ucn-1028-c and thiazolyl-blue

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

The light-activated protein kinase C inhibitor, calphostin C, is shown to inhibit the ability of IL-3-dependent 32D cells to reduce the tetrazolium salt, MTT. To determine whether this inhibition was mediated through mitochondria which have been implicated in MTT reduction, isolated mitochondria were treated with calphostin C in the presence of various substrates for mitochondrial electron transport and EDTA (to exclude PKC involvement). Calphostin C extensively inhibited succinate-dependent MTT reduction (IC50 = 110nM) but had little effect on either NADH- or NADPH-dependent MTT reduction. An alternative protein kinase C inhibitor, H7, did not affect succinate-dependent mitochondrial MTT reduction, and the protein kinase A inhibitor, KT5720, had little effect on either cellular or mitochondrial MTT reduction. These results show that in addition to its role as a PKC inhibitor, calphostin C is also a potent inhibitor of succinate-dependent mitochondrial electron transport.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Bone Marrow; Carbazoles; Cell Division; Cell Line; Coloring Agents; DNA Replication; Indoles; Isoquinolines; Kinetics; Mice; Mice, Inbred C3H; Mitochondria; Naphthalenes; Oxidation-Reduction; Piperazines; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Succinates; Tetrazolium Salts; Thiazoles; Thymidine