Hepatitis B Surface Antigen Interacts With Enoyl–coenzyme A Hydratase(ECHS1) And Induces Apoptosis In Hepatoma Cells

Objective: To further examine the interaction between SHBs and ECHS1, study thebiologic funcation of the interaction and to explore the pathological significance of theover production of HBsAg.Methods: In the current study, GST pull-down, co-immunoprecipitation and laserscanning confocal assays were used to further examine the interaction between SHBsand ECHS1. Western Blotting was used to quantify ECHS1levels in transientSHBs-expressing Huh7cells and in stable SHBs-expressing HepG2cells. Weinvestigated Camptothecin-induced apoptosis in stable SHBs-expressing HepG2cellsand control cell lines using flow cytometry assay. To confirm the role of thedown-regulation of ECHS1by SHBs in apoptosis, we used the siRNA strategy todeplete ECHS1or transiently transfected with expression vectors pcDNA3.1-ECHS1to over-express ECHS1in stable SHBs-expressing HepG2cells. Thirty-six hours later,cells underwent Camptothecin for24hours. Then We investigated apoptosis ratiosusing flow cytometry assay.Results: The direct interaction between SHBs and ECHS1existed both in vitro and invivo. In GST pull-down assays, GST-ECHS1protein specifically pulled-down SHBs.The result indicated that SHBs could interact with ECHS1directly in vitro. Inco-immunoprecipition assays, anti-ECHS1antibody precipitated SHBs. Results ofreverse co-immunoprecipition showed that anti-HBs antibody precipitated ECHS1.These findings suggested that SHBs interacted ECHS1in vivo. We examined subcellular localization of SHBs and ECHS1by immunofluorescent staining and livecell confocal imaging. Merging of the images revealed co-localization between SHBsand ECHS1in cytoplasm of HepG2cells.2) SHBs modulates ECHS1levels ofprotein in hepatoma cells. We found that the protein levels of ECHS1decreasedsignificantly in transient SHBs-expressing Huh7cells and in stable SHBs-expressingHepG2cells.3) SHBs sensitizes hepatocytes to Camptothecin-induced apoptosis. Thenumber of apoptotic cells was significantly higher in HepG2-SHBs as compared withHepG2-neo.4) ECHS1may play a key role in apoptosis induced by camptothecin inHepG2cells expressing SHBs. We used the siRNA strategy to deplete ECHS1inHepG2cells expressing SHBs. ECHS1protein levels were significantly suppressed insiRNA clones. More importantly, depletion of ECHS1with concomitant camptothecintreatment led to significant increase in the number of apoptotic cells. Over-expressionof ECHS1induced less reduction in the number of apoptotic cells compared to cellstransfected with empty vector in the presence of camptothecin.Conclusions: SHBs interacts with and modulates ECHS1expression in hepatomacells. SHBs sensitized hepatocytes to Camptothecin-induced apoptosis. ECHS1mayplay a key role in apoptosis induced by camptothecin in HepG2cells expressingSHBs. HBsAg may be play a very important role in the pathogenesis of HBVinfection.