calcimycin and sodium-arsenite

Licofelone is a dual inhibitor of the cyclooxygenase and 5-lipoxygenase (5-LO) pathway, and has been developed for the treatment of inflammatory diseases. Here, we investigated the molecular mechanisms underlying the inhibition by licofelone of the formation of 5-LO products.. The efficacy of licofelone to inhibit the formation of 5-LO products was analysed in human isolated polymorphonuclear leukocytes (PMNL) or transfected HeLa cells, as well as in cell-free assays using respective cell homogenates or purified recombinant 5-LO. Moreover, the effects of licofelone on the subcellular redistribution of 5-LO were studied.. Licofelone potently blocked synthesis of 5-LO products in Ca(2+)-ionophore-activated PMNL (IC(50)=1.7 microM) but was a weak inhibitor of 5-LO activity in cell-free assays (IC(50)>>10 microM). The structures of licofelone and MK-886, an inhibitor of the 5-LO-activating protein (FLAP), were superimposable. The potencies of both licofelone and MK-886 in ionophore-activated PMNL were impaired upon increasing the concentration of arachidonic acid, or under conditions where 5-LO product formation was evoked by genotoxic, oxidative or hyperosmotic stress. Furthermore, licofelone prevented nuclear redistribution of 5-LO in ionophore-activated PMNL, as had been observed for FLAP inhibitors. Finally, licofelone as well as MK-886 caused only moderate inhibition of the synthesis of 5-LO products in HeLa cells, unless FLAP was co-transfected.. Our data suggest that the potent inhibition of the biosynthesis of 5-LO products by licofelone requires an intact cellular environment and appears to be due to interference with FLAP.

Topics: 5-Lipoxygenase-Activating Proteins; Acetates; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arsenites; Bridged Bicyclo Compounds; Calcimycin; Calcium; Carrier Proteins; Cell-Free System; Cells, Cultured; Dithiothreitol; Dose-Response Relationship, Drug; HeLa Cells; Humans; Indoles; Leukotriene Antagonists; Leukotrienes; Lipoxygenase Inhibitors; Membrane Proteins; Molecular Structure; Neutrophils; Nuclear Envelope; Pyrroles; Quinolines; Sodium Compounds; Transfection

Nonredox-type 5-lipoxygenase (5-LO) inhibitors such as ZM230487 or L-739.010 potently suppress leukotriene biosynthesis at low cellular peroxide tone. Here, we show that inhibition of 5-LO product formation by nonredox-type 5-LO inhibitors in human isolated polymorphonuclear leukocytes (PMNL) depends on the activation pathway of 5-LO. Thus, compared with 5-LO product synthesis induced by the Ca2+-mobilizing agent ionophore A23187, cell stress-induced 5-LO product formation involving 5-LO kinase pathways required ~10- to 100-fold higher concentrations of ZM230487 or L-739.010 for comparable 5-LO inhibition. No such differences were observed for the iron ligand-type 5-LO inhibitor BWA4C or the novel-type 5-LO inhibitors hyperforin and 3-O-acetyl-11-keto-boswellic acid. Experiments using purified 5-LO revealed that Ca2+ is no prerequisite for potent enzyme inhibition by ZM230487, and exposure of PMNL to the combination of ionophore and cell stress did not restore potent 5-LO suppression. Intriguingly, a significant difference in the potency of nonredox-type inhibitors (but not of BWA4C) was determined between wild-type 5-LO and the mutant S271A/S663A-5-LO (lacking phosphorylation sites for ERK1/2 and MAPKAPK-2) in HeLa cells. Collectively, our data suggest that compared with Ca2+-mediated 5-LO product formation, enzyme activation involving 5-LO phosphorylation events specifically and strongly alters the susceptibility of 5-LO toward nonredox-type inhibitors in intact cells.

Topics: Arachidonate 5-Lipoxygenase; Arsenites; Benzeneacetamides; Bridged Bicyclo Compounds; Calcimycin; Calcium; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; HeLa Cells; Humans; Hydroxamic Acids; Lipoxygenase Inhibitors; Mitogen-Activated Protein Kinases; Mutation, Missense; Neutrophils; Peroxides; Phloroglucinol; Phosphorylation; Pyrans; Quinolines; Quinolones; Sodium Chloride; Sodium Compounds; Terpenes

In mammalian cells, gene regulation by amino acid deprivation is poorly understood. Here, we examined the signaling pathways involved in the induction of the C/EBP homologous protein (CHOP) by amino acid starvation. CHOP is a transcription factor that heterodimerizes with other C/EBP family members and may inhibit or activate the transcription of target genes depending on their sequence-specific elements. Amino acid deficiency, when accompanied by insulin-like growth factor I signaling, results in the accumulation of CHOP messenger RNA and protein in AKR-2B and NIH-3T3 cells. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 are able to block CHOP induction in response to amino acid deprivation. Rapamycin is also able to abrogate CHOP expression, suggesting that the mammalian target of rapamycin is involved in CHOP induction by amino acid deficiency. LY294002 and rapamycin are also able to block CHOP induction by hydrogen peroxide, but do not affect expression induced by sodium arsenite or A23187. This is the first evidence that the insulin-like growth factor I/phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway is required for gene regulation by amino acid deprivation and that this pathway is involved in the induction of CHOP by both amino acid deficiency and oxidative stress by hydrogen peroxide.

Topics: 3T3 Cells; Amino Acids; Androstadienes; Animals; Arsenites; Calcimycin; CCAAT-Enhancer-Binding Proteins; Cell Line; Chromones; Culture Media; Dimerization; Enzyme Inhibitors; Gene Expression Regulation; Hydrogen Peroxide; Insulin-Like Growth Factor I; Mammals; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sirolimus; Sodium Compounds; Transcription Factor CHOP; Transcription Factors; Transcription, Genetic; Wortmannin

The Maf protein family consists of eight transcription factors containing a basic-leucine zipper (bZIP) domain. We have previously reported that the mRNA to one of these members, mafG/adapt66, is induced by oxidative stress in hamster HA-1 cells. It has subsequently been reported that mafG is induced bystress that activates the expression of genes under the control of the antioxidant/electrophile response element (ARE/EpRE), and that small Maf proteins are present in ARE/EpRE-protein complexes. Here we extend these studies to assess the effects of various types of stress on maf mRNA induction in human cells. The oxidative stressor cadmium, and the heavy metals cadmium, zinc, and arsenite induced mafG RNA levels within two hours, and maximally at five hours for cadmium and zinc. This induction was observed for multiple transcripts including two not normally associated with mafG, suggesting that these stress agents induced the expression of other related maf family RNAs. Modest induction of mafG mRNA was also observed with heat shock but not calcium elevation. These results suggest that mafG is a human stress-response gene induced by multiple stress, and that several maf (proto-)oncogene members play an important role in cellular stress response.

Topics: Animals; Arsenites; Cadmium Chloride; Calcimycin; Chlorides; Cricetinae; DNA-Binding Proteins; Gene Expression Regulation; HeLa Cells; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Kinetics; MafG Transcription Factor; Metals, Heavy; Oxidative Stress; Proto-Oncogene Mas; Repressor Proteins; RNA, Messenger; Sodium Compounds; Transcription, Genetic; Zinc Compounds

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arsenites; B-Lymphocytes; Calcimycin; Calcium; Cell Line; Cell-Free System; Enzyme Activation; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertonic Solutions; Imidazoles; Interleukin-1; Intracellular Signaling Peptides and Proteins; Ionophores; Leukotrienes; Lipoxygenase Inhibitors; Mitogen-Activated Protein Kinases; Osmotic Pressure; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Pyridines; Sodium Compounds; Sorbitol; Subcellular Fractions; Thapsigargin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vanadates

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Topics: Amino Acid Sequence; Animals; Arachidonate 5-Lipoxygenase; Arsenites; Calcimycin; Cell Line; Cricetinae; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Mice; Mitogen-Activated Protein Kinases; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sodium Compounds; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Necrosis Factor-alpha

This study was designed to test the hypothesis that the antioxidant taurine may modulate human endothelial cell (EC) death (apoptosis versus necrosis). Sodium arsenite (80 microM) alone and in combination with tumor necrosis factor-alpha (25 ng/mL) caused EC apoptosis after 24 h of treatment. Taurine (.5 mg/mL) added at 0 and 6 h could significantly attenuate EC apoptosis, and oxidative state in response to lipopolysaccharide and tumor necrosis factor-alpha stimulation. EC necrosis was induced by activated neutrophils (PMNs). Taurine reduced PMN-mediated EC necrosis in a dose-dependent manner. Moreover, treatment of ECs with a calcium ionophore, A23187 (1.0-4.0 microM), resulted in both EC apoptosis and necrosis. Taurine significantly abrogated A23187-mediated intracellular calcium elevation and EC death. These data indicate that taurine, possibly through its antioxidant activity and regulation of intracellular calcium flux, can prevent EC dysfunction and cell death, which may have implications for the application of this amino acid in the amelioration of acute lung injury during systemic inflammatory response syndrome.

Topics: Apoptosis; Arsenites; Calcimycin; Calcium; Cell Death; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Electrophoresis; Endothelium, Vascular; Flow Cytometry; Humans; Intercellular Adhesion Molecule-1; Ionophores; Necrosis; Neutrophils; Oxidative Stress; Sodium Compounds; Taurine; Tumor Necrosis Factor-alpha

In this report we show that the adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite, which have been defined previously as inhibitors of cytolysis, inhibit the tumor necrosis factor-alpha (TNF)-induced release of 3H-arachidonic acid from cycloheximide-sensitized C3HA fibroblasts. Since the A23187-induced release of 3H-a.a. was unaffected, our results suggest that these inhibitors provide resistance to lysis by selectively interfering with the lytic response pathway. Our results also show that heat and sodium arsenite can themselves induce the release of 3H-arachidonic acid. These results raise the possibility that stressor-induced resistance to TNF results from the selective desensitization of phospholipase A2.

Topics: Adenovirus E3 Proteins; Animals; Arachidonic Acid; Arsenic; Arsenites; Calcimycin; Cell Line; Cycloheximide; Fibroblasts; Hot Temperature; Kinetics; Mice; Mice, Inbred C3H; Recombinant Proteins; Sodium Compounds; Transfection; Tumor Necrosis Factor-alpha

The stress, or heat shock response of eukaryotic cells is characterized by dramatic changes in the metabolism of responding cells, most notably the increased synthesis of a group of proteins known as heat shock proteins. In this study, we examined the relationship of prostaglandin synthesis/release to the stress response. Stress protein synthesis was induced with sodium arsenite, and prostaglandin E2 and prostacyclin (measured as 6-keto PGF1 alpha) levels were determined by enzyme immunoassay. The stress response was monitored by the incorporation of [35S]methionine and separation of protein by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Prostaglandin synthesis and the stress response were both induced by sodium arsenite. However, aspirin, a cyclooxygenase inhibitor, inhibited arsenite-induced prostaglandin synthesis but did not inhibit stress protein synthesis. Conversely, the calcium ionophore A23187 also stimulated prostaglandin synthesis, but did not induce the stress response. The results of this study indicate that sodium arsenite, a stress response inducer, stimulates prostaglandin production, but this appears to be a correlative rather than causative occurrence in the stress response. Determination of the cytotoxicity of arsenite indicated a high correlation of stimulation of prostaglandin release with cytotoxicity.

Topics: 6-Ketoprostaglandin F1 alpha; Arsenic; Arsenites; Aspirin; Calcimycin; Cell Line; Cell Survival; Dinoprostone; Heat-Shock Proteins; Humans; Kinetics; Sodium Compounds

We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.

Topics: Animals; Arsenic; Arsenites; Calcimycin; Cell Survival; Cells, Cultured; Cerebellum; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Heat-Shock Proteins; Hot Temperature; Immunoblotting; Immunohistochemistry; L-Lactate Dehydrogenase; Neurons; Rats; Sodium Compounds

Challenge of human or murine melanoma cells with sodium arsenite, heavy metals (Zn2+, Cu2+ and Cd2+), or thiol-reactive agents (p-chloromercuribenzoate and iodoacetamide) induced the synthesis of four stress proteins with molecular masses of 100, 90, 72 (a doublet), and 32 (human) or 34 (murine) kDa. Enhanced expression of the 32- and 34-kDa polypeptides (p32 and p34) preceded or paralleled the synthesis of the other stress proteins. Hyperthermia, the calcium ionophore A23187, and amino acid analogs (L-azetidine-2-carboxylic acid and L-canavanine) induced the formation of the major stress proteins, but failed to increase synthesis of p32 and p34. Characterization of the dose and time dependence of p32 and p34 synthesis in human (A375) and murine (B16-F10) melanoma cells, respectively, indicated that these proteins were subject to similar regulatory mechanisms. Electrophoretic analysis of stressed cells pulsed with different metabolic precursors revealed that p32 and p34 were radiolabeled with [35S]methionine or 3H-amino acids but not by [3H]mannose or [35S]cysteine. Polyclonal antibodies raised against human p32 cross-reacted with murine p34. These data suggest that p32 and p34 are closely regulated human and murine gene products, respectively, whose synthesis can be modulated by thiol-reactive reagents. Induction of p32 and p34 by these agents, but not by heat shock, suggests that these proteins are a subset of stress-inducible gene products.

Topics: Amino Acids; Animals; Arsenic; Arsenites; Cadmium; Calcimycin; Chloromercuribenzoates; Copper; Cycloheximide; Dactinomycin; Heat-Shock Proteins; Hot Temperature; Humans; Iodoacetamide; Melanoma; Metals; Mice; Molecular Weight; Sodium Compounds; Sulfhydryl Reagents; Zinc