The Effects Of Inostitol Hexaphosphate (IP₆) On Apoptosis By The Mitochondrial Pathway In Human Heptoma Cell Line HepG2

Objective:To Investigate the role of mitochondrial pathway in which caspase-3 Cytochrome-c (Cyt-C) participated in inositol hexaphosphate (IP6) induced apoptosis of human heptoma cell line HepG2.Methods:HepG2 cells were treated with various concentrations of IP6 cultured in vitro. Its effects on the cells were evaluated by following indexes:1 To detect the change of HepG2 cell with transmission electron microscopy (TEM).2 Performed the Hoechst 33258 method to determine cell apoptosis.3 Designed flow cytometry analysis to investigate the status of the mitochondrial membrane potential.4 Immunocytochemical stain was used to detect the expression of Bcl-2 and Bax protein.5 RT-PCR (reverse transcription polymerase chain reaction) was adopted to detect the expression of caspase-3 mRNA.6 Western Blot was used to detect the expression of Cyt-c protein.Results:1 Morphological examination by transmission electron microscopy(TEM)showed cells shrinkage, and the presense of'apoptopic bodies".2 Using Hoechst 33258 staining the cell apoptosis morphological changes was observed. The IP6-treated cells were fewer and smaller than control group. It illustrate that IP6 could induce cell apoptosis of HepG2 cells.3 Compared with control group, IP6 obviously decreased the expression of mitochondrial membrane potential and the effect was manifest with concentration rising (F =14802.610, q=101.531-209.237, p<0.05)and had a dose-dependent relationship(q= 22.344-107.706,p<0.05).4 Compared with the control group, different concentration of IP6 decreased the expression of Bcl-2 protein (F=110.21; q=5.88—16.92; p<0.05)and strongly increased the expression of Bax (F=26.00;q=2.97—8.19;p<0.05).5 Compared with the control group, different concentration of IP6 decreased the expression of caspase-3 mRNA(F=30.474, q=3.406～9.103, P> 0.05).6 Compared with the control group, the expressions of Cyt-c protein were up-regulated in different concentration of IP6(F=87.194, q=5.246～15.218, P<0.05). Conclusion:1 IP6 could induce apoptosis in vitro;2 IP6 could down-regulation of the Bcl-2 protein expression and up-regulation of the expression of Bax, Cyt-c and Caspase-3 protein induced apoptosis of HepG2 cells.