HIF-1α抑制剂YC-1对肝癌细胞增殖凋亡的影响及机制

EFFECTS AND MECHANISM OF YC-1 ON THE CELL PROLIFERATION AND THE APOPTOSIS OF HUMAN HEPATOCELLULAR CARCINOMA CELL

目的应用缺氧诱因因子-1 α(hypoxia inducible factor-1 α,HIF-1α)抑制剂YC-1处理SMMC-7721肝癌细胞,观察不同药物浓度对SMMC-7721肝癌细胞增殖的作用和对细胞周期及凋亡率的影响及其机制。方法肝癌细胞系SMMC-7721分对照组和实验组。对照组细胞常规培养,实验组细胞施加HIF-1α抑制剂YC-1干预,设浓度梯度和时间梯度。检测不同药物浓度、不同时间对SMMC-7721细胞增殖的作用及对细胞周期和凋亡率的影响。对bc1-2、bax、Survivin mRNA表达产物相对定量,并对SMMC-7721细胞胞浆bcl-2、bax、Survivin蛋白进行相对定量分析。结果①与对照组相比,各实验组OD值均有显著下降,细胞数量降低(P<0.05),且呈明显的时间-剂量依赖效应。②不同浓度YC-1处理细胞后,均可使各处理组G_0/G_1期细胞增多,S期细胞减少,且呈时间-剂量依赖效应(P<0.05)。③不同浓度YC-1处理细胞不同时间后,与对照组相比,各实验组bcl-2mRNA和Survivin mRNA表达均有显著下降(P<0.05),而bax mRNA无明显变化趋势(P>0.05)。④不同浓度YC-1处理细胞不同时间后,与对照组相比,各实验组bcl-2和Survivin的蛋白表达显著下降(P<0.05),bax蛋白表达显著增加(P<0.05)。结论 YC-1可以将SMMC-7721肝癌细胞株阻滞于G_0/G_1期,影响细胞周期进程,抑制细胞增殖,促进细胞凋亡。HIF-1α促进增殖抑制凋亡的机制是上调肝癌细胞Survivin和bcl-2的表达,下调bax的表达。HIF-1 α抑制剂的应用为肝癌的治疗提供了新的思路。

Objective To explore the effects and mechanism of YC - 1 on the cell proliferation and the apoptosis of human hepatocellular carcinoma cell line SMMC -7721. Methods SMMC -7721 cells were divided as control group and experiment group. The former were cultured with normal condition, the later were treated by YC - 1 with Various concentration and time. Changes of cell cycle and apoptosis rate after treated by YC - 1 with various concentration and time were detected. The expression of bcl-2,bax,Survivin mRNA level in cultured SMMC -7721 was evaluated by RT- PCR. The bcl- 2,bax, Survivin protein in cytoplasm was evaluated by western blot. Results ①After treated with YC - 1, the OD values of treated groups decreased compared with control group, and there was statistically significant difference between control group and every treatment group （P 〈 0.05 ）. The inhibition of the proliferation of SMMC - 7721 cells by YC - 1 displayed a dose - and - time - dependent manner （ P 〈 0. 05）. ②The distribution of cell cycle by flow cvtometrv indicates that after treated with YC -1,the number of cells in GO/G1 phase increased gradually, while the number of cells in S phase decreased grudually,which was in a dose - and - time - dependent manner. ③Following the increasing of the concentration of YC - 1, the expression of bcl - 2 mRNA and Survivin mRNA decreased gradually compared with control group. But there was no obvious change of the bax mRNA （ P 〉 0.05 ）. ④Following the increasing of the concentration of YC - 1, expression of bcl - 2 protein and Survivin protein decreased gradually,butbax protein increased gradually. Conclusion The YC -1 can inhibit the proliferation and affect the cell cycle by inhibiting the G1 period of SMMC -7721 cell. The mechanisms were supposed to be that HIF - 1α up - regulates the expression of Survivin and bcl - 2 and down - regulates the expression of bax. The effects were in time - dependent pattern. The application of the HIF - 1α inhibitor offer a new way for the treatment of the HCC.