3-溴丙酮酸对肠癌HCT116细胞的增殖抑制作用及其机制

Inhibitory effect of 3-bromopyruvate on proliferation of colon carcinoma HCT116 cells and its mechanism

目的:研究3-溴丙酮酸(3-BP)对肠癌细胞HCT116增殖的抑制作用并初步探讨其抑制作用的机制,为3-BP的临床应用提供依据。方法:将体外培养的HCT116细胞分为对照组和不同浓度3-BP组(3-BP的终浓度分别为25、50、75和100mg.L-1)。采用Western blotting法检测己糖激酶Ⅱ(HKⅡ)蛋白表达水平;采用6-磷酸葡萄糖(G-6-P)检测试剂盒检测培养液中G-6-P的浓度;采用MTT比色法测定细胞增殖活性;采用流式细胞术检测细胞周期。结果:与对照组比较,25mg.L-1 3-BP组HKⅡ蛋白表达水平无明显变化(P>0.05),与对照组和25mg.L-1 3-BP组比较,50、75和100 mg.L-1 3-BP组HKⅡ蛋白表达水平均显著下调(P<0.01)。与对照组比较,25 mg.L-1 3-BP组培养液中G-6-P浓度无明显变化(P>0.05);与对照组和25mg.L-1 3-BP组比较,50、75和100mg.L-1 3-BP组培养液中G-6-P的浓度均显著下降(P<0.01)。MTT法,与对照组比较,25mg.L-1 3-BP组细胞存活率无明显变化(P>0.05);与对照组和25mg.L-1 3-BP组比较,50、75和100mg.L-1 3-BP组细胞存活率明显降低(P<0.05)。流式细胞术,与对照组比较,25mg.L-13-BP组G1和S期细胞所占比例无明显变化(P>0.05);与对照组和25 mg.L-1 3-BP组比较,50、75和100mg.L-1 3-BP组S期细胞所占比例逐渐减少(P<0.05),G1期细胞则明显增加(P<0.01),细胞滞留在G1期。结论:3-BP可显著抑制HCT116细胞增殖,其机制可能与3-BP抑制HKⅡ活性、减少细胞对葡萄糖的利用有关。

Objective To study the inhibitory effect of 3-bromopyruvate（3-BP） on proliferation of colon carcinoma HCT116 cells and to explore its mechanism,and to provide evidence for the clinical application of 3-BP.Methods HCT116 cells were cultivated in vitro and divided into control group and different concentrations of 3-BP groups（25,50,75,and 100 mg·L-1）.The hexokinaseⅡ（HKⅡ） protein expression levels in various groups were determined by Western blotting method;the concentrations of glucose-6-phosphatase（G-6-P） were determined by G-6-P assay kit;the proliferation activities of the cells were observed by MTT method;the cell cycle was measured by flow cytometry.Results Compared with control group,the HKⅡ protein level in 25 mg·L-1 3-BP group did not change significantly（P0.05）;compared with control group and 25 mg·L-1 3-BP group,the HKⅡprotein levels in 50,75,and 100 mg·L-1 3-BP groups were decreased significantly（P0.01）.Compared with control group,the concentration of G-6-P in culture medium in 25 mg·L-1 3-BP group did not change significantly（P0.05）;compared with control group and 25 mg·L-1 3-BP group,the concentration of G-6-P in culture medium in 50,75,and 100 mg·L-1 3-BP groups were decreased significantly（P0.01）.The MTT results showed that compared with control group,the survival rate in 25 mg·L-1 3-BP group did not change significantly（P0.05）;compared with control group and 25 mg·L-1 3-BP group,the survival rates in 50,75,and 100 mg·L-1 3-BP groups were decreased markedly（P0.05）.The flow cytometry analysis results showed that compared with control group,the percentages of the cells at G1 phase and S phase in 25 mg·L-1 3-BP group did not change significantly（P0.05）;compared with control group and 25 mg·L-1 3-BP group,the percentages of the cells at G1 phase in 50,75 and 100 mg·L-1 3-BP groups were increased significantly（P0.01） and the percentages of the cells at S phase were decreased（P0.05）.The cell cycle was arrested at G1 stage.Conclusion 3-BP could inhibit the proliferation of HCT116 cells significantly,which may be related to the decreasing of HKⅡ protein expression and use of glucose.