Nakao, Kazuhiko

AbstractBackground and AimElimination of hepatitis B virus (HBV) is infrequently achieved with current therapies. Therefore, more effective anti‐HBV therapy is needed. We previously reported that geranylgeranylacetone (GGA) showed anti‐hepatitis C virus activity in human hepatoma cells. In this study, we examined the anti‐HBV activity of GGA.MethodsWe used HepG2.2.15.7 cells, PXB cells infected with HBV, Huh7 cells transfected with linear HBV, and PLC/PRF/5 cells as HBV‐infected hepatocyte models. After GGA treatment, HBV‐related antigen was measured by chemiluminescent immunoassay. HBV‐related mRNA was examined by Northern blot. cccDNA and endoplasmic reticulum stress markers were measured by real‐time polymerase chain reaction. The activities of HBV promoters and enhancer regions were examined using luciferase vectors.ResultsAfter GGA treatment, hepatitis B surface antigen and hepatitis B e antigen secretion was decreased in all HBV‐infected hepatocyte models. HBV‐related mRNA was also decreased by GGA treatment, although cccDNA levels were not affected. Additionally, the activity of HBV S1 and S2 promoter region and Enhancer 1/Enhancer 2/core promoter region was reduced by GGA treatment. The mRNA expression of the main transcription factors, hepatocyte nuclear factor 3 and 4 and CCAAT/enhancer binding protein, was also decreased. Further, the expression levels of endoplasmic reticulum stress markers were increased by GGA treatment, which reflected the change in HBV‐related antigen secretion.ConclusionsGeranylgeranylacetone treatment reduces HBV‐related protein levels by suppressing comprehensive downregulation of HBV promoter and enhancer activity, which might be caused by decreased hepatic transcription factor expression. GGA treatment may enhance anti‐HBV effects in combination with other therapies.

Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.