可诱导型及Bak基因过度表达对多柔比星诱导HCC-9204细胞凋亡的增敏作用(英文)

目的:研究Bak基因过度表达在HCC-9204细胞凋亡途径中的作用以及对多柔比星和长春瑞滨的可能的增敏作用.方法:采用MT-Ⅱ可调控性表达载体系统,通过外加ZnSO_4(100 μmol/L)诱导Bak基因表达,并获得稳定转染子.以形态学标准并结合TUNEL或流式细胞仪检测细胞凋亡.克隆形成实验反映克隆细胞存活率,MTF法检测细胞活力.结果:Bak基因过度表达的细胞出现显著的细胞死亡,TUNEL证实为一种凋亡性细胞死亡.流式细胞仪的结果显示Bak能够显著地诱导细胞在G_1期聚积并在阿霉素诱导后24 h 19.26％的细胞发生凋亡.Bak基因过度表达只能显著降低阿霉素处理组的克隆存活率,而对长春花碱组没有显著效果.MTT实验的结果类似,提示Bak基因能够选择性对阿霉素诱导的细胞死亡具有增敏作用,而对长春花碱没有这种作用.结论:Bak基因的过度表达使HCC-9204细胞的细胞周期在G_1延长,导致细胞凋亡并选择性对化疗药物具有增敏作用.

Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin

AIM: To investigate the role of overexpression of Bak in apoptotic pathways and drug susceptibility using doxorubicin and vinorelbine in human HCC-9204 cells. METHODS: An inducible system, MT-II regulatory system which allowed controlled expression of protein upon addition of ZnSO4(100 mumol/L) as an external inducer was used. Stable transfection of pMD-Bak gene was performed on HCC-9204 cells. Apoptotic cells were measured by morphological criteria, as well as by TUNEL assay and flow cytometry. The ability of Bak to decrease clonogenic cell survival was studied by colony-forming assays, while decrease in cell viability was assessed by MTT assay. RESULTS: Cells overexpressing Bak showed extensive cell death with nucleus fragmentation detected by TUNEL assay. FACS analyses showed that Bak could induce significant G1 accumulation and apoptosis in 19.29% cells 24 h after induction. Bak significantly decreased the clonogenic survival following exposure to adriamycin, but not vinorelbine. Furthermore, the time-course of cell viability rates following exposure of HCC-9204/Bak cells to adriamycin and vinorelbine was in agreement with the above findings. Bak selectively sensitized HCC-9204 cells to death induced by adriamycin while resisted to vinorelbine. CONCLUSION: Bak may prolong cell cycle in G1 phase, leading to apoptosis and decrease clonogenic survival of HCC-9204 cells in a drug-specific manner.