In addition to hypoxia, HIF-1alpha is shown to be activated by several factors including protein Kinase C(PKC). However, the precise molecular mechanisms and PKC isoform(s) involved in the HIF-1alpha activation process remained unclear. Recently, we

have reported that menatetrenone, a vitamin K2 analogue, inhibited the NF-kappaB activation through the suppression of PKC activity. In this study, we investigated the roles of PKC isoforms in the HIF-1alpha activation and effects of VK2 on the activation of HIF-1alpha. Ibrutinib concentration Human HCC-derived Huh7 cells were cultured in normoxia and hypoxia (1% O2) with or without the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate(TPA). The expression, transcriptional activity and nuclear translocation of HIF-1alpha were examined by Western blot and lucifer-ase assay respectively. To explore the role of PKC isoform, PKC inhibitors (Ro-31-8425, Gö6976 and Rottlerin) or siR-NAs against each PKC isoforms,

and VK2 were employed. To explore the nuclear translocation of HIF-1alpha, GFP-tagged HIF-1alpha cDNA was introduced into the cells and distribution of GFP was observed under fluorescence microscope. ChIP assay was performed to identify the recruitment of HIF-1alpha to VEGF promoter region. Hypoxia led to an increased expression of HIF-1alpha protein and stimulated the luciferase activity of HIF-1alpha more than 10 times. selleck products TPA increased the HIF-1al-pha luciferase activity several times under both normoxia and hypoxia. Although knockdown of PKC-alpha or -epsilon did not affect the HIF-1alpha transcriptional activity and protein amounts, PKC-delta siRNA-mediated knockdown and rottlerin (PKC-delta inhibitor) suppressed the transcriptional activity of HIF-1alpha and HIF-1alpha protein expression while HIF-1al-pha mRNA was not affected. Pan-PKC inhibitor (Ro-31-8425) showed similar

effects. ChIP assay showed the decreased Sucrase recruitment of HIF-1alpha to VEGF promoter when cells were treated with PKC-delta siRNA, but not with PKC-alpah or -epsilon siRNA. VK2 significantly inhibited the TPA-induced HIF-1alpha transcriptional activity in a dose-dependent manner. Moreover, VK2 suppressed the expression and nuclear translocation of HIF-1alpha induced by TPA as same as PKC-delta knockdown did in HCC cells. PKC-delta contributes to enhance the HIF-1al-pha transcriptional activity by stabilizing and increasing the nuclear translocation and recruitment of HIF-1alpha protein to target gene promoter. Therefore inhibition of PKC-delta might contribute to suppress the HIF-1alpha activity in HCC cells and might be a potential therapeutic target of HCC.