葫芦素B调控糖酵解抑制HuCCT1细胞增殖的作用机制

Mechanism of Cucurbitacin B in Regulating Glycolysis and Inhibiting Proliferation of HuCCT1 Cells

目的:探究葫芦素B(CuB)抑制细胞增殖和糖酵解的作用机制。方法:采用细胞增殖与活性检测-8(CCK-8)法检测CuB(0、40、80、120、160、200、400、800 nmol·L^(-1))对HuCCT1细胞增殖的影响;采用平板克隆实验检测CuB(50、100、200 nmol·L^(-1))对HuCCT1细胞集落形成能力的影响;采用流式细胞术检测CuB(50、100、200 nmol·L^(-1))对HuCCT1细胞周期的影响;采用可见分光光度法检测CuB(50、100、200 nmol·L^(-1))给药后HuCCT1细胞中糖酵解关键酶己糖激酶(HK)、丙酮酸激酶(PK)活性,及葡萄糖消耗量、乳酸生成量和三磷酸腺苷(ATP)生成量的变化;采用蛋白免疫印迹法(Western blot)检测CuB对细胞周期相关蛋白、增殖相关蛋白、糖酵解关键蛋白及蛋白激酶B/哺乳动物雷帕霉素靶蛋白(Akt/mTOR)通路相关蛋白表达情况的影响。结果:与空白组比较,给药24 h,160~800 nmol·L^(-1)CuB,给药48 h后80~800 nmol·L^(-1)可以抑制HuCCT1细胞增殖(P<0.05,P<0.01),并呈时间和浓度依赖性,给药48 h半数抑制浓度为200 nmol·L^(-1);CuB可以呈浓度依赖性的抑制HuCCT1细胞集落形成能力(P<0.01);CuB能够将HuCCT1细胞周期阻滞在G2期(P<0.05,P<0.01);CuB(100、200 nmol·L^(-1))可以抑制糖酵解关键酶HK、PK的活性,并降低细胞葡萄糖消耗量及乳酸和ATP的生成量(P<0.05,P<0.01)。Western blot结果显示,CuB(100、200 nmol·L^(-1))可以抑制周期相关蛋白Cyclin B1、增殖细胞核抗原(PCNA)、己糖激酶1(HK1)、己糖激酶2(HK2)、丙酮酸激酶1(PKM1)、丙酮酸激酶2(PKM2)、磷酸化蛋白激酶B(p-Akt)、磷酸化mTOR(p-mTOR)及磷酸化核糖体蛋白(p-RPS6)的蛋白表达水平(P<0.05,P<0.01)。结论:CuB可能通过调控Akt/mTOR通路抑制HuCCT1细胞糖酵解进而影响细胞增殖。

Objective:To explore the mechanism of cucurbitacin B(CuB)in inhibiting cell proliferation and glycolysis.Method:Cell counting kit-8(CCK-8)was applied to investigate the effect of different concentrations of CuB(0,40,80,120,160,200,400,and 800 nmol·L^(-1))on the proliferation of HuCCT1 cells.The effect of different concentrations of CuB(50,100,and 200 nmol·L^(-1))on the colony formation ability of HuCCT1 cells was detected by plate cloning assay.The effect of different concentrations of CuB(50,100,200 nmol·L^(-1))on the HuCCT1 cell cycle was analyzed by flow cytometry.Visible spectrophotometry was employed to detect the activity of key glycolytic enzymes hexokinase(HK)and pyruvate kinase(PK))and changes in glucose consumption,lactate production,and adenosine triphosphate(ATP)production in HuCCT1 cells after administration of different concentrations of CuB(50,100,200 nmol·L^(-1)).Western blotting was used to assay the effect of CuB on the expression of cell cycle-related proteins,proliferation-related proteins,key glycolytic proteins,and Akt/mammalian target of rapamycin(mTOR)pathway-related proteins.Result:As compared with the blank group,CuB at dose of 160-800 nmol·L^(-1) after 24 h administration and CuB at dose of 80-800 nmol·L^(-1) after 48 h administration inhibited the proliferation of HuCCT1 cells in a time-and dose-dependent manner(P<0.05,P<0.01),and the median inhibitory concentration was 200 nmol·L^(-1)48 h after administration.CuB can restrain the colony formation ability of HuCCT1 cells in a dose-dependent manner(P<0.01),and block HuCCT1 cell cycle in G2 phase(P<0.05,P<0.01).CuB(100 and 200 nmol·L^(-1))can suppress the activities of HK and PK and reduce cell glucose consumption and production of lactate and ATP(P<0.05,P<0.01).Western blot results showed that CuB(100 and 200 nmol·L^(-1))can inhibit the protein levels of cycle-related protein Cyclin B1,proliferating cell nuclear antigen(PCNA),HK1,HK2,PKM1,PKM2,phosphorylated Akt(p-Akt),phosphorylated mTOR(p-mTOR),and phosphorylated ribosomal protein S6(p-RPS6)(P<0.05,P<0.01).Conclusion:CuB can inhibit aerobic glycolysis in HuCCT1 cells via the Akt/mTOR pathway,thereby affecting cell proliferation.