| Objective:To investigate the effects and explore the related mechanisms of Inositol Hexaphosphate (IP6) on human liver cancer cell line HepG2 in vitro.Methods:HepG2 cells were cultured in vitro and treated with different concentrations of IP6 Its effects on the cells were evaluated by the follow experiments:1 MTT assay was used to evaluate the effect of IP6 on cells proliferation.2 The plate colony-forming efficiency was detected by colony-forming assay.3 Immunocytochemical stain was used to detect the expression of PCNA, p53 protein, p21 protein.4 Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the expression of oncogene Mdm2 mRNA in cells.5 Flow cytometry analysis were designed to investigate the effect on apoptosis of HepG2 cells.6 Western Blot was used to detect the expression of nuclear transcription factor NF-kB P65.Results:1 MTT assay indicated IP6 could inhibit the proliferation of HepG2 cells:at the same reaction time IP6 could reduce the OD value with increased concentration (F=4.214,4.952, q=1.562～3.784, p<0.05) and at the dose IP6 could reduce the OD value with the extension of time (F=6.798,q=3.687, p<0.05)2 Plate colony-forming assay showed that the cloning efficiency decline in IP6 treated HepG2 cells(F=122.287, q=2.595～17.103, p<0.05).3 The immunocytochemical stain showed that different concentration of IP6 could decrease PCNA(F=21.491, q=2.243～7.341, p< 0.05) and the abnormal expression of p53 gene (F=51.927, q=3.057～10.894, p<0.05), and induced the expression of p21 gene (F=52.520, q=2.473～11.631, p<0.05).4 Reverse transcription polymerase chain reaction(RT-PCR) showed that IP6 decreased the expression of Mdm2 mRNA of HepG2 cells, and the difference was significant (F=13.698, q=2.843～5.556, p<0.05).5 Flow cytometry showed that different concentration of IP6 increased the rate of cells apoptosis(F=342.15;q=2.98-28.53; p<0.05).6 Western Blot showed that IP6 reduced the expression of NF-kB p65 protein (F=8.472, q=2.679～4.770,p<0.05).Conclusion:IP6 could inhibit the proliferation of HepG2 cells, and reduce their colony formation ability. The growth inhibition of IP6 on HepG2 cells may be related to the inhibition of proliferating cell nuclear antigen PCNA and p53 protein, and the promotion of the expression of p21 protein. IP6 may inhibit the expression of oncogenes Mdm2 and NF-kB p65 signaling pathway, thus inhibit cancer cell proliferation and promote apoptosis. |
