辛伐他汀通过抑制Warburg效应改善索拉非尼耐药的肝癌细胞转移侵袭的作用机制

Simvastatin Inhibits the Warburg Effect and Improves the Mechanism of Metastasis and Invasion of Sorafenib-resistant Liver Cancer Cells

目的探索辛伐他汀通过抑制Warburg效应改善索拉非尼耐药的肝癌细胞转移侵袭的作用机制。方法将索拉非尼耐药的人肝癌细胞株SMMC-7721/S(5μmol·L^(−1)的索拉非尼耐药)分为对照组、索拉非尼组、辛伐他汀组。索拉非尼组为5μmol·L^(−1)的索拉非尼干预,辛伐他汀组用10μmol·L^(−1)和20μmol·L^(−1)的辛伐他汀干预。CCK-8法检测SMMC-7721/S细胞活力的改变,PI染色后流式细胞术检测细胞周期的变化,体外细胞划痕试验检测细胞迁移能力,Transwell小室试验检测细胞侵袭和转移能力,Western blotting法检测细胞中Warburg效应关键酶己糖激酶、丙酮酸激酶、乳酸脱氢酶、丙酮酸脱氢酶以及柠檬酸合酶的表达变化,试剂盒检测培养基中乳酸产生的水平,ECAR检测细胞糖酵解的压力的改变,929 oxygen system检测细胞对氧气的吸收水平。结果细胞活力检测结果显示索拉非尼组细胞活力与对照组比较无显著改变,同时细胞周期也无明显差异,细胞中己糖激酶、丙酮酸激酶、乳酸脱氢酶、丙酮酸脱氢酶以及柠檬酸合酶的表达无显著变化,同时培养基中乳酸水平以及氧气吸收也无显著差别。辛伐他汀组中细胞活力显著下调,同时细胞周期阻滞在G0/G1期,相比索拉非尼组差异具有统计学意义(P<0.05),而细胞迁移能力、转移能力和侵袭能力相比索拉非尼组显著减弱(P<0.05),细胞中己糖激酶、丙酮酸激酶、乳酸脱氢酶、丙酮酸脱氢酶以及柠檬酸合酶的水平低于索拉非尼组(P<0.05),辛伐他汀组培养基中乳酸的水平下调,氧气的吸收水平上调,相比索拉非尼组差异显著(P<0.05)。结论辛伐他汀可以通过改善耐药肝癌细胞的Warburg效应,抑制肝癌细胞的转移和侵袭。

OBJECTIVE To explore the mechanism of simvastatin to improve the metastatic invasion of sorafenib-resistant liver cancer cells after inhibiting the Warburg effect.METHODS Sorafenib-resistant human liver cancer cell line SMMC-7721/S(5μmol·L^(-1) sorafenib-resistant)was divided into control group,sorafenib group and simvastatin group.The sorafenib group was treated with 5μmol·L^(−1) sorafenib,and the simvastatin group was treated with 10μmol·L^(−1) and 20μmol·L^(−1) simvastatin.CCK-8 method was used to detect changes in SMMC-7721/S cell viability.Flow cytometry after PI staining was used to detect cell cycle changes.In vitro cell scratch test was used to detect cell migration.Transwell cell test was used to detect cell invasion and metastasis.Western blotting was used to detect the key enzymes of Warburg effect,hexokinase,pyruvate kinase,lactate dehydrogenase,pyruvate dehydrogenase and citrate synthase expression changes.The kit detected the level of lactic acid production in the culture medium.ECAR detected the change in the pressure of glycolysis in the cell.And the 929 oxygen system detected the level of oxygen absorption by the cell.RESULTS The cell viability test results showed that the cell viability of the sorafenib group was not significantly changed compared with the control group,and there was no significant difference in the cell cycle.The expression of hexokinase,pyruvate kinase,lactate dehydrogenase,pyruvate dehydrogenase and citrate synthase enzymes in the cells did not change,and there was no significant difference in the level of lactic acid and oxygen absorption in the medium.The cell viability was significantly down-regulated in the simvastatin group,and the cell cycle was blocked at the G0/G1 phase,which was significantly different from the sorafenib group(P<0.05),while the cell migration,metastasis,and invasion ability was significantly weakened(P<0.05),the levels of hexokinase,pyruvate kinase,lactate dehydrogenase,pyruvate dehydrogenase and enzymes in the cells were lower than those in the sorafenib group(P<0.05),the level of lactic acid in the medium of the simvastatin group was reduced,and the level of oxygen absorption was increased,which was significantly different from the sorafenib group(P<0.05).CONCLUSION Simvastatin can inhibit the metastasis and invasion of liver cancer cells by improving the Warburg effect of drug-resistant liver cancer cells.