Effects of 1,25(OH)2D3 and its combination with LY294002 on proliferation and invasion of human hepatocellular carcinoma HepG2 cells
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摘要: 目的 探讨1,25二羟维生素D3[1,25(OH)2D3]及PI3K/AKT信号通路抑制剂(LY294002)对人肝癌HepG2细胞的增殖、侵袭影响及作用机制。方法 实验设10-8、10-7、10-6 mol/L 1,25(OH)2D3组及2.5、5.0、10.0、20.0、40.0、80.0μmol/L LY294002组,10-7 mol/L 1,25(OH)2D3+5μmol/L LY294002联合组,噻唑蓝法检测人肝癌HepG2细胞增殖抑制率;CompuSyn软件计算联合指数;Tanswell小室检测HepG2细胞侵袭数;Western blot检测HepG2细胞增殖细胞核抗原(PCNA),细胞基质金属蛋白酶9(MMP-9)、磷酸化丝氨酸苏氨酸蛋白激酶(p-AKT)、10号染色体缺失且与张力蛋白同源物磷酸脂酶基因(PTEN)蛋白。结果 1,25(OH)2D3及LY294002对人肝癌HepG2细胞增殖抑制率呈时间-剂量依赖效应(P<0.05);1,25(OH)2D3联合LY294002组细胞增殖抑制率明显高于二者单独处理组(P<0.05),联合指数=0.728,2者具有协同效应;10-7 mol/L 1,25(OH)2D3组、5μmol/L LY294002组以及联合组HepG2细胞侵袭数[分别为(45.9±6.4)、(49.9±6.0)、(27.8±4.0)个]明显低于对照组[(64.6±8.0)个](P<0.05)。与对照组比较,10-7 mol/L 1,25(OH)2D3组及联合组HepG2细胞PTEN蛋白表达量增加,差异有统计学意义(P<0.05),10-7 mol/L 1,25(OH)2D3组、5μmol/L LY294002组及联合组HepG2细胞PCNA、MMP-9、p-AKT蛋白表达均降低,差异有统计学意义(P<0.05),且联合组蛋白表达量低于二者单独处理组(P<0.05)。结论 1,25(OH)2D3可抑制人肝癌HepG2细胞增殖、侵袭,其机制可能与上调PTEN表达,抑制PI3K/AKT信号通路活性,下调PCNA、MMP-9蛋白有关;与LY294002合用具有协同效应。
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关键词:
- 1,25(OH)2D3 /
- HepG2细胞 /
- 增殖 /
- 侵袭 /
- PI3K/AKT
Abstract: Objective To explore effects and mechanism of 1,25-dihydroxyvitamin D3(1,25[OH]2D3) and LY294002(an inhibitor of phosphatidylinositol 3-kinase/serine/threonine protein kinase[PI3K/AKT]signal pathway) on proliferation,migration potentiality and metastasis of human hepatocellular carcinoma HepG2 cells.Methods HepG2 cells were treated with 1,25(OH)2D3(10-8,10-7,10-6 mol/L),LY294002(2.5,5,10,20,40,80 umol/L) and the combination of 1,25(OH)2D3(10-7 mol/L) and LY294002(5 umol/L),repectively.Cell proliferation was assayed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) method;invasion ability of HepG2 cells was detected by transwell assay;expressions of proliferating cell nuclear antigen(PCNA),matrix metalloprotein-9(MMP-9),phosphorylase protein serine threonine kinase(p-AKT),and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in HepG2 cells were assessed by Western blot;the combination indexes were calculated with CompuSyn software.Results The inhibitive effect of 1,25(OH)2D3 and LY294002 on HepG2 cells' proliferation showed a time-dose dependent manner(P<0.05).The proliferation inhibitive rate of HepG2 cells with combined treatment of 1,25(OH)2D3 and LY294002 was significantly higher than that of the cells treated by 1,25(OH)2D3 or LY294002 separately(both P<0.05),with a combination index of 0.728 and suggesting a synergistic effect of the two agents.The numbers of invasion cells were 45.9±6.4,49.9±6.0,and 27.8±4.0 for the HepG2 cells treated with 1,25(OH)2D3(10-7 mol/L),LY294002(5 umol/L),and the two agents combined at the same doses,which were significantly lower than that (64.6±8.0) of the control HepG2 cells(P<0.05 for all).The expression level of PTEN in HepG2 cells treated with 1,25(OH)2D3(10-7 mol/L) and 1,25(OH)2D3(10-7 mol/L) combined with LY294002(5 μmol/L) was up-regulated significantly(both P<0.05),while the expressions of PCNA,MMP-9,and p-AKT in HepG2 cells treated with 1,25(OH)2D3(10-7 mol/L),LY294002(5 μmol/L),and the two agents combined at the same doses were significantly down-regulated(P<0.05 for all) and the expressions of the proteins in HepG2 cells with the combined treatment of the two agents were significantly higher that those in the cells with separate treatment of 1,25(OH)2D3 or LY294002(both P<0.05).Conclusion The results of the study suggest that 1,25(OH)2D3 can inhibit the proliferation and invasion ability of human hepatocellular carcinoma HepG2 cells and the mechanism may be correlated to the up-regulation of PTEN,the inhibition of PI3K/AKT signal,and down-regulation of PCNA and MMP-9 protein;the results also demonstrate that combined treatment of 1,25(OH)2D3 and LY294002 has a synergistic effect on proliferation and invasion of HepG2 cells. -
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