新藤黄酸诱导人结肠癌HCT116细胞凋亡的作用机制研究

Study on the mechanism of gambogenic acid-induced apoptosis of human colon cancer HCT116 cells

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.