13-hydroperoxy-9-11-octadecadienoic-acid and 15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid

Topics: Animals; Arteriosclerosis; Cardiovascular System; Cornea; Erythropoiesis; Leukotrienes; Linoleic Acids; Lipid Peroxides; Mammals; Skin; Vasoconstrictor Agents

Recent studies using 3D scaffolds have emphasized the importance of the surrounding stroma on chemoresistance in drug efficacy screenings. Since 15-lipoxygenase (15-LOX) metabolites reduced growth of breast, colon, prostate, lung and leukemia cancer cells in 2D cell culture, we were intrigued by the direct comparison of 15-LOX metabolite efficacy in 2D and 3D culture including a stroma equivalent. Herein, we studied the effects of 15-LOX metabolites 13-HpOTrE, 13-HpODE, and 15-HpETE on cutaneous squamous cell carcinoma cells. All metabolites reduced the viability of cancer cells in 2D culture below 10% at 100 µM of each substance. 13-HpOTrE, being the most active agent with respect to cytotoxicity and apoptosis was selected for further experiments. Other than with the 2D culture, we did not obverse cell death, neither from lactate dehydrogenase release, nor from morphology when applying 13-HpOTrE onto the surface of the 3D tumor constructs for one week. Next, we investigated the protein expression of peroxisome proliferator activated receptor gamma, for which the ligand is 13-HpOTrE, and Bcl-2 protein, an apoptosis regulator, but did not find any change following 13-HpOTrE administration. However, 13-HpOTrE treatment reduced the release of interleukin-6, bringing it closer to the level of tumor-free constructs. In conclusion, 13-HpOTrE reduces viability of skin cancer cells in 2D cultures only but modulates inflammatory cytokine levels in the corresponding 3D tumor constructs, too. These studies highlight the need for screening of anticancer drugs employing 3D tumors and including tumor microenvironment in the screening process to increase the low success rate of clinical trials in oncology.

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Arachidonate 15-Lipoxygenase; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Leukotrienes; Linoleic Acids; Lipid Peroxides; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Tumor Microenvironment

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-​hydroperoxy-​5Z,​8Z,​11Z,​13E-​eicosatetraenoic acid (15-HpETE) and 13S-​hydroperoxy-​9Z,​11E-​octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Glutathione Peroxidase; Hypochlorous Acid; Leukotrienes; Linoleic Acids; Lipid Peroxides; Oxidation-Reduction; Peroxidase; Spectrometry, Mass, Electrospray Ionization

Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Allosteric Regulation; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Biocatalysis; Blood Platelets; Carbon Isotopes; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Recombinant Proteins; Reticulocytes; Solvents; Temperature

There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase.

Topics: Arginine; Binding Sites; Catalysis; Computational Biology; Computer Simulation; Deuterium Exchange Measurement; Enzyme Activation; Glycine max; Kinetics; Leukotrienes; Ligands; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Protein Binding; Substrate Specificity; Tryptophan

Apolipoprotein A-I (apoA-I) and an apoA-I peptide mimetic removed seeding molecules from human low density lipoprotein (LDL) and rendered the LDL resistant to oxidation by human artery wall cells. The apoA-I-associated seeding molecules included hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). LDL from mice genetically susceptible to fatty streak lesion formation was highly susceptible to oxidation by artery wall cells and was rendered resistant to oxidation after incubation with apoA-I in vitro. Injection of apoA-I (but not apoA-II or murine serum albumin) into mice rendered their LDL resistant to oxidation within 3 h. Infusion of apoA-I into humans rendered their LDL resistant to oxidation within 6 h. We conclude that 1) oxidation of LDL by artery wall cells requires seeding molecules that include HPODE and HPETE; 2) LDL from mice genetically susceptible to atherogenesis is more readily oxidized by artery wall cells; and 3) normal HDL and its components can remove or inhibit the activity of lipids in freshly isolated LDL that are required for oxidation by human artery wall cells.

Topics: Animals; Aorta; Apolipoprotein A-I; Cell Adhesion; Cells, Cultured; Chemotaxis, Leukocyte; Endothelium, Vascular; Glycine max; Humans; Leukotrienes; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Monocytes; Muscle, Smooth, Vascular; Oxidation-Reduction

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.

Topics: Aorta; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arteriosclerosis; Aryldialkylphosphatase; Cells, Cultured; Chemotaxis, Leukocyte; Coculture Techniques; Coronary Disease; Endothelium, Vascular; Esterases; Female; Humans; Hydrogen Peroxide; Leukotrienes; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Male; Models, Cardiovascular; Monocytes; Muscle, Smooth, Vascular; Oligodeoxyribonucleotides, Antisense; Oxidation-Reduction; Phospholipids; Reference Values

The reaction of lipid hydroperoxide with protein was investigated using an antibody, which was prepared using 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified keyhole limpet hemocyanin as an immunogen. The obtained antibody recognized not only 15-HPETE-modified bovine serum albumin (BSA) but also 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified BSA. Glutaroyl-BSA adduct, which was prepared by the reaction of glutaric anhydride with protein, was also recognized by the antibody. The results revealed that the carboxyl terminus of lipid moiety in adducts was required for an appearance of the antigenicity. The cross-reactivity of phosphatidylcholine hydroperoxide-modified BSA (PCAOOH-BSA) with the antibody was examined. The antibody could not recognize the intact PCAOOH-BSA, whereas alkaline-treated modified BSA revealed the antigenicity. Furthermore, stearic acid at the 1 position in the phospholipid was liberated from the PCAOOH-BSA following treatment with 0.25 N NaOH. The result showed that the phospholipid moiety could be covalently bound to the protein molecule. The formation of esterified fatty acid-protein adduct during oxidation was confirmed using low-density lipoprotein (LDL). During oxidation of LDL by copper ion or 2,2'-azo-bis(2-amidinopropane)dihydrochloride, the formation of antigenic materials was observed in a time- or dose-dependent fashion. The antigenicity was significantly enhanced by the alkaline treatment on the oxidized LDL, suggesting that considerable amounts of oxidized esterified fatty acids can covalently react with apoprotein B-100 in oxidatively modified LDL.

Topics: Amidines; Antibody Specificity; Antigens; Enzyme-Linked Immunosorbent Assay; Esterification; Fatty Acids; Hemocyanins; Humans; Leukotrienes; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Lipoproteins, LDL; Oxidants; Proteins; Serum Albumin, Bovine; Thiobarbituric Acid Reactive Substances

The microsomal fraction of homogenate of garlic (Allium sativum L.) bulbs contains a divinyl ether synthase which catalyzes conversion of (9Z,11E,13S)-13-hydroperoxy-9, 11-octadecadienoic acid and (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatri eno ic acid into (9Z,11E,1'E,)-12-(1'-hexenyloxy)-9,11-dodecadienoic acid (etherolenic acid) and (9Z,11E,1'E,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dode cadienoic acid (etherolenic acid), respectively. Two isomers of etherolenic acid were isolated. As shown by NMR spectrometry, the double bond configurations of these compounds were (9E,11E,1'E) and (9Z,11Z,1'E). Experiments with linoleic acid (13R,S)-hydroperoxide demonstrated that the S enantiomer was a much better substrate for the divinyl ether synthase compared to the R enantiomer. Incubation of (9Z,11E,13S)-[18O2]hydroperoxy-9,11-octadecadienoic acid led to the formation of etherolenic acid which retained 18O in the ether oxygen. An intermediary role of an epoxyallylic cation in etherolenic acid biosynthesis is postulated.

Topics: Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Garlic; Isomerism; Leukotrienes; Linoleic Acids; Lipid Peroxides; Magnetic Resonance Spectroscopy; Microsomes; Oxidoreductases; Plant Proteins; Plants, Medicinal; Substrate Specificity

Topics: Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Garlic; Leukotrienes; Linoleic Acids; Lipid Peroxides; Molecular Structure; Oxidoreductases; Plant Proteins; Plants, Medicinal; Substrate Specificity

Mammalian 15-lipoxygenases undergo a characteristic self-inactivation. The oxygenation of a single methionine to methionine sulfoxide, by 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), was previously suggested as the cause of the inactivation of rabbit reticulocyte lipoxygenase. The site of oxygenation is potentially near the enzyme's active site; however, the specific location of the modified amino acid residue has not been identified. To determine which of the methionine residues is oxygenated, we inactivated both human and rabbit 15-lipoxygenases with 13-HPODE and sequentially denatured, reduced, carboxymethylated, and digested the enzymes with trypsin. The digested mixtures were analyzed by reverse-phase HPLC chromatography. Mass spectrometric analysis of each of the methionine-containing fractions enabled us to locate the peptide segments containing the oxidized methionine in both enzymes separately. Tandem electrospray mass spectrometry identified the oxidized methionine residues to be amino acid 590 in the human enzyme and 591 in the rabbit enzyme. To investigate the significance of this oxygenation, Met590 in human 15-lipoxygenase was substituted with leucine by site-directed mutagenesis. The mutant protein was inactivated by 13-HPODE, yet no oxygenated peptide or other modified peptide could be identified by HPLC-MS analysis. We also found that human 15-lipoxygenase was inactivated during arachidonate oxidation and by the reaction product 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), and no modified peptide was detected. Thus, methionine oxygenation is not essential for the inactivation of human 15-lipoxygenase. We suggest, however, that Met590 is an amino acid in the substrate binding pocket of human 15-lipoxygenase and interacts with the enzyme product 13-HPODE.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Base Sequence; Chromatography, High Pressure Liquid; Enzyme Activation; Humans; Iron; Leukotrienes; Linoleic Acids; Lipid Peroxides; Lipoxygenase Inhibitors; Mass Spectrometry; Methionine; Molecular Sequence Data; Oligodeoxyribonucleotides; Oxygen; Peptide Mapping; Rabbits; Trypsin

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) or 13-hydroperoxydodecadienoic acid (13-HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15-HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl-2 proto-oncogene were 6- to 8-fold more resistant to apoptotic killing by tumor necrosis factor-alpha, but only 0.4-fold more resistant to 15-HPETE. Thus, Bcl-2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

Topics: Apoptosis; Calcium; Cell Nucleus; Cells, Cultured; Dose-Response Relationship, Drug; Egtazic Acid; Humans; Leukotrienes; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; T-Lymphocytes

In a previous paper we reported that arachidonic acid (20:4(n-6] strongly enhances the endothelial cell synthesis of prostaglandin I3 (PGI3) from eicosapentaenoic acid (20:5(n-3], in stimulating the cyclooxygenase rather than the prostacyclin synthase (Bordet et al. (1986) Biochem. Biophys. Res. Commun. 135, 403-410). In the present study, endothelial cell monolayers were co-incubated with exogenous 20:5(n-3) or docosatetraenoic acid (22:4(n-6], and n-6 lipoxygenase products of 20:4(n-6) or linoleic acid (18:2(n-6], namely 15-HPETE and 13-HPOD, respectively. Prostaglandins or dihomoprostaglandins were then measured by gas chromatography-mass spectrometry. Both hydroperoxides, up to 20 microM, stimulated the cyclooxygenation of 20:5(n-3) and 22:4(n-6), in particular the formation of PGI3 and dihomo-PGI2, respectively. Higher concentrations inhibited prostacyclin synthetase. In contrast, the reduced products of hydroperoxides, 15-HETE and 13-HOD, failed to stimulate these cyclooxygenations, 13-HPOD appeared more potent than 15-HPETE and the cyclooxygenation of 22:4(n-6) seemed to require higher amounts of hydroperoxides to be efficiently metabolized than 20:5(n-3). These data suggest that prostacyclin potential of endothelium might be enhanced by raising the peroxide tone.

Topics: Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Endothelium, Vascular; Epoprostenol; Erucic Acids; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase; Mass Spectrometry; Prostaglandins F; Umbilical Veins

Soybean lipoxygenase is rapidly inactivated when incubated with arachidonic acid and any of several lipoxygenase inhibitors, including NDGA, the aminopyrazolines BW 755C and BW 540C, and the acetohydroxamic acid derivatives BW A4C and BW A137C. Little or no inactivation was found when the enzyme was incubated with substrate or with inhibitors alone. 15-HPETE was as effective as arachidonic acid in promoting inactivation, but linoleic acid and 13-HPOD were much less effective. The UV absorption at 235 nm, due to the conjugated diene in 15-HPETE or 13-HPOD, was rapidly destroyed in the presence of soybean lipoxygenase and inhibitor in a presumed pseudoperoxidase reaction. The products of the reaction between linoleic acid, BW A137C and soybean lipoxygenase have been partially characterized. A derivative of arachidonic acid is postulated to be the inactivating agent.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Arachidonic Acid; Arachidonic Acids; Benzeneacetamides; Ethanol; Ethylene Glycol; Ethylene Glycols; Glycine max; Hydroxamic Acids; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoxygenase Inhibitors; Masoprocol; Pyrazoles