l-663536 and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

Neuroinflammation and oxidative stress are important factors that induce neurodegeneration in age-related neurological disorders. 5-Lipoxygenase (5-LOX) is the enzyme responsible for catalysing the synthesis of leukotriene or 5-HETE from arachidonic acid. 5-LOX is expressed in the central nervous system and may cause neurodegenerative disease. In this study, we investigated the effect of the pharmacological inhibition of 5-lipoxygenase on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP(+)-induced dopaminergic neuronal death in midbrain neuron-glia co-cultures and in mice. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into C57BL6 mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [(3)H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP(+) treatment. In addition, LTB₄, one of 5-LOX's downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB₄ but not LTD₄ and 5-HETE enhanced MPP(+)-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. Furthermore, the MPTP-induced upregulation of LTB₄ in the striatum and substantia nigra was antagonised by MK-886. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB₄ may play an important pathological role in Parkinson's disease.

Topics: 5-Lipoxygenase-Activating Protein Inhibitors; 5-Lipoxygenase-Activating Proteins; Animals; Arachidonate 5-Lipoxygenase; Astrocytes; Cell Death; Coculture Techniques; Corpus Striatum; Dopamine; Dopaminergic Neurons; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene B4; Leukotriene D4; Male; Mesencephalon; Mice; MPTP Poisoning; Neuroprotective Agents; Substantia Nigra

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Aspirin; Benzoates; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Indoles; Inflammation; Lipopolysaccharides; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Urea

Neutrophil apoptosis is an important physiological process in the resolution of pulmonary inflammation. Previous studies have shown that eicosapentaenoic acid (EPA; 20:5n-3) increases the rate of apoptosis in a concentration- and time-dependent manner in HL-60 cells. However, it is not known if the EPA-induced apoptosis involves the lipoxygenase (LO) and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effects of inhibitors LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells.. Cells were incubated with 50 mum EPA in the presence of an enzyme inhibitor (1-10 microm) for 12 h. Compounds were used to inhibit COX 1 and 2 (ibuprofen), 5-, 12-, 15-LO (NDGA), 12-LO (baicalein), 5-LO (AA-861), and 5-LO activating protein (MK-886). Eicosanoid (0.001-1.0 mum) add-back experiments were also conducted; LTB(4) and 5-HETE with 5-LO inhibition and 12-HETE with 12-LO inhibition. Flow cytometry was used to assess apoptosis.. Inhibition of COX 1 and 2 had no effect on apoptosis. Inhibition of 5-LO and 12-LO significantly increased apoptosis in EPA-treated HL-60 cells. Addition of LTB(4) reduced apoptosis to levels significantly lower than in HL-60 cells treated with EPA alone; 5-HETE and 12-HETE also lowered apoptosis to control levels.. These data indicate that inhibition of LO, particularly 5-LO, increased apoptosis in EPA-treated HL-60 cells. Furthermore, this study demonstrated that the products of the LO enzymes, particularly LTB(4), are critical in the regulation of apoptosis in EPA-treated HL-60 cells.

Topics: Apoptosis; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cyclooxygenase Inhibitors; Eicosapentaenoic Acid; Enzyme Inhibitors; Fatty Acids, Unsaturated; Flavanones; Guaiacol; HL-60 Cells; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Leukotriene B4; Lignans; Lipoxygenase Inhibitors; Neutrophils; Respiratory Distress Syndrome

The arachidonic acid-metabolizing enzymes cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX) are overexpressed during lung carcinogenesis and their end products (e.g.; PGE2, 5-HETE, and LTB4) have been implicated in tumor development. Recently, COX-2 inhibitors (e.g.; celecoxib) and 5-LOX inhibitors (e.g.; MK886 and REV5901) used as single agents have shown promising activities in the treatment and chemoprevention of cancer. However, little is known about the effects of combinations of these inhibitors. We found that simultaneous treatment of premalignant and malignant human lung cell lines with celecoxib, MK886, and REV5901 is more potent in growth suppression and induction of cell death than single or dual combination of these agents. However, their sensitivity to the inhibitors was not directly associated with the expression of COX-2, 5-LOX, or 5-LOX-activating protein (FLAP), but correlated with the production of corresponding metabolites. Furthermore, partial protection of cell death was observed when PGE2 and/or 5-HETE was added to cell cultures treated with celecoxib, MK886, and REV5901 simultaneously. Our data indicate that a triple drug combination of distinct inhibitors of the eicosanoid metabolism at clinically feasible concentrations were more effective than each agent alone suggesting further investigations.

Topics: 5-Lipoxygenase-Activating Proteins; Apoptosis; Blotting, Western; Carrier Proteins; Celecoxib; Cell Cycle; Cell Death; Cell Division; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene B4; Lipoxygenase Inhibitors; Lung Neoplasms; Membrane Proteins; Pyrazoles; Quinolines; Spectrometry, Mass, Electrospray Ionization; Sulfonamides

5(S)-Hydroxy-6,8,11,14-E,Z,Z,Z-eicosatetraenoate (5-HETE) causes PC3 cells to grow by an unknown mechanism. We find that it also induces the cells to activate extracellular signal-regulated kinases and Akt. Pertussis toxin inhibits both responses. 5-HETE, 5-oxo-6,8,11,14-E,Z,Z,Z-eicosatetraenoate, and 5-oxo-15-hydroxy-eicosatetraenoate are known to stimulate leukocytes by a receptor coupled to pertussis toxin-sensitive G proteins. Their respective relative potencies in leukocytes are 1, 10, and 3. In PC3 cells, however, these values are 10, 1, and 0. PC3 cells, we propose, express a non-leukocyte-type, G protein-coupled, 5-HETE receptor. This novel receptor and the extracellular signal-regulated kinase and Akt pathways it recruits may contribute to the progression of prostate adenocarcinoma.

Topics: Benzoquinones; GTP-Binding Proteins; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Eicosanoid; Stimulation, Chemical; Tumor Cells, Cultured

Many arachidonic acid metabolites function in growth signaling for epithelial cells, and we previously reported the expression of the major arachidonic acid enzymes in human breast cancer cell lines. To evaluate the role of the 5-lipoxygenase (5-LO) pathway on breast cancer growth regulation, we exposed cells to insulinlike growth factor-1 or transferrin, which increased the levels of the 5-LO metabolite, 5(S)-hydrooxyeicosa-6E,8C,11Z,14Z-tetraenoic acid (5-HETE), by radioimmunoassay and high-performance liquid chromatography. Addition of 5-HETE to breast cancer cells resulted in growth stimulation, whereas selective biochemical inhibitors of 5-LO reduced the levels of 5-HETE and related metabolites. Application of 5-LO or 5-LO activating protein-directed inhibitors, but not a cyclooxygenase inhibitor, reduced growth, increased apoptosis, down-regulated bcl-2, up-regulated bax, and increased G1 arrest. Exposure of breast cancer cells to a 5-LO inhibitor up-regulated peroxisome proliferator-activated receptor (PPAR)a and PPARg expression, and these same cells were growth inhibited when exposed to relevant PPAR agonists. These results suggest that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. Additional experiments suggest that this involves the interplay of several factors, including the loss of growth stimulation by 5-LO products, the induction of PPARg, and the potential activation of PPARg by interactions with shunted endoperoxides.

Topics: Apoptosis; Arachidonic Acid; bcl-2-Associated X Protein; Breast Neoplasms; Cell Division; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Insulin-Like Growth Factor I; Ligands; Lipoxygenase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antineoplastic Agents; Apoptosis; Cell Membrane; Dietary Fats; Drug Combinations; Drugs, Chinese Herbal; Flavanones; Flavonoids; Glycyrrhiza; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mitochondria; Models, Biological; Nucleosomes; Oxidative Stress; Paeonia; Permeability; Phosphatidylserines; Prostatic Neoplasms; Tumor Cells, Cultured

Arachidonic acid (5,8,11,14-eicosatetraenoic acid), a member of the omega-6 poly-unsaturated fatty acids, was found to be an effective stimulator of human prostate cancer cell growth in vitro at micromolar concentrations. Selective blockade of the different metabolic pathways of arachidonic acid (e.g. ibuprofen for cyclooxygenase, SKF-525A for cytochrome P-450, baicalein and BHPP for 12-lipoxygenase, AA861 and MK886 for 5-lipoxygenase, etc.) revealed that the growth stimulatory effect of arachidonic acid is inhibited by the 5-lipoxygenase specific inhibitors, AA861 and MK886, but not by others. Addition of the eicosatetraenoid products of 5-lipoxygenase (5-HETEs) showed stimulation of prostate cancer cell growth similar to that of arachidonic acid, whereas the leukotrienes were ineffective. Moreover, the 5-series of eicosatetraenoids could reverse the growth inhibitory effect of MK886. Finally, prostate cancer cells fed with arachidonic acid showed a dramatic increase in the production of 5-HETEs which is effectively blocked by MK886. These experimental observations suggest that arachidonic acid needs to be metabolized through the 5-lipoxygenase pathway to produce 5-HETE series of eicosatetraenoids for its growth stimulatory effects on human prostate cancer cells.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Benzoquinones; Cell Division; Cyclooxygenase Inhibitors; Eicosanoids; Enzyme Inhibitors; Flavanones; Flavonoids; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indoles; Lipoxygenase Inhibitors; Male; Masoprocol; Proadifen; Prostatic Neoplasms; Tumor Cells, Cultured

Topics: Aminobenzoates; Animals; Aristolochic Acids; Chickens; Chlorobenzoates; Cinnamates; Cyclooxygenase 2; Dinoprostone; DNA Replication; Enzyme Induction; Enzyme Inhibitors; Fatty Acids, Unsaturated; Female; Gene Expression Regulation; Granulosa Cells; Hydroxyeicosatetraenoic Acids; Indoles; Isoenzymes; Leukotrienes; Lipoxygenase Inhibitors; Lysophospholipids; Masoprocol; Mitosis; ortho-Aminobenzoates; Ovarian Follicle; Phenanthrenes; Phospholipases A; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Transforming Growth Factor alpha

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Arachidonic Acid; Biological Transport; Calcimycin; Diglycerides; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Tetradecanoylphorbol Acetate

BAY X 1005 ((R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) has been demonstrated to be a potent inhibitor of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis in various in vitro systems. Using mainly human polymorphonuclear leukocytes (PMNL) this study elucidates the mechanism of inhibition of 5-lipoxygenase (5-LOX, EC 1.13.11.34)-derived arachidonic acid metabolites by BAY X 1005. At concentrations of BAY X 1005 which almost totally inhibited the formation of 5-LOX-derived metabolites, both arachidonic acid release and platelet-activating factor synthesis were only modestly affected. This suggests that the inhibitory effect of BAY X 1005 is not due to a limitation of substrate availability for 5-LOX. Compared to the inhibition of leukotriene synthesis in intact human PMNL about 800-fold higher concentrations of BAY X 1005 were required to inhibit leukotriene formation in a cell-free system suggesting that the inhibitory effect of BAY X 1005 cannot be explained by a direct effect on 5-LOX. In an attempt to identify possible target proteins of BAY X 1005, [14C]BAY X 1005 was used in binding studies under equilibrium conditions. The quantitative analysis of specific binding in intact human PMNL revealed two binding sites for BAY X 1005. Upon subcellular fractionation of these cells the BAY X 1005 high affinity binding site was localized in the microsomal fraction whereas the low affinity binding site was localized in the granule fraction. The Kd for BAY X 1005 binding to the high affinity binding site (0.165 mumol/L) was almost identical to the IC50 value for inhibition of LTB4 synthesis (0.22 mumol/L). Furthermore, the IC50 values for competition of BAY X 1005 binding at the high affinity binding site were almost identical to the IC50 values for inhibition of LTB4 synthesis in the case of BAY X 1005, 12 other structurally related quinoline derivatives and the reference compounds REV-5901, WY-50,295 and MK-886, but not in the case of the direct 5-LOX inhibitors A-64077 and AA-861. The analysis of BAY X 1005 binding in rat PMNL also revealed two binding sites. Whereas the low affinity binding site in rat PMNL exhibited a Kd similar to the human, the rat high affinity binding site showed a 5.5-fold higher affinity for BAY X 1005 compared to the human. This correlates well with the 8.5-fold higher sensitivity of rat versus human PMNL concerning inhibition of LTB4 synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Arachidonic Acid; Binding Sites; Binding, Competitive; Cell-Free System; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Kinetics; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophils; Platelet Activating Factor; Quinolines; Rats; Structure-Activity Relationship; Subcellular Fractions