Identification of a protein kinase C (PKC) activator, daphnoretin, that suppresses hepatitis B virus gene expression in human hepatoma cells

We examined the antiviral activity of a crude extract prepared from a Chinese medicinal herb Wikstroemia indica C.A. Mey. One active component, daphnoretin (7-hydroxy-6-methoxy-3,7'-dicoumarylether), was identified, which showed strong suppressive effects on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells. To examine the signaling pathway of daphnoretin on the Hep3B cells, we pretreated Hep3B cells with 12-O-tetradecanoylphorbol-13-O-acetate (200 nM) for 24 hr to down-regulate intracellular protein kinase C (PKC) levels and found that the PKC-down-regulated Hep3B cells did not respond at all to daphnoretin. Furthermore, daphnoretin induced translocation of PKC from the cytosol to the membrane and down regulated intracellular PKC levels in the Hep3B cells, indicating that it may directly activate PKC. This hypothesis was supported by the observation that daphnoretin directly competed with [H-3]phorbol dibutyrate for the binding of PKC in the whole cell and activated purified PKC activity in vitro. Our results demonstrated that daphnoretin, with a structure distinct from phorbol ester, is a PKC activator and has suppressive effects on HBsAg gene expression in human hepatoma cells.