The effect of overexpressed daxx in liver tumor cells on the apoptosis induced by oxidative stress

In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG 2 to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG 2 cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG 2/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG 2/GFP-Daxx cells). After incubation with hydrogen peroxide (H 2O 2) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG 2/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG 2 cells and observed that the hydrogen peroxide decreased the viability of HepG 2 cells in concentration-dependent pattern. The IC 50 values in three groups (Normal cells, HepG 2GFP cells and HepG 2/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG 2/GFP-Daxx cells as compared to the other two groups. HepG 2GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG 2 cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.