3-溴丙酮酸联合索拉菲尼对人肝癌细胞增殖和凋亡的影响

Effects of 3-bromopyruvate combined with sorafenib on proliferation and apoptosis of human hepatic carcinoma cells in vitro

目的探讨3-溴丙酮酸(3-BrPA)联合索拉菲尼(SOR)对人肝癌细胞SMMC7721、HepG2增殖和凋亡的影响及其相关分子机制。方法首先采用MTT法检测不同浓度3-BrPA(20,40,80,160,320μmol/L)、索拉菲尼(2,4,8,16,32μmol/L)以及100μmol/L 3-BrPA与索拉菲尼(2,4,8,16,32μmol/L)联合对肝癌细胞SMMC7721、HepG2作用24 h的增殖抑制作用。之后将肝癌细胞分为对照组、3-BrPA组、SOR组、3-BrPA+SOR组,PI单染流式细胞术检测肝癌细胞SMMC7721、HepG2凋亡;DAPI染色检测SMMC7721、HepG2细胞核的变化;Western blot检测SMMC7721、HepG2细胞Bcl-2和Bax蛋白表达。结果 3-BrPA、索拉菲尼以及两药联合作用于SMMC7721、HepG2细胞24 h细胞存活率随浓度增高而降低(P<0.05)。100μmol/L3-BrPA与索拉菲尼联合作用于SMMC7721、HepG2细胞24 h的存活率显著高于同浓度的索拉菲尼单独处理(P<0.05)。100μmol/L 3-BrPA与16μmol/L索拉菲尼联合诱导肝癌细胞SMMC7721、HepG2的凋亡率为(47.4±5.02)%、(46.2±4.14)%,显著高于对照组和单独用药组(P<0.05)。DAPI染色结果显示3-BrPA+SOR组细胞表现为细胞核皱缩浓集,呈现亮蓝色荧光,或细胞核呈分叶、碎片状,且明显多于单独用药组;Western blot结果显示3-BrPA+SOR组明显下调SMMC7721细胞Bcl-2的表达(P<0.05),但对促凋亡蛋白Bax的表达无明显影响。结论 3-BrPA能增强索拉菲尼对肝癌细胞SMMC7721、HepG2的增殖抑制作用,且能增强索拉菲尼诱导肝癌细胞的凋亡,其机制可能与下调抑制凋亡蛋白Bcl-2的表达有关。

Objective To investigate the effects of 3-bromopyruvate（3-BrPA）combined with sorafenib（SOR） on the proliferation and apoptosis of human hepatic carcinoma cells in vitro and its related molecular mechanism. Methods MTT assay was used to detect the viability of hepatic carcinoma HepG2 and SMMC7721 cells after exposed to different concentrations of 3-BrPA （ 20,40,80,160,320 μmol/L）, SOR（2,4,8,16,32 μmol/L）, and 100 μmol/L 3-BrPA combined with SOR （ 2 ,g, 8,16,32 μmol/L）. Then the experiment was divided into four groups ： control group, 3-BrPA group, SOR group, and 3-BrPA ＋ SOR group. The apoptosis rate of cells was as- sessed using flow eytometry with PI staining, DAPI staining was performed to demonstrate the cell nucleus changes, and the expression of Bcl-2 and Bax was analyzed using Western blot. Results The cell viability of HepG2 and SMMC7721 cells decreased in a concen- tration-dependent manner after treated with 3-BrPA, SOR, and 100 μmol/L 3-BrPA combined with SOR, respectively （ P 〈 0.05 ）. The survival rates of HepG2 and SMMC7721 ceils after treated with I00 μmol/L 3-BrPA combined with SOR for 24 h were lower than that of the single dose of SOR （P 〈 0.05 ）. The apoptotic rate was （47.4 ± 5.02） % in SMMC7721 ceils and （46.2 ± 4.14 ） % in HepG2 cells after combined treatment for 24 h, which were also significantly higher than that of cells treated with 3-BrPA or SOR （ P 〈 0.05 ）. DAPI staining showed that HepG2 and SMMC7721 cell nucleus became significantly condensed and fragmented in 3-BrPA ＋ SOR group. Bcl-2 protein in SMMC7721 cells was down-regulated clearly in 3-BrPA ＋ SOR group （P 〈 0. 05）. Conclusion 3-BrPA can inhibit the proliferation of HepG2 and SMMC7721 cells and enhance sorafenib-induced cell death, which may be caused by down-regu- lating Bcl-2.