h-89 and Carcinoma--Hepatocellular

Chronic hepatitis C virus (HCV) infection causes hepatocellular carcinoma (HCC). The HCC risk, while decreased compared with active HCV infection, persists in HCV-cured patients by direct-acting antiviral agents (DAA). We previously demonstrated that Wnt/β-catenin signaling remained activated after DAA-mediated HCV eradication. Developing therapeutic strategies to both eradicate HCV and reverse Wnt/β-catenin signaling is needed.. Cell-based HCV long term infection was established. Chronically HCV infected cells were treated with DAA, protein kinase A (PKA) inhibitor H89 and endoplasmic reticulum (ER) stress inhibitor tauroursodeoxycholic acid (TUDCA). Western blotting analysis and fluorescence microscopy were performed to determine HCV levels and component levels involved in ER stress/PKA/glycogen synthase kinase-3β (GSK-3β)/β-catenin pathway. Meanwhile, the effects of H89 and TUDCA were determined on HCV infection.. Both chronic HCV infection and replicon-induced Wnt/β-catenin signaling remained activated after HCV and replicon eradication by DAA. HCV infection activated PKA activity and PKA/GSK-3β-mediated Wnt/β-catenin signaling. Inhibition of PKA with H89 both repressed HCV and replicon replication and reversed PKA/GSK-3β-mediated Wnt/β-catenin signaling in both chronic HCV infection and replicon. Both chronic HCV infection and replicon induced ER stress. Inhibition of ER stress with TUDCA both repressed HCV and replicon replication and reversed ER stress/PKA/GSK-3β-dependent Wnt/β-catenin signaling. Inhibition of either PKA or ER stress both inhibited extracellular HCV infection.. Targeting ER stress/PKA/GSK-3β-dependent Wnt/β-catenin signaling with PKA inhibitor could be a novel therapeutic strategy for HCV-infected patients to overcomes the issue of remaining activated Wnt/β-catenin signaling by DAA treatment. Video Abstract.

Topics: Antiviral Agents; beta Catenin; Carcinoma, Hepatocellular; Cells, Cultured; Endoplasmic Reticulum Stress; Glycogen Synthase Kinase 3 beta; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Neoplasms; Protein Kinase Inhibitors

Nonalcoholic fatty liver is characterized by the abnormal accumulation of triglycerides within hepatocytes, resulting in a steatotic liver. Glucagon-like peptide 1 and its analog exendin-4 can ameliorate certain aspects of this syndrome by inducing weight loss and reducing hepatic triglyceride accumulation, but it is unclear whether these effects result from the effects of glucagon-like peptide 1 on the pancreas, or from direct action on the liver. This study investigated the direct action and putative cellular mechanism of exendin-4 on steatotic hepatocytes in culture. Steatosis was induced in cultured HepG2 human hepatoma cells by incubation in media supplemented with 2 mM each of linoleic acid and oleic acid. Steatotic hepatocytes were then pre-incubated in the protein kinase A inhibitor H89 for 30 min, then treated with exendin-4 over a period of 24 h. Cell viability and triglyceride content were characterized by a TUNEL assay and AdipoRed staining, respectively. Our results showed that steatotic cells maintained high levels of intracellular triglycerides (80%) compared to lean controls (25%). Exendin-4 treatment caused a significant reduction in intracellular triglyceride content after 12 h that persisted through 24 h, while protein kinase A inhibitors abolished the effects of exendin-4. The results demonstrate the exendin-4 induces a partial reduction in triglycerides in steatotic hepatocytes within 12 h via the GLP-1 receptor-mediated activation of protein kinase A. Thus, the reduction in hepatocyte triglyceride accumulation is likely driven primarily by downregulation of lipogenesis and upregulation of β-oxidation of free fatty acids.

Topics: Carcinoma, Hepatocellular; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Exenatide; Fatty Liver; Glucagon-Like Peptide 1; Hep G2 Cells; Hepatocytes; Humans; Isoquinolines; Linoleic Acid; Lipogenesis; Liver Neoplasms; Oleic Acid; Pancreas; Peptides; Sulfonamides; Triglycerides; Venoms

Serum amyloid A (SAA) reduces fat deposition in adipocytes and hepatoma cells. Human SAA1 mRNA is increased by docosahexaenoic acid (DHA) treatment in human cells. These studies asked whether DHA decreases fat deposition through SAA1 and explored the mechanisms involved. We demonstrated that DHA increased human SAA1 and C/EBPbeta mRNA expression in human hepatoma cells, SK-HEP-1. Utilizing a promoter deletion assay, we found that a CCAAT/enhancer-binding protein beta (C/EBPbeta)-binding site in the SAA1 promoter region between -242 and -102 bp was critical for DHA-mediated SAA1 expression. Mutation of the putative C/EBPbeta-binding site suppressed the DHA-induced SAA1 promoter activity. The addition of the protein kinase A inhibitor H89 negated the DHA-induced increase in C/EBPbeta protein expression. The up-regulation of SAA1 mRNA and protein by DHA was also inhibited by H89. We also demonstrated that DHA increased protein kinase A (PKA) activities. These data suggest that C/EBPbeta is involved in the DHA-regulated increase in SAA1 expression via PKA-dependent mechanisms. Furthermore, the suppressive effect of DHA on triacylglycerol accumulation was abolished by H89 in SK-HEP-1 cells and adipocytes, indicating that DHA also reduces lipid accumulation via PKA. The observation of increased SAA1 expression coupled with reduced fat accumulation mediated by DHA via PKA suggests that SAA1 is involved in DHA-induced triacylglycerol breakdown. These findings provide new insights into the complicated regulatory network in DHA-mediated lipid metabolism and are useful in developing new approaches to reduce body fat deposition and fatty liver.

Topics: Adipocytes; Adipose Tissue; Adult; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Docosahexaenoic Acids; Enzyme Inhibitors; Female; Humans; Isoquinolines; Lipids; Middle Aged; Molecular Sequence Data; Serum Amyloid A Protein; Sulfonamides

Keratins are members of a diverse group of tissue-specific cytoskeletal components known as intermediate filaments. Regulation of the structure and intracellular distribution of intermediate filaments is known to be related to the phosphorylation state of their structural subunits. It also is known that disruption of the keratin filaments of hepatocytes in response to chronic ethanol ingestion is characteristic of alcoholic liver disease.. To characterize the mechanism of ethanol-induced keratin filament reorganization and dephosphorylation, cells were grown in culture with and without ethanol, and then were treated at the end of the incubation period for 1 hr with either 8-bromo-adenosine 3':5'-cyclic monophosphate (8Br), water-soluble forskolin (ws-forskolin), H-89 diHCL, or okadaic acid. Morphology of the cells was examined by immunofluorescence microscopy, and keratin phosphorylation levels were determined by analysis of 32p labeling.. We found that treatment of hepatoma cells with 300 mM ethanol results in disruption and aggregation of the keratin network in the vicinity of the nucleus as well as a hypophosphorylation of keratin subunits from ethanol-treated cells compared with non-ethanol-treated controls. 8Br and ws-forskolin treatment of ethanol groups restored keratin phosphorylation to control levels and reversed the ethanol-induced aggregation of keratin filaments. When H-89, an inhibitor of A-kinase, was added to control cells, keratin filament disorganization and dephosphorylation was observed. H-89 produced only a slight additional decrease in keratin phosphorylation in ethanol-treated cells, with no change in keratin distribution. Okadaic acid treatment of control cells produced hyperphosphorylation and filament network disruption, whereas in ethanol groups a reversal of the ethanol-mediated hypophosphorylation was observed but without reversal of the keratin filament aggregation.. These results suggest that site-specific phosphorylation of keratin filaments is important in maintaining their integrity and that activation of the A-kinase system can antagonize the effects of ethanol, whereas its inhibition results in filament dephosphorylation and reorganization, mimicking effects of ethanol treatment.

Topics: Animals; Carcinoma, Hepatocellular; Central Nervous System Depressants; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Ethanol; Hepatocytes; Intermediate Filaments; Isoquinolines; Keratins; Phosphorylation; Rats; Sulfonamides; Tumor Cells, Cultured