Apoptosis Induction Of Sodium Butyrate On Human Hepatoma Cell Line 7721

Objective As one of the most malignant cancers with poor prognosis, hepatocellular carcinoma (HCC) is detected each year in approximately 125,000 new patients in the world. However, the methods for the treatment of the patients with HCC are less effective. A new method with inhibiting HDAC is used to modify the chromatin constitution. A variety of studies conformed that sodium butyrate is an inhibitor that can induce differentiation and apoptosis in many tumor cells. The purpose of this experiment is to determine the optimal concentration and mechanism of the apoptosis induction of Sodium Butyrate on human hepatoma cell line 7721.Methods The hepatoma cells were treated with various concentrations of sodium butyrate in different duration in vitro. The suitable concentration of sodium butyrate was chosen by Hua's optimal selection method, which are 0.10, 0.65, 1.00, 1.70, 2.15, 2.85, 4.00, 7.70, 10.00mmol/L, separately. Proliferation suppression was observed by MTT method and invert microscope, while morphological changes of apoptosis were ascertained by cytochemical staining (Hochest33258) and transmission electron microscopy (TEM). Flow cytometry (FCM) was used to investigate the apoptosis rate and the cell cycle. The expression of p21 mRNA and protein were detected by semi-quantitative RT-PCR and Western-blot method, respectively.Results The sodium butyrate inhibited the growth of hepatoma cells in the dose-dependent and time-dependent manner. But the cells treated with high concentration sodium butyrate (^4mmol/L) exhibited the characteristics of cell necrosis including cell membrane effacement , swelling and fragmentation of mitochondrion within 12h. Fluorescent staining and TEM showed that the cells treated with sodium butyrate exhibited characteristics of apoptosis including cell membrane shrinkage, condensation and fragmentation of nuclear chromatin and formation of apoptotic bodies. FCM analysis showed a decrease in S phase and an increase in GO/G1 phase as well as a decrease of the exponent of cell proliferation. Semi-quantitative RT-PCR and western-blot indicated that the expression of p21 mRNA and protein significantly increased in experiment group.Conclusion Proliferation inhibition and apoptosis induction of hepatoma cells could be detected when they were treated with sodium butyrate, which were correlated with the up-regulation of p21 gene. The cells cultured for five days in vitro showed that the optimal concentration of sodium butyrate for inducing apoptosis on hepatoma cells was 2.85mmol/L.