ucn-1028-c and Liver-Neoplasms

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

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