柠檬酸盐对人胃癌细胞株SGC7901糖酵解的抑制作用及其机制研究

Inhibitory Effect and Mechanism of Citrate on Glycolysis in Human Gastric Cancer Cell Line SGC7901

背景:糖酵解是肿瘤细胞的主要能量来源。柠檬酸作为三羧酸循环的中间产物,对糖酵解具有抑制作用。目的:研究柠檬酸盐对人胃癌细胞糖酵解的抑制作用及其机制。方法:柠檬酸三钠以不同浓度、不同作用时间处理人胃癌细胞株SGC7901,以生化法检测细胞培养液乳酸含量,CCK-8法检测细胞增殖率,Hoechst凋亡染色观察细胞凋亡情况,ELISA法检测细胞内磷酸果糖激酶(PFK)含量,蛋白质印迹法检测caspase-3、-9、cleaved caspase-3、Bcl-2、细胞色素(Cyt)、缺氧诱导因子-1α(HIF-1α)、葡萄糖转运蛋白-1(GLUT-1)蛋白表达,定量RT-PCR法检测PFK亚型mRNA表达。结果:在糖培养条件下,经不同浓度、不同作用时间柠檬酸三钠处理的SGC7901细胞,细胞培养液乳酸含量、细胞增殖率、细胞内PFK含量、caspase-3、-9、Bcl-2、HIF-1α、GLUT-1蛋白表达、肌肉型PFK(PFK-M)比例较空白对照组显著降低,cleaved caspase-3、Cyt蛋白表达较空白对照组显著增高,细胞内可见典型凋亡征象,上述作用多呈时间和浓度依赖性。结论:柠檬酸盐对人胃癌细胞糖酵解的抑制作用与抑制PFK、HIF-1α、GLUT-1表达相关。柠檬酸盐可通过启动内源性凋亡途径诱导人胃癌细胞凋亡。

Background： Cancer cells are mainly dependent on glycolysis to generate energy. As an intermediate of tricarboxylic acid cycle, citrate exerts an inhibitory effect on glycolysis. Aims： To investigate the inhibitory effect and mechanism of citrate on glycolysis in human gastric cancer cells. Methods： Human gastric cancer cell line SGC7901 was treated with different concentrations and time duration of citrate. The content of lactic acid in cell culture was determined by biochemical method. The cell proliferation rate was measured by CCK-8 assay. The cell apoptosis was determined by Hoechst apoptosis staining. The content of intracellular phosphofrnctokinase （PFK） was determined by ELISA. The protein expressions of caspase-3, -9, cleaved caspase-3, Bcl-2, cytochrome （Cyt）, hypoxia-inducible factor-1α （HIF-1α）, glucose transporter-1 （ GLUT-1 ） were determined by Western blotting. The mRNA expression of intracellular PFK isoforms was determined by real time RT-PCR. Results： Under sugar cultural condition, content of lactic acid in cell culture, cell proliferation rate, content of intracellular PFK, protein expressions of caspase-3, -9, Bcl-2, HIF-1 α and GLUT-1, as well as proportion of PFK muscle type （PFK-M） were significantly decreased, while protein expressions of cleaved caspase-3 and Cyt were significantly increased in SGC7901 cells treated with different concentrations and time duration of citrate as compared with blank control group. Typical cell apoptotic signs were observed. The effect of citrate showed a time- and concentration-dependent manner. Conclusions ： The inhibitory effect of citrate on glycolysis in human gastric cancer cells is correlated with the inhibition of expressions of PFK, HIF-1α and GLUT-1. Citrate can induce cell apoptosis by activating the intrinsic apoptotic pathway in human gastric cancer cells.