner. （4） MOG activated caspases-3/7 in 6-12 hours, and both caspases activation and apoptosis could be completely prevented with the general caspase inhibitor of Z-VAD-FMK. （5） The antioxidant NAC could inhibit the decrement of △ψm, apoptosis and caspases-3 activation induced by MOG. Conclusion： MOG can inhibit significantly tumor cell proliferation and exert anti-cancer effect by inducing cell apoptosis. These results indicated that the possible mechanism of MOG-inducing cell apoptosis was associated with imbalance of oxidation-reduction state, destruction of mitochondrial function and caspase pathway activation.