摘要
目的:探讨新藤黄酸诱导人结肠癌HCT116细胞凋亡的作用及其可能的分子机制。方法:采用MTT法检测新藤黄酸对人结肠癌HCT116细胞增殖的抑制作用;DAPI染色及荧光显微镜观察细胞凋亡情况;FCM法检测细胞周期分布;蛋白质印迹法检测新藤黄酸对cyclin D1、cyclin E、P21、P27和多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]蛋白表达的影响。结果:新藤黄酸对HCT116细胞增殖有明显的抑制作用,并呈浓度和时间依赖性。DAPI染色后荧光显微镜观察发现,新藤黄酸能明显诱导HCT116细胞凋亡。FCM法检测发现,新藤黄酸可以使HCT116细胞G0/G1期比例显著升高,S期比例相应降低,表明细胞周期阻滞于G0/G1期。蛋白质印迹法检测结果表明,新藤黄酸下调了细胞周期蛋白cyclin D1和cyclin E的表达,上调了P21和P27的蛋白表达,诱导了PARP蛋白的剪切。结论:新藤黄酸通过下调细胞周期蛋白cyclin D1、cyclin E的表达和上调P21、P27的表达,使HCT116细胞阻滞在G0/G1期,进而显著抑制HCT116细胞的增殖,并诱导其凋亡。
Objective: To investigate the pro-apoptotic effect of gambogenic acid (GNA) on human colonic carcinoma HCT116 cells, and to explore the possible molecular mechanism. Methods: MTT assay was performed to detect proliferation inhibition effect of GNA on HCT116 cells. After staining with DAPI, the pro-apoptotic effect of GNA on HCT116 cells was observed under a fluorescence microscope. The changes in cell cycle distribution of HCT116 cells induced by GNA were examined by FCM. The expressions of cyclin D1, cyclin E, P21, P27 and poly (ADP-ribose) polymerase (PARP) proteins in HCT116 cells were detected by Western blotting. Results: GNA exerted an significant inhibitory effect on HCT116 cell proliferation in a time-and dose-dependent manner, and it could induce the typical nuclear apoptotic morphology. Under a fluorescence microscope, DAPI staining results showed that GNA could obviously induce the apoptosis of HCT116 cells. FCM showed that GNA could significantly increase the percentage of cells at Go/G1 phase, whereas obviously decrease the percentage of cells at S phase, which indicated that the cell cycle was arrested at Go/G1 phase. Western blotting analysis showed that GNA could efficiently down-regulate the expression levels of cyclin D1 and cyclin E proteins, whereas up-regulate the expression levels of P21 and P27 proteins, and also promote the cleavage of PARP. Conclusion: GNA can induce the cell cycle arrest at Go/G1 phase by down-regulating the expression levels of cyclin D1 and cyclin E, and up-regulating the expression levels of P21 and P27. Therefore, GNA can efficiently promote the cell proliferation inhibition and the apoptosis of HCT116 cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2011年第7期580-584,共5页
Tumor
基金
国家科技重大新药创制专项课题(编号:2009ZX09103-399)
安徽省自然基金资助项目(编号:070413128)