尿石素A通过调控有氧糖酵解抑制肝癌细胞生长的机制

Mechanism of Urolithin A Inhibiting the Growth of Hepatoma Cells by Regulating Aerobic Glycolysis

关键词:  尿石素  肝癌  糖酵解  细胞生长

Key words:  urolithin  hepatoma  glycolysis  cells growth

摘要:

目的 探讨尿石素抑制肝癌细胞生长的分子机制。方法 不同浓度尿石素A(urolithin A，Uro-A)处理肝癌Huh-7细胞，CCK-8法检测细胞抑制率，并计算 IC₅₀，克隆形成试验检测细胞增殖能力，细胞划痕试验评估细胞迁移率，比色法检测细胞培养液中葡萄糖摄取量和乳酸水平，化学发光法检测细胞内ATP含量；Western blotting检测各浓度Uro-A对葡萄糖转运载体GLUT1、糖酵解关键酶(HK2、PKM2、LDHA)、p53、p-p38和Bcl-2蛋白表达的影响，流式细胞术和TUNEL法检测细胞凋亡率。结果 CCK-8 检测结果显示，Uro-A能明显抑制Huh-7细胞的增殖，IC₅₀为(48.54±1.21)μmol·L^-1。Uro-A处理后细胞克隆形成量减少，迁移率降低。不同浓度Uro-A作用后细胞葡萄糖摄取量、乳酸水平和ATP生成减少，糖酵解相关蛋白GLUT1、PKM2、LDHA和HK2表达量下降；Western blotting显示p53和p-p38被激活，Bcl-2表达降低；流式细胞术和TUNEL法显示细胞凋亡率显著升高。结论 Uro-A可抑制Huh-7细胞增殖和迁移，其机制可能是通过p53、p-p38和Bcl-2抑制糖酵解，使细胞生长受阻，诱导细胞凋亡。

Abstract:

OBJECTIVE To explore the molecular mechanism of urolithin A inhibition of human hepatoma cells growth. METHODS Hepatoma Huh-7 cells were treated with different concentrations of urolithin A(Uro-A). The inhibition rate of Huh-7 cells survival was detected by CCK-8 assay and the IC₅₀ was calculated. Cell proliferation was detected by colony formation assay and cell migration ability was assessed by cell wound healing experiment. Glucose uptake and lactate level in culture medium through colorimetry and the ATP production in cell through chemiluminescence method was analyzed. Western blotting was applied to detect protein expression levels of glucose transporter(GLUT1), key enzymes of glycolysis(HK2, PFKM, LDHA), p53, p-p38 and Bcl-2 after treatment with different concentrations of Uro-A. Flow cytometry and TUNEL method were used to detect apoptosis rate. RESULTS The results of CCK-8 showed that Uro-A significantly inhibited the proliferation of Huh-7 cells，and the IC₅₀ was(48.54±1.21) μmol·L^-1. The ability of clone formation and migration decreased after Uro-A treatment. Cellular glucose uptake and level of lactic acid and ATP production were down regulated in Huh-7 cells treated with Uro-A. The results showed that expression of glycolytic key proteins GLUT1, PKM2, LDHA and HK2 decreased. Western Blotting further research indicated that the p53 and p-p38 were activated, while the Bcl-2 was down-regulated. Flow cytometry data and TUNEL method revealed that the induction of apoptosis by Uro-A was remarkably increased. CONCLUSION These findings suggest that Uro-A can suppress Huh-7 cell proliferation and migration. The possible mechanism is the inhibition of glycolysis by p53, p-p38 and Bcl-2, which prevent cell growth and finally induce apoptosis.