12-LOX Activity Research Articles

Abstract 40: Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation

Introduction: Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis which is a major cause of myocardial infarction and stroke. Following activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation as inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Objectives: Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. Methods: To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured in human platelets including platelet aggregation, calcium mobilization, integrin activation, and phosphorylation of PKC substrates. Results: Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ 3 activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Additionally, direct PKC activation rescued deficits in phosphorylation of pleckstrin and other PKC substrates mediated by inhibition of 12-LOX. Finally, inhibition of 12-LOX had no effect on PKC-mediated aggregation indicating that 12-LOX is upstream of PKC. Conclusions: These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for anti-platelet therapy.

Salmonella Typhimurium induces HXA3 secretion by decreasing cellular levels and activity of the oxido‐reductase enzymes GPX 1 and PHGPX

Salmonella Typhimurium (S. Typhimurium)‐induced transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously reported that hepoxilin A3 (HXA3) mediates the final step in this process. HXA3 is a derivative of arachidonic acid formed from the enzymatic action of the 12‐lipoxygenase (12‐LOX) pathway. However, the molecular mechanisms regulating the activity/expression of 12‐LOX during S. Typhimurium‐induced intestinal inflammation remains unknown. Thus, we sought to decipher the mechanisms regulating 12‐LOX and increased HXA3 secretion during S. Typhimurium infection. We found that the protein expressions of Glutathione peroxidase (GPX) 1 and PHGPX (GPX 4), known to have an inhibitory effect on 12‐LOX activity, are significantly decreased during S. Typhimurium infection as well as an ~20% decrease in total cellular GPX activity. We also show that inhibition of total cellular GPX activity by iodoacatetate treatment, which increases 12‐LOX activity, resulted in a significant increase in neutrophil transmigration during S. Typhimurium infection. These results suggest that S. Typhimurium induces apical secretion of HXA3 by decreasing the expression of cellular GPX, which in turn leads to an increase in 12‐LOX activity. We are currently performing RNAi studies to determine which GPX isoform is responsible for these functions.

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.

Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation

Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.

Abstract 2902: Phosphorylation of 12 lipoxygenase at Tyrosines 19 and 614 regulates its activity during beta4 integrin signaling

Abstract 12-Lipoxygenase (12-LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE, an eicosanoid implicated in tumor angiogenesis, growth, and metastasis. We have demonstrated previously that 12-LOX interacts with integrin beta4 cytoplasmic domain by using yeast two hybrid system. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. Increased expression of 12-LOX and beta4 integrin have been documented in a number of carcinomas. The purpose of this study is to find out the mechanisms underlying the signaling pathways in 12-LOX mediated cancer progression. The phosphorylation of beta4 integrin, Src, and 12-LOX were analyzed using specific antibodies against phospho-tyrosine, phospho-beta4 integrin, Phospho-Src (pSrc). c-Src activity and association was determined in beta4 integrin immunoprecipitate. The HEK293 cells transiently transfected with wild type or dominant negative (DN) Src were used as in vitro models to study the regulation of 12-LOX activity. The 12-LOX product 12(S)-HETE was analyzed by using an analytical liquid chromatography-tandem mass spectrometry.12-LOX tyrosine phosphorylation sites mutants viz., 19, 295, and 614 were used to study the effect on its activity upon beta4 ligation. We show here that the cytoplasmic domain of beta4 integrin, can interact specifically with pSrc after beta4 integrin ligation in A431 cells. Herein we report that beta4 integrin ligation resulted in significant increase in 12(S)-HETE level in A431 cells, compared to that of unligated beta4 integrin. An increase in Src kinase activity was found in beta4 integrin ligation of A431 cells and inhibition of Src kinase by PP2 or DN Src significantly reduced 12(S)-HETE production. The ligation of beta4 integrin induces src activation, which resulted in tyrosine phosphorylation of 12-LOX. The residues that are important for 12-LOX activity and tyrosine phosphorylation were mapped to Y19 and Y614. We examined that 12-LOX mutants (Y19F and Y614F), showed 70% less enzymatic activity, compared to wild type 12-LOX. Furthermore, we showed that the 12-LOX activity modulated by these residues impacts migration in HUVEC cells. To our knowledge, this is the first report to show that Src kinase activity is required for beta4 integrin mediated regulation of 12-LOX, which may in turn regulate metastatic potential of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2011-2902

Abstract 2890: A novel interaction between 12-lipoxygenase and integrin subunit Beta4 plays a role in tumor migration, invasion and metastasis

Abstract This study has the potential to establish a new paradigm in adhesion-mediated control of eicosanoid production and could be relevant to tumor migration, invasion, and metastasis. 12-Lipoxygenase metabolizes arachidonic acid to form 12(S)-HETE, a metabolite known to play a significant role in the process of tumor-induced angiogenesis and metastasis. The role of 12-LOX in proliferation, survival, and motility of tumor cells as well as in angiogenesis has been described for a variety of cancers. Similarly Alpha6Beta4 integrin also plays a role in both angiogenesis and tumorigenesis in addition to having a role in the formation of hemidesmosomes (HD). Evidence suggests a correlation between 12-LOX and integrin Beta4 since they play a similar role in cancer progression. The physical interaction between 12-LOX and Beta4 was first identified by a yeast two-hybrid screen using 12-LOX as bait against a library made from A431 cells that are known to have endogenous expression of both proteins. We verified this interaction by co-immunoprecipitation from A431 cells, and recapitulated it in CHO and PC-3 cells ectopically expressing 12-LOX and the cytoplasmic tail of Beta4 integrin. Additionally our data shows that ligation or clustering of Alpha6Beta4 integrin by the natural ligand laminin or an antibody mimic of laminin, 3E1, caused an increase in translocation of 12-LOX from the cytosol to membrane and an increase in 12-LOX enzymatic activities as assayed by immunofluorescence and 12(S)-HETE measurements respectively. 3E1-stimulated A431 cells showed increased cell migration but there was 40% decrease in presence of 12-LOX inhibitor BMD122 which confirms the role of 12-LOX in cell invasion. Here we describe for the first time the interaction domains that unite Beta4 integrin and 12-LOX. We mapped the domains in the cytoplasmic tail of Beta4 critical for interacting with 12-LOX and found that the 1126-1157 domain in the cytoplasmic tail of Beta4 is important for binding of 12-LOX. Also we have shown the mutant fragment (1126-1315) interacts with 12-lipoxygenase in the cytosol and prevent its binding to native, full-length membrane-associated Beta4 and thereby prevents 12-LOX activation. This physical interaction has functional implications with regard to decreased cell survival and migration. Determination of the structural basis for protein-protein interaction may provide potential means to manipulate or block such interaction, which may yield therapeutic benefits. We propose that the results obtained from these studies will provide a novel, and important dimension to both integrin and eicosanoid biology. Acknowledgements: This project is supported by NIH: NCI, 2 R01 CA029997-17A1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2011-2890

Distinct role of nitric oxide and peroxynitrite in mediating oligodendrocyte toxicity in culture and in experimental autoimmune encephalomyelitis

Nitric oxide has been implicated in the pathogenesis of multiple sclerosis. However, it is still unclear whether nitric oxide plays a protective role or is deleterious. We have previously shown that peroxynitrite, a reaction product of nitric oxide and superoxide, is toxic to mature oligodendrocytes (OLs). The toxicity is mediated by intracellular zinc release, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), activation of 12-lipoxygenase (12-LOX) and the formation of reactive oxygen species (ROS). In this study, we found that the donors of nitric oxide, dipropylenetriamine NONOate (DPT NONOate) and diethylenetriamine NONOate (DETA NONOate), protected OLs from peroxynitrite or zinc-induced toxicity. The protective mechanisms appear to be attributable to their inhibition of peroxynitrite- or zinc-induced ERK1/2 phosphorylation and 12-LOX activation. In cultures of mature OLs exposed to lipopolysaccharide (LPS), induction of inducible nitric oxide synthase (iNOS) generated nitric oxide and rendered OLs resistant to peroxynitrite-induced toxicity. The protection was eliminated when 1400W, a specific inhibitor of iNOS, was co-applied with LPS. Using MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we found that nitrotyrosine immunoreactivity, an indicator of peroxynitrite formation, was increased in the spinal cord white matter, which correlated with the loss of mature OLs. Targeted gene deletion of the NADPH oxidase component gp91phox reduced clinical scores, the formation of nitrotyrosine and the loss of mature OLs. These results suggest that blocking the formation specifically of peroxynitrite, rather than nitric oxide, may be a protective strategy against oxidative stress induced toxicity to OLs.

Vitamin K prevents oxidative cell death by inhibiting activation of 12‐lipoxygenase in developing oligodendrocytes

Oxidative mechanisms of injury are important in many neurological disorders. Developing oligodendrocytes (pre-OLs) are particularly sensitive to oxidative stress-mediated injury. We previously demonstrated a novel function of phylloquinone (vitamin K(1)) and menaquinone 4 (MK-4; a major form of vitamin K2) in protecting pre-OLs and immature neurons against glutathione depletion-induced oxidative damage (Li et al. [ 2003] J. Neurosci. 23:5816-5826). Here we report that vitamin K at nanomolar concentrations prevents arachidonic acid-induced oxidative injury to pre-OLs through blocking the activation of 12-lipoxygenase (12-LOX). Arachidonic acid metabolism is a potential source for reactive oxygen species (ROS) generation during ischemia and reperfusion. Exposure of pre-OLs to arachidonic acid resulted in oxidative cell death in a concentration-dependent manner. Administration of vitamin K (K(1) and MK-4) completely prevented the toxicity. Consistent with our previous findings, inhibitors of 12-LOX abolished ROS production and cell death, indicating that activation of 12-LOX is a key event in arachidonic acid-induced pre-OL death. Vitamin K(1) and MK-4 significantly blocked 12-LOX activation and prevented ROS accumulation in pre-OLs challenged with arachidonic acid. However, vitamin K itself did not directly inhibit 12-LOX enzymatic activity when assayed with purified 12-LOX in vitro. These results suggest that vitamin K, or likely its metabolites, acts upstream of activation of 12-LOX in pre-OLs. In summary, our data indicate that vitamin K prevents oxidative cell death by blocking activation of 12-LOX and ROS generation.

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

Platelet 12-lipoxygenase Arg261Gln polymorphism: functional characterization and association with risk of esophageal squamous cell carcinoma in combination with COX-2 polymorphisms

Aberrant arachidonic acid metabolism by 12-lipoxygenase (12-LOX) and cyclooxygenase-2 (COX-2) has been implicated in human carcinogenesis. Inherited polymorphisms in 12-LOX and COX-2 contributed to differential expression or activity of these enzymes might confer interindividual susceptibility to cancer. To examine the functional significance of 12-LOX 261 Arg> Gln polymorphism and its association, alone and in combination with COX-2 -1195G > A and -765G > C polymorphisms, with risk of developing esophageal squamous cell carcinoma (ESCC). The platelet 12-LOX activity was measured by quantifying 12-HETE in the lipoxygenation reaction. Genotypes of 12-LOX261Arg>Gln and COX-2 -1195G>A and -765G>C polymorphisms were determined in a case-control study consisting of 1026 patients and 1270 controls. Associations with the risk of ESCC were estimated by logistic regression. Subjects with the 12-LOX Gln/Gln genotype had higher platelet 12-LOX activity (mean+/-SEM nmol/mg/min) than those with the Arg/Arg genotype (0.405+/-0.047 [n=10] versus 0.136+/-0.022 [n=6]; P=0.001). Genotyping data showed that the 12-LOX Gln/Gln genotype was associated with increased risk of developing ESCC (odds ratio [OR]=1.42, 95% confidence interval [CI]=1.12-1.81), compared with the Arg/Arg genotype adjusted for sex, age, and smoking. An increased risk of ESCC was also associated with the COX-2 -1195GA (OR=1.34, 95% CI=1.08-1.68; P=0.008), -1195AA (OR=1.72, 95% CI=1.35-2.20; P=<0.001), and -765GC (OR=2.24, 95% CI=1.59-3.16; P<0.001) genotypes. Furthermore, a multiplicative interaction between the 12-LOX Gln/Gln and COX-2 -1195AA or -765GC genotype in intensifying risk of ESCC was observed, with the ORs for the presence of both 12-LOX Gln/Gln and COX-2 -1195AA or -765GC genotypes being 3.21 (95% CI=1.93-5.34) and 3.33 (95% CI=1.59-6.98). A multiplicative interaction between the -765GC genotype and smoking was also evident (OR=4.45, 95% CI=2.71-7.29). These observations suggest that inherited polymorphisms in arachidonic acid-metabolizing enzymes, which result in heightened gene expression or enzymatic activity, may confer host susceptibility to ESCC.

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.

Intracellular Zinc Release and ERK Phosphorylation Are Required Upstream of 12-Lipoxygenase Activation in Peroxynitrite Toxicity to Mature Rat Oligodendrocytes

Peroxynitrite toxicity has been implicated in the pathogenesis of white matter injury. The mechanisms of peroxynitrite toxicity to oligodendrocytes (OLs), the major cell type of the white matter, are unknown. Using primary cultures of mature OLs that express myelin basic protein, we found that 3-morpholinosydnonimine, a peroxynitrite generator, caused toxicity to OLs. N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a zinc chelator, completely blocked peroxynitrite-induced toxicity. Use of FluoZin-3, a specific fluorescence zinc indicator, demonstrated the liberation of zinc from intracellular stores by peroxynitrite. Peroxynitrite caused the sequential activation of extracellular signal-regulated kinase 42/44 (ERK42/44), 12-lipoxygenase, and generation of reactive oxygen species, which were all dependent upon the intracellular release of zinc. The same cell death pathway was also activated when exogenous zinc was used. These results suggest that in addition to preventing the formation of peroxynitrite, useful strategies in preventing disease progression in pathologies in which peroxynitrite toxicity plays a critical role might include maintaining intracellular zinc homeostasis, blocking phosphorylation of ERK42/44, inhibiting activation of 12-lipoxygenase, and eliminating the accumulation of reactive oxygen species.

Zaltoprofen inhibits bradykinin-induced responses by blocking the activation of second messenger signaling cascades in rat dorsal root ganglion cells

Bradykinin interacts with the bradykinin B 2 receptor on dorsal root ganglion (DRG) neurons, setting off a series of reactions inside the cells that ultimately make the vanilloid receptor 1 more sensitive to a normal stimulus by activating various enzymes coupled with second messenger signaling cascades. Zaltoprofen, a propionic acid derivative non-steroidal anti-inflammatory drug (NSAID), was proved to inhibit bradykinin-induced pain responses in vivo experimental systems more potently than indomethacin or other NSAIDs, but the molecular mechanisms underlying its action are not yet fully understood. Currently it appears unlikely that zaltoprofen binds to specific sites on the protein of the bradykinin B 2 receptor, hence we have examined the effect of zaltoprofen on bradykinin-induced responses of adult DRG neurons to investigate possible interaction sites. Compared with several other NSAIDs, such as indomethacin, loxoprofen and diclofenac, zaltoprofen most potently inhibits bradykinin-enhancement of capsaicin-induced 45Ca 2+ uptake into DRG neurons. Zaltoprofen also significantly inhibits bradykinin-induced 12-lipoxygenase (12-LOX) activity and the slow bradykinin-induced onset of substance P release from DRG neurons. These data indicate zaltoprofen may produce its analgesic effects through the inhibition of bradykinin B 2 receptor-mediated bradykinin responses of not only cyclooxygenases (COXs) but also bradykinin induced 12-LOX inhibitors.

Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death.

Dual inhibition of cyclooxygenase-1 and 5-lipoxygenase by aerial part of Bupleurum fruticescens methanol extract

The effect of the methanol extract from aerial parts of Bupleurum fruticescens on the release of eicosanoids and hydrolytic enzymes was determined on in vitro cell systems . The extract had a significant effect on 5-lipoxygenase (5-LOX) activity, inhibiting both LTB 4 and 5( S)-HETE production with IC 50 values of 112 μg/ml and 95 μg/ml, respectively. At concentrations of 200 μg/ml, the extract also inhibited cyclooxygenase-1 (90%) and elastase activities (54%). The 12-LOX activity in intact platelets was not affected; a fact, which suggests that phospholipase A 2 (PLA 2) activity, is not modified by the extract.

Molecular Basis of Vitamin E Action: TOCOTRIENOL MODULATES 12-LIPOXYGENASE, A KEY MEDIATOR OF GLUTAMATE-INDUCED NEURODEGENERATION

Vitamin E is a generic term for tocopherols and tocotrienols. This work is based on our striking evidence that, in neuronal cells, nanomolar concentrations of alpha-tocotrienol, but not alpha-tocopherol, block glutamate-induced death by suppressing early activation of c-Src kinase (Sen, C. K., Khanna, S., Roy, S., and Packer, L. (2000) J. Biol. Chem. 275, 13049-13055). This study on HT4 and immature primary cortical neurons suggests a central role of 12-lipoxygenase (12-LOX) in executing glutamate-induced neurodegeneration. BL15, an inhibitor of 12-LOX, prevented glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOX-deficient mice were observed to be resistant to glutamate-induced death. In the presence of nanomolar alpha-tocotrienol, neurons were resistant to glutamate-, homocysteine-, and l-buthionine sulfoximine-induced toxicity. Long-term time-lapse imaging studies revealed that neurons and their axo-dendritic network are fairly motile under standard culture conditions. Such motility was arrested in response to glutamate challenge. Tocotrienol-treated primary neurons maintained healthy growth and motility even in the presence of excess glutamate. The study of 12-LOX activity and metabolism revealed that this key mediator of glutamate-induced neurodegeneration is subject to control by the nutrient alpha-tocotrienol. In silico docking studies indicated that alpha-tocotrienol may hinder the access of arachidonic acid to the catalytic site of 12-LOX by binding to the opening of a solvent cavity close to the active site. These findings lend further support to alpha-tocotrienol as a potent neuroprotective form of vitamin E.

Nitric Oxide Triggers the Toxicity due to Glutathione Depletion in Midbrain Cultures through 12-Lipoxygenase

Glutathione (GSH) depletion is the earliest biochemical alteration shown to date in brains of Parkinson's disease patients. However, data from animal models show that GSH depletion by itself is not sufficient to induce nigral degeneration. We have previously shown that non-toxic inhibition of GSH synthesis with l-buthionine-(S,R)-sulfoximine in primary midbrain cultures transforms a nitric oxide (NO) neurotrophic effect, selective for dopamine neurons, into a toxic effect with participation of guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG) (Canals, S., Casarejos, M. J., de Bernardo, S., Rodríguez-Martín, E., and Mena, M. A. (2001) J. Neurochem. 79, 1183-1195). Here we demonstrate that arachidonic acid (AA) metabolism through the 12-lipoxygenase (12-LOX) pathway is also central for this GSH-NO interaction. LOX inhibitors (nordihydroguaiaretic acid and baicalein), but not cyclooxygenase (indomethacin) or epoxygenase (clotrimazole) ones, prevent cell death in the culture, even when added 10 h after NO treatment. Furthermore, the addition of AA to GSH-depleted cultures precipitates a cell death process that is indistinguishable from that initiated by NO in its morphology, time course, and 12-LOX, GC, and PKG dependence. The first AA metabolite through the 12-LOX enzyme, 12-hydroperoxyeicosatetraenoic acid, induces cell death in the culture, and its toxicity is greatly enhanced by GSH depletion. In addition we show that if GSH synthesis inhibition persists for up to 4 days without any additional treatment, it will induce a cell death process that also depends on 12-LOX, GC, and PKG activation. In this study, therefore, we show that the signaling pathway AA/12-LOX/12-HPETE/GC/PKG may be important in several pathologies in which GSH decrease has been documented, such as Parkinson's disease. The potentiating effect of NO over such a signaling pathway may be of relevance as part of the cascade of events leading to and sustaining nerve cell death.

Growth factor-induced proliferation in corneal epithelial cells is mediated by 12(S)-HETE

Purpose. Previous studies in our laboratory have shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a product of 12-lipoxygenase (12-LOX) activity, is the predominant metabolite formed in rabbit corneas after injury. The present study was undertaken to investigate the effects of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF) on 12-LOX expression and activity. We also investigated whether 12(S)-HETE mediated the growth factor-induced proliferation of corneal epithelial cells. Methods. Rabbit corneas were stimulated with EGF, HGF, and KGF (10 ng ml −1) for different times. 12-LOX activity was assayed by incubating corneal microsomal preparations with radiolabeled arachidonic acid (AA) as substrate. For inhibitor studies, the microsomes were pretreated with 12-LOX-specific inhibitors baicalein (BC) or cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC). Lipid extracts were injected onto an Ultramex 5 μm C 18 column and radioactivity was monitored online by a Radiomatic Flo-One Beta detector. Stereochemical analysis of 12-HETE product was determined by chiral-phase HPLC. To evaluate the effects of growth factors on 12-LOX mRNA expression, mRNA was extracted at several time points (12, 24, 36, 48 hr) and subjected to real-time PCR. For 12-LOX protein expression, microsomal preparations from 24- and 48-hr incubations were analyzed by Western blot. In cell-proliferation studies, epithelial cells treated with EGF, HGF, or KGF for 24, 48, and 72 hr were measured with a CyQUANT cell-proliferation assay kit. To determine the role of growth factor-induced 12(S)-HETE synthesis on corneal epithelial cell proliferation, cells were pretreated with 12-LOX-specific inhibitors BC or CDC prior to growth-factor supplementation. Results. Stimulation with EGF, HGF, or KGF for 12 hr induced 12-LOX mRNA expression in rabbit corneal epithelial cells. This gene induction was followed by an increase in protein expression at 24 and 48 hr and a marked increase in 12(S)-HETE synthesis when compared to untreated controls. At 24-hr incubations, KGF showed a greater capacity than did EGF and HGF to stimulate microsomal 12-LOX activity, while at 48 hr 12(S)-HETE synthesis was significantly greater in EGF-treated cells as compared to that of HGF- and KGF-treated cells. Pretreatment with 12-LOX inhibitors blocked the growth factor-induced increase in 12(S)-HETE synthesis. Stimulation with growth factors or 12(S)-HETE for 24, 48, and 72 hr produced a significant increase in corneal epithelial proliferation, which was partially inhibited by pretreatment of cells with 12-LOX-specific inhibitors. Conclusion. These findings suggest that EGF, HGF, and KGF stimulate 12(S)-HETE production in rabbit corneal epithelial cells through gene induction of 12-LOX. Furthermore, 12(S)-HETE may play a role in regulating epithelial cell proliferation and the rate of corneal re-epithelialization following an injury.

Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase.

Arachidonate 12-lipoxygenase (LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (HETE), a bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. Alteration in 12-LOX expression or activity has been reported in various carcinomas including prostate carcinoma. However, little is known about the impact of the altered expression or activity of 12-LOX on tumor metastasis. In the present study, we examined whether or not an increase in 12-LOX expression in human prostate carcinoma cells can modulate their metastatic potential. We report that increased expression of 12-LOX in PC-3 cells caused a significant change in cell adhesiveness, spreading, motility, and invasiveness. Specifically 12-LOX transfected PC-3 cells were more adhesive toward vitronectin, type I and IV collagen, but not to fibronectin or laminin, than cells transfected with control vector. Increased spreading on vitronectin, fibronectin, collagen type I and IV also was observed in 12-LOX transfected PC-3 cells when compared to control PC-3 cells. The increased spreading of 12-LOX transfected PC-3 cells was blocked by treatment with 12-LOX inhibitors, baicalein and CDC. 12-LOX transfected PC-3 cells were more invasive through Matrigel than cells transfected with control vector. In vivo, tumor cell invasion to surrounding muscle or fat tissues was more frequent in nude mice bearing s.c. tumors from 12-LOX transfected PC-3 cells than in those from control vector transfected cells. When injected via the tail vein into SCID mice with implanted human bone fragments, there was an increase in tumor metastasis to human bone by 12-LOX transfected PC-3 cells in comparison to control vector transfected cells. Taken together, our data suggest that an increase in 12-LOX expression enhances the metastatic potential of human prostate cancer cells.

Kinetics of inhibition of leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.

Lipoxygenases (LOXs) are a ubiquitous family of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Their role in a diverse range of biological processes has prompted the development of a large number of lipoxygenase inhibitors of possible therapeutic and probative value. The isoform-selective inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was previously shown to inhibit leukocyte-type 12-LOX by a novel mechanism in which it binds to both the ferrous and ferric forms of the enzyme. The current study provides a detailed kinetic model of this inhibition. Nonlinear regression analysis of OPP's inhibition of arachidonic acid dioxygenation indicated mixed inhibition toward the ferric form of 12-LOX with apparent K(I) values in the low micromolar range: 2.0 +/- 0.2 microM for the free enzyme and 4.5 +/- 0.7 microM for the substrate-bound form of the enzyme. Rapid kinetic techniques allowed OPP's inhibition of the activation of the enzyme from the ferrous to the ferric form to be investigated. Titration of ferrous 12-LOX with OPP indicated that it bound to the ferrous form with an apparent K(I) value of 70 +/- 20 nM, suggesting a significantly higher affinity for the ferrous form than for the ferric form of the enzyme. Investigation of the LOX inhibitors nordihydroguaiaretic acid, N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, BWA137C, and eicosatetraynoic acid revealed that eicosatetraynoic acid also inhibited the activation of 12-LOX. These results demonstrate that LOX inhibitors are capable of binding to multiple forms of LOXs with high affinity and suggest that inhibition of enzyme activation may be an unrecognized mechanism of inhibition of additional LOX inhibitors.