表没食子儿茶素没食子酸酯通过Hedgehog信号诱导结肠癌HCT-116细胞凋亡的机制研究

Mechanism Study of Epigallocatechin-3-Gallate on Inducing Human Colon Cancer HCT-116 Cell Apoptosis through Hedgehog Pathway

目的:研究表没食子儿茶素没食子酸酯(EGCG)对人结肠癌HCT-116细胞的抑制作用和机制。方法:MTT实验检测细胞活力,并计算表没食子儿茶素没食子酸酯的IC50值,确定有效给药剂量。采用5-氟尿嘧啶(5-FU)为阳性对照药;EdU掺入法测定细胞增殖情况;JC-1染色测定线粒体膜电位;流式细胞术测定肿瘤细胞凋亡;RT-PCR法测定凋亡相关基因B细胞淋巴瘤因子-2(Bcl-2)、Bcl-2相关x蛋白(Bax)mRNA表达;Western blot测定Smo、Gli-1、半胱天冬氨酸蛋白酶-3(Caspase-3)和核凋亡诱导因子(AIF)蛋白表达情况。结果:与模型对照组比较,32μmol/L和64μmol/L的表没食子儿茶素没食子酸酯显著抑制结肠癌HCT-116细胞的增殖(P<0.01),诱导线粒体膜电位显著降低(P<0.01),显著诱导细胞凋亡(P<0.01)。与模型对照组比较,表没食子儿茶素没食子酸酯的应用在mRNA水平下调Bcl-2表达,上调Bax表达(P<0.01),在蛋白水平上调Caspase-3和AIF表达(P<0.01),下调Smo和Gli-1表达(P<0.01)。结论:表没食子儿茶素没食子酸酯抑制结肠癌HCT-116细胞增殖,诱导其凋亡,其机制可能通过抑制Smo/Gli-1信号通路的表达实现。

Objective: To investigate the inhibitory effect of epigallocatechin-3-gallate(EGCG) on HCT-116 cells and its mechanism. Methods: MTT assay was used to detect the cell viability, and IC50 of EGCG was calculated to determine the effective dosage. 5-fluorouracil(5-FU) was used as the positive control drug. Proliferation was measured by EdU incorporation assay. The MMP was measured by JC-1 staining. Apoptosis was determined by AV/PI staining. The mRNA expressions of B cell lymphoma factor-2(Bcl-2), Bcl-2 related x protein(Bax) were determined by RT-PCR. The protein expressions of Smo、Gli-1、Caspase-3 and apoptosis inducing factor(AIF) were detected by western blotting.Results: Compared with the model group, 32 μmol/L and 64 μmol/L EGCG significantly inhibited the proliferation of colon cancer HCT-116 cells(P<0.01), decreased the MMP(P<0.01), and induced the cell apoptosis(P<0.01). Compared with the model group, EGCG down regulated the mRNA expression of Bcl-2 and increased the expression of Bax(P<0.01),up regulated the expressions of Caspase-3 and AIF(P<0.01),while down regulated the expressions of SMO and Gli-1(P<0.01). Conclusions: EGCG can inhibit the proliferation and induce the apoptosis of HCT-116 cells, its mechanism may be achieved by inhibiting the expression of SMO/Gli-1 signaling pathway.