The Effect Of DAXX Overexpressed On The Apoptosis Of HepG₂ Cells Induced By Oxidative Stress

Background : liver cancer is a common threat to our people's health and it's mortality rate have been placed the third in cancer. However, therapeutic selectivity and drug resistance are two major issues in liver cancer chemotherapy. Although drugs can target different ways to promote apoptosis, apoptosis between cancer cells and normal cells is not similar.Evidence from recent studies suggests that cancer cells, compared to normal cells, are increased with oncogenes expression and activity, which can prevent tumor cells apoptosis and lower sensitivity to drugs. Reconstruction apoptosis signal transmitting system or promotion death gene expression in tumor cells is another strong target that we should aim at in the research of cancer therapeutic drugs.Death domain-associated protein (Daxx) mediated apoptosis not only through Fas-Daxx-JNK pathway but also served as tumor growth factor (TGF-beta) receptor to promote apoptosis. Daxx can sensitized apoptosis of tumor cells, However, the impact of Daxx on the liver tumor cell line HepG₂ has not been reported. This experiment is to study the effects and mechanisms of Daxx overexpressed in HepG₂ on drug sensitivity, and in order to provide theoretic target for cancer chemotherapy.Methods : HepG₂ cells were transfected using lipofectamine 2000,selected by treatment with G418. The stable cell lines were assessed for vector transfection by reverse transcriptase polymerase chain reaction(RT-PCR), the groups is as follows : (1) the control group (non-transfected cells); (2) transfected with empty vector (HepG₂/GFP cells); (3) transfected with pEGFP-C1-Daxx (HepG₂/GFP-Daxx cells ). After incubated with hydrogen peroxide for 24 hours, cellular activity was analyzed by MTT;cellular apoptosis was measured by flow cytometric analysis. Protein expression was detected by Western blot.Results : The RT-PCR results showed the cells transfected with pEGFP-C1-Daxx was increased Daxx RNA significantly compare with the HepG₂/GFP cells, and at the same time, Fluorescence microscopy show that Daxx protein localized in the nuclei of cells. We used hydrogen peroxide to induce apoptosis of HepG₂ cells and observed that the hydrogen peroxide inhibited the activity of HepG₂ cells in concentration-dependent way. The IC50 value of three groups cells (Normal cells, HepG₂/ GFP cells and HepG₂/ GFP-Daxx cells) were 0.72,0.76, 0.49 mmol/L respectively. We detected apoptosical ratio was significantly higher in HepG₂/GFP-Daxx cells compare with another groups(42.9±8.42 vs 27.3±6.38 or 28.5±4 . 71), which suggested that Daxx improve cellular sensitivity to hydrogen peroxide.We also observed activation of caspase 3 by the Western-blot.the Expression of caspase 3 was increased strongly induced by hydrogen peroxide in HepG₂/GFP-Daxx cells(204.66±19.68%), control cells and the cells transfected with empty vector were 100±3.1% and 107.39±20.1% respectively. The result further illustrate the Daxx increase the apoptosis of HepG₂ cells induced by hydrogen peroxide. The JNK phosphorylation of Daxx transfected cells was significantly higher than that of the cells transfected with empty vector or without.Conclusion : 1. Daxx can increase HepG₂ apoptosis induced by hydrogen peroxide; 2. That Daxx protein sensitized HepG₂ cell apoptosis induced by hydrogen peroxide may be a synergistic increase of JNK activity.