Study on relationship of intracellular Ca2+ and apoptosis in liver cancer cell line HepG-2 treated with all-trans retinoic acid and (or) As2O3

Abstract

Objective To observe inhibitory effects and the induction of differentiation on the human liver cancer cell line HepG-2 in vitro after induced by As2O3 alone or combined with all-trans retinoic acid (ATRA),and the change of intracellular Ca2+.Methods In vitro,HepG-2 cells were treated with ATRA (0.1,1.0,10.0 μ mol/L) and (or) As2O3(0.5,1.0,2.0 μmol/L).At24,48,and72 h,the cell growth inhibition rate was assayed by methyl thiazol tetrazolium (MTT) method.The apoptosis rate of HepG-2 cells was examined by using flow cytometry (FCM) with Annexin V/propidium iodide (PI) double stainings.The intracellular Ca2+ levels were determined under laser scanning confocal microscope.Results Mter induction by ATRA (0.1,1.0 and 10.0 mol/L) and (or) As2O3 (0.5,1.0 and 2.0 mol/L),the cell proliferation was inhibited in time-and dose-dependent manners.As compared with As2O3 or ATRA used alone,ATRA combined with As2O3 significantly inhibited the growth of HepG-2 cells.The apoptosis morphologic changes of HepG-2 cells treated with As2O3 and (or) ATRA were observed through Annexin V-PI staining.AnnexinV-PI staining showed that both As2O3 and ATRA could induce HepG-2 cell apoptosis and the apoptosis ratio was time-dependent (P ＜0.05).The combined use of As2O3 and ATRT could significantly increase the apoptosis rate (P ＜0.05).The concentration of intracellular Ca2+ in HepG-2 cells treated with As2O3 and (or) ATRA for 24 and 48 h was statistically different from that in the controls (P＜0.01).Laser scanning confocal microscopy revealed that both As2O3 and ATRA could induce HepG-2 cell apoptosis and the apoptosis rate was time-dependent.The combined use of As2 O3 and ATRT could significantly increase the concentration,but after 72 h,the concentration of intracellular Ca2 + was not stastically different from that in controls (P ＞ 0.05).Conclusion ATRA or As2O3 could inhibit the growth of human liver cancer cell line HepG-2 in a dose-and-time dependent manner.The molecular mechanism of inducing apoptosis of HepG-2 cells may be related to up-regulation of hypertonic solution on calcium in endothelial cells.ATRA combined with As2O3 could significantly increase anti-liver cancer effect synergically. Key words: Carcinoma, hepatocellular; All-trans retinoic acid ; Arsenic trioxide ; Calcium