ucn-1028-c and Carcinoma--Hepatocellular

Metabolism of arachidonic acid (AA) is known to induce in different cell types an oxidative stress via the production of reactive oxygen species. As these latter may be scavenged by antioxidant enzymes as manganese and copper/zinc-dependent superoxide dismutase (MnSOD and Cu/ZnSOD, respectively), we investigated the effects of AA on their expression in human HepG2 hepatoma cells. RT-PCR and Western blot data revealed that AA induced an increase in the MnSOD, but not Cu/ZnSOD, expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in MnSOD activity. The AA-induced MnSOD expression required de novo transcription as demonstrated by cotreatment of HepG2 cells with AA and actinomycin D. The fact that MnSOD expression was not induced when HepG2 cells were cultured with 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolizable analog of AA, or with different inhibitors of the AA metabolism pathways suggested that the metabolism of AA was required. Further investigations into the mechanisms by which AA induced MnSOD expression showed that superoxide anions released from AA metabolism act as second messengers via a signal-controlling pathway involving protein kinase C and p38 mitogen activated protein kinase (MAPK). These results define a novel role of p38 MAPK dependent-pathway in the regulation of MnSOD gene.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Arachidonic Acid; Blotting, Western; Carcinoma, Hepatocellular; Cell Division; DNA Primers; Enzyme Induction; Enzyme Inhibitors; Flow Cytometry; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Superoxide Dismutase; Superoxides; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Hepatocyte growth factor (HGF) is known to be a potent mitogen and motogen for epithelial cells. Hepatocellular carcinoma (HCC) often metastasizes, and the c-Met/HGF receptor is highly expressed by HCC cells. The aim of this study was to investigate the signaling pathways associated with the motogenic effect of HGF on HCC cells via c-Met. HCC cell lines (Hep3B, HepG2, PLC, and Huh-7) and HCC cells harvested from patients were used for the Boyden chamber assay of chemotactic activity as well as for immunoprecipitation and immunoblotting studies. HGF stimulated the motility of Hep3B, HepG2, and Huh-7 cells in a dose-dependent manner in association with tyrosine phosphorylation of c-Met and activation of phosphatidylinositol 3-kinase (PI3-K). A tyrosine kinase inhibitor (genistein) and a PI3-K inhibitor (wortmannin) prevented the migration of HCC cells. However, migration was not prevented by calphostin C, an inhibitor of protein kinase C (PKC), which is a downstream target of phospholipase Cgamma (PLCgamma). HGF also stimulated the migration of HCC cells obtained from three patients, while wortmannin prevented the migration of these cells. These results indicate that HGF stimulates the migration of HCC cells through the tyrosine phosphorylation of c-Met via activation of PI3-K.

Topics: Androstadienes; Carcinoma, Hepatocellular; Cell Adhesion Molecules; Cell Movement; Enzyme Induction; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genistein; Hepatocyte Growth Factor; Humans; Isoenzymes; Naphthalenes; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phospholipase C gamma; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-met; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine; Wortmannin

Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-alpha and PKC-zeta were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-zeta predominantly attaches to the trans-Golgi region, whereas PKC-alpha binds to the cis- and trans-Golgi area.

Topics: Carcinoma, Hepatocellular; Cell Fractionation; Cell Line; Cell-Free System; Cytoplasmic Granules; Egtazic Acid; Enzyme Inhibitors; Golgi Apparatus; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Indoles; Intracellular Membranes; Isoenzymes; Kinetics; Liver Neoplasms; Naphthalenes; Protein Binding; Protein Kinase C; Protein Kinase C-alpha; Proteoglycans

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified potent mitogen for smooth muscle cells and fibroblasts. HB-EGF has been shown to be an EGF receptor ligand, and also to stimulate epithelial cell growth. A human hepatoma-derived cell line, Mahlavu, was analyzed for the production of HB-EGF mRNA and active HB-EGF protein. It was found that the cell line synthesized very low or undetectable basal level of HB-EGF mRNA. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a rapid and transient rise in HB-EGF mRNA level. HB-EGF in Mahlavu cells appears to be regulated by a protein kinase C (PKC) pathway, since PKC inhibitors, H7, staurosporin, and calphostin C, abrogated the induction of HB-EGF mRNA by TPA. Unlike vascular smooth muscle cells, induction of HB-EGF gene transcription by TPA was blocked completely by incubation with cycloheximide, suggesting that protein synthesis may be a prerequisite for HB-EGF gene transcription in Mahlavu cells. Mahlavu cells were also found to release a bioactive HB-EGF-like protein into conditioned medium which stimulates DNA synthesis in EP170.7 cells. This activity was neutralized by an anti-HB-EGF antibody. These results indicate that HB-EGF gene transcription is regulated via a PKC pathway, resulting in secretion of active HB-EGF into the culture medium of hepatoma-derived Mahlavu cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Carcinoma, Hepatocellular; Cycloheximide; Epidermal Growth Factor; Gene Expression Regulation; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Naphthalenes; Protein Kinase C; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured