Effects of 1,25(OH)2D3 and its combination with LY294002 on proliferation and invasion of human hepatocellular carcinoma HepG2 cells

Objective To explore effects and mechanism of 1,25-dihydroxyvitamin D3(1,25OH₂D₃) and LY294002(an inhibitor of phosphatidylinositol 3-kinase/serine/threonine protein kinasePI3K/AKTsignal 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.