Apoptosis of human colon cancer cells induced by steroidal saponin YB16 of Ypsilandra thibetica

Abstract

Objective To explore the effect and mechanism of steroidal saponin from Ypsilandra thibetica (YB16) on apoptosis of HCT15 human colon cancer cells.

Methods HCT15 cells were cultured in vitro and treated with YB16 at doses of 0.125 – 16.000 mol/L for 24 hours. The survival rate of HCT15 cells was measured with methyl thiazolyl tetrazolium (MTT) assay. The morphological changes of cells were observed under light microscope. Apoptosis was observed with acridine orange (AO) staining. Changes in cell membrane potential were observed with JC-1 staining. Western blot was used to detect protein expression of B lymphocyto tumor-2 (Bcl-2), poly(ADP-ribose) polymerase (PARP), cleaved-PARP, caspase-3, and cleaved-caspase-3.

Results Compared with that of the control cells, the survival rates of HCT15 cells exposed to various doses of YB16 decreased significantly in a dose-effect manner ( P < 0.05) and significantly increased number of the cells with apoptotic characteristics was also observed for the YB16 treated HCT15 cells. Significantly increased expression of Bcl-2 protein (0.542 ± 0.04, 0.351 ± 0.03) and increased expressions of cleaved-PARP (0.647 ± 0.05, 0.686 ± 0.03) and cleaved-caspase-3 protein (0.677 ± 0.03, 0.762 ± 0.04) were detected in the HCT15 cells treated with 0.500 and 1.000 μmol/L YB16 in comparison with those in the control cells (all P < 0.05).

Conclusion YB16 can inhibit the proliferation and promote cell apoptosis of HCT15 cells, which may be related to down-regulation of Bcl-2 protein expression and up-regulation of cleaved-PARP and cleaved-caspase-3 protein expression.

Abstract

【摘 要】 目的 探讨丫蕊花甾体皂苷（YB16）对人结肠癌细胞 HCT15 凋亡的作用及其机制。 方法 体外培养 HCT15 细胞，以不同浓度的 YB16（0.125～16.000 μmol/L）干预 24 h，噻唑蓝（MTT）法检测 HCT15 细胞存活率，光镜下观察细胞形态变化，吖啶橙（AO）染色观察细胞凋亡情况，JC-1 染色观察细胞膜电位改变，蛋白免疫印迹检测细胞内 B 淋巴细胞瘤 – 2（Bcl-2）、多聚 ADP – 核糖聚合酶（PARP）、激活型 PARP（cleaved-PARP）、半胱氨酸蛋白酶 – 3 （caspase-3）、激活型 caspase-3（cleaved-caspase-3）蛋白表达。 结果 与对照组比较，各剂量 YB16 组细胞存活率明显下降，呈剂量效应关系（ P < 0.05）；与对照组比较，YB16 组细胞出现凋亡特征性改变，凋亡细胞明显增多；与对照组比较，0.500、1.000 μmol/L YB16 组 HCT15 细胞内 Bcl-2 蛋白表达[分别为（0.542 ± 0.04）、（0.351 ± 0.03）]明显下降， cleaved-PARP、cleaved-caspase-3 蛋白表达[分别为（0.647 ± 0.05）、（0.686 ± 0.03）和（0.677 ± 0.03）、（0.762 ± 0.04）]均显著上升，差异有统计学意义（均 P < 0.05）。 结论 YB16 可抑制 HCT15 细胞增殖、促进细胞凋亡，其机制可能与下调 Bcl-2 蛋白表达、上调 cleaved-PARP、cleaved-caspase-3 蛋白表达有关。