5-LOX Activity Research Articles

Targeting 5-Lipoxygenase for Prevention and Treatment of Cancer

Aberrant arachidonic acid metabolism has been recognized as a dominant mechanism underlying the development and progression of a range of human cancers. Metabolism of arachidonic acid through the 5-lipoxygenase (5-Lox) pathway generates an array of highly reactive eicosanoids categorized into two distinct groups; the leukotrienes (LTs) and the 5-hydroxyeicosatetraenoids (5-HETEs). Evidence documenting prominent roles of these two groups of eicosanoids in the development and progression of cancer are accumulating rapidly both by in vitro cell culture experiments using cancer cells originating from many tissues, and by in vivo tumor model studies. Moreover, significantly higher levels of expression of 5-Lox has been observed in patient tumor specimens in grade-specific manner, and production of elevated levels of 5-Lox metabolites were observed in cancer cells compared to normal counterparts. Thus, 5-Lox has emerged as a new potential target for the prevention and treatment of cancer. Metabolites of 5-Lox are potent signaling molecules and elicit diverse biological activities that are involved in neoplastic transformation and progression, such as cell proliferation, invasion and motility, and apoptosis-resistance. Blocking 5-Lox activity by pharmacological agents will deprive cancer cells of the critical metabolites for their survival and growth, and thus may be effective in the prevention as well as treatment of cancer. Indeed some studies have already shown promise for using 5-Lox inhibitors for the prevention and treatment of cancers of the prostate, lung, pancreas, brain, esophagus, oral cavity, etc.

Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species

Continuous recruitment and inappropriate activity of platelets in the airways may contribute to airway remodeling, a characteristic feature of inflammatory airway diseases that includes increased proliferation of the smooth muscle. The aim of the present investigation was to examine the effect of platelets on proliferation of airway smooth muscle cells (ASMC) in culture and to determine the possible role of 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) in this context. ASMC obtained from guinea pigs were cultured and co-incubated with washed platelets for 24 hours. Thereafter, the proliferation was measured with the MTS-assay; the results were also verified by using thymidine incorporation, DNA measurements and manual counting. The interaction between platelets and ASMC was visualized with fluorescence microscopy. We found that platelets bind to the ASMC and the presence of platelets caused a significant dose-dependent increase in ASMC proliferation. Co-incubation of ASMC with platelets also increased ROS-production, detected by the fluorescent probe DCFDA. Furthermore, the platelet-induced proliferation was reduced in the presence of the NADPH-oxidase inhibitors DPI and apocynin. A possible role of 5-LOX in platelet-induced proliferation and ROS-generation was evaluated by using the 5-LOX inhibitor AA-861 and the PLA2-inhibitor ATK. The results showed that inhibition of these enzymes significantly reduced the platelet-induced proliferation. Moreover, Western blot analysis revealed that the ASMC but not the platelets express 5-LOX. In addition, our experiments revealed that the presence of AA-861 and ATK significantly inhibited the ROS-production generated upon co-incubation of platelets and ASMC. In conclusion, we show that platelets have a marked capacity to induce ASMC proliferation. Furthermore, our study indicates that the interaction between platelets and ASMC leads to activation of 5-LOX in the ASMC followed by an increased ROS-production, events resulting in enhanced ASMC proliferation. The new findings are of importance in understanding possible mechanisms contributing to airway remodeling.

In vitro and in vivo effects of Ranunculus peltatus subsp. baudotii methanol extract on models of eicosanoid production and contact dermatitis

Ranunculus (Crowfoot) species are numerous and they are all reputed to be counter-irritants and are used in several topical conditions. In order to study the pharmacological mechanisms of action underlying this popular use, a methanol extract of Ranunculus peltatus was tested in vitro in various assays involving eicosanoid and human elastase release by intact cells as well as in vivo, with models of delayed-type hypersensitivity (DTH) contact dermatitis. The extract proved to be a selective inhibitor of the cyclooxygenase-1 pathway, producing the total inhibition of 12-(S)-HHTrE release at 200 microg/mL, while leaving both 5-lipoxygenase and 12-lipoxygenase activities unaffected at the same dose. The n-hexane, chloroform and ethyl acetate fractions of the crude methanol extract inhibited LTB(4) release by intact rat peritoneal neutrophils, but more polar fractions were inactive and did not increase the 5-LOX activity as seen previously for extracts of other Ranunculus species. In the in vivo models, the methanol extract reduced the dinitrofluorobenzene (DNFB)-induced oedema by 40%, but failed to inhibit the oedema brought on by oxazolone. The results agree with the age-old assertion that Water Crowfoot species can be used as a topical antiinflammatory remedy without the prominent irritant action that accompanies the application of non-aquatic Ranunculus species.

Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect the arachidonic acid cascade

Nowadays, no data are available concerning the potential use of dual cyclooxygenase (COX)/5-lipoxygenase (LOX) inhibitors as anticancer agents in colon cancer treatment. Here, we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose- and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADP-ribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full-length p21(Bax) into p18(Bax), a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid (AA) cascade, as it blocked the activity of 5-LOX and COX enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A(2) (cPLA(2)), the release of unesterified AA from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the AA cascade, since neither exogenous prostaglandin E(2) and leukotriene B(4) addition, nor pharmacological inhibition of cPLA(2), was able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggests that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.

Effects of naturally occurring dihydroflavonols from Inula viscosa on inflammation and enzymes involved in the arachidonic acid metabolism

The anti-inflammatory properties of three flavanones isolated from Inula viscosa, sakuranetin, 7- O-methylaromadendrin, and 3-acetyl-7- O-methylaromadendrin, have been tested both in vitro and in vivo. Acute inflammation in vivo was induced by means of topical application of 12- O-tetradecanoylphorbol 13-acetate (TPA) to mouse ears or by subcutaneous injection of phospholipase A 2 (PLA 2) into mouse paws. The test compounds were evaluated in vitro for their effect on both the metabolism of arachidonic acid and on the release and/or activity of enzymes involved in the inflammatory response such as elastase, myeloperoxidase (MPO), and protein kinase C (PKC). The most active compounds in vivo against PLA 2-induced paw oedema were 7- O-methylaromadendrin (ED 50 = 8 mg/kg) and sakuranetin (ED 50 = 18 mg/kg). In contrast, the most potent compound against TPA-induced ear oedema was 3-acetyl-7- O-methylaromadendrin (ED 50 = 185 μg/ear), followed by sakuranetin (ED 50 = 205 μg/ear). In vitro, the latter compound was the most potent inhibitor of leukotriene (LT) B 4 production by peritoneal rat neutrophils (IC 50 = 9 μM) and it was also the only compound that directly inhibited the activity of 5-lipoxygenase (5-LOX). 3-Acetyl-7- O-methylaromadendrin also inhibited LTB 4 production (IC 50 = 15 μM), but had no effect on 5-LOX activity. The only flavanone that inhibited the secretory PLA 2 activity in vitro was 7- O-methylaromadendrin. This finding may partly explain the anti-inflammatory effect observed in vivo, although other mechanisms such as the inhibition of histamine release by mast cells may also be implicated. Sakuranetin at 100 μM was found to inhibit elastase release, although this result is partly due to direct inhibition of the enzyme itself. At the same concentration, 7- O-methylaromadendrin only affected the enzyme release. Finally, none of the flavanones exhibited any effect on MPO or PKC activities. Taken together, these findings indicate that sakuranetin may be a selective inhibitor of 5-LOX.

Inhibitory Activities and Attenuated Expressions of 5-LOX with Red Ginseng in Helicobacter pylori-Infected Gastric Epithelial Cells

Our recent studies documented that red ginseng extract (RGE, isolates from steamed and dried Panax ginseng, C.A. Meyer) can inhibit Helicobacter pylori-induced mitogen-activated protein kinase (MAPK) signaling with repressing either nuclear factor (NF)-kappaB-DNA binding activity or releases of IL-8 and COX-2 in gastric epithelial cells (Dig Dis Sci 50:1218-1227, 2005). We extended the experiment to prove whether RGE influences 5-lipoxygenase (5-LOX) pathway, thereby suppressing the biosynthesis of 5(S)-HETE. The 5-LOX enzyme activities were measured by thin layer chromatography using (14)C-labeled arachidonic acid (AA) and quantified by reverse phase-high performance liquid chromatography in human gastric adenocarcinoma (AGS) cells cocultured with H pylori (ATCC 43504 strain) with or without pretreatment of RGE. Western blotting analyses for MAPK signaling and 5-LOX, reverse transcriptase polymerase chain reaction for interleukin-8, and electrophoretic mobility shift assay for NF-kappaB-DNA binding were done, respectively. H pylori infection increased exclusively 5-LOX enzyme activity and RGE inhibited H pylori-stimulated 5-LOX activity, resulting in suppression of 5(S)-HETE generations from AA. RGE inactivated c-jun phosphorylation and repressed redox-sensitive transcriptional activation, led to reduced expression of IL-8 and 5-LOX mRNA in gastric mucosal cells, of which action was very similar to known LOX inhibitor, 200 mumol of geraniin. RGE could be phytoceutical against H pylori infection-associated gastric inflammation through its LOX-inhibiting actions, inhibitory 5-LOX enzyme activity, and attenuating its expression.

Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A 4 (LTA 4) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC 50 values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5( S)-hydroperoxyeicosatetraenoic acid (5-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX.

Induction of apoptosis in an estrogen-responsive mouse Leydig tumor cell by leukotriene

For estrogen-responsive B-1F cells, established from estrogen-responsive mouse Leydig cell tumor, it has been reported that the 5-lipoxygenase (5-LOX) metabolic pathway appears to be associated with cell growth. The addition of 5-LOX inhibitor 2-(12-hydroxydodeca-5,10-diyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861) to the medium resulted in a dose-dependent increase in cell yield as described previously. When the growth of the palpable tumors was measured, AA861 had stimulated in vivo tumor growth in adult male mouse inoculated B-1F cells. The effects of AA861 and 17beta-estradiol (E2) on the contents of various arachidonic acid metabolites in B-1F cells and their conditioned medium were examined. Although AA861 and E2 decreased the contents of leukotrienes (LTs), the two did not significantly change those of prostaglandins, thromboxan, prostacyclin, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE. In immunohistochemical study B-1F cells show positive staining for 5-LOX in the E2-depleted condition, while E2 decreased the expression of 5-LOX. The decrease of the intensities of 79-kDa 5-LOX protein and 403-bp RT-PCR product bands was observed. The growth of Morpholino-anti oligo delivered B-1F cells was higher than that of Standard control oligo delivered cells. The delivery of Morpholino-anti oligo into B-1F cells caused the decrease of contents of LTs and 5-HETE in the cells and medium, and the reduction of 5-LOX activity. When LTD4 was added in the culture medium, the increasing concentrations of LTD4 resulted in a significant inhibition of cell yields of E2-treated B-1F cells. Morphological changes such as nuclear condensation and fragmentation, and DNA ladder pattern were demonstrated in E2-stimulated B-1F cells treated with LTD4 as well as in control cells cultured in the basal medium. These results implicate that 5-LOX at least plays an important role in the growth of B-1F cells and LD4 induces the apoptosis of B-1F cells.

Baicalin attenuates oxygen–glucose deprivation-induced injury via inhibiting NMDA receptor-mediated 5-lipoxygenase activation in rat cortical neurons

The flavonoid baicalin exerts neuroprotective effects but the mechanism is not fully clarified. On the other hand, 5-lipoxygenase (5-LOX) activation is involved in ischemic neuronal injury. In this study, we determined whether baicalin protects rat cortical neurons against oxygen–glucose deprivation (OGD)-induced ischemic-like injury, if so, whether this effect relates to 5-LOX activation. After the neurons were injured by 1.5-h OGD and 24-h recovery, their viability reduced and necrosis occurred; these injuries were attenuated by baicalin (1 and 5 μM) as well as caffeic acid (a 5-LOX inhibitor, 5 and 25 μM) and MK-801 (an NMDA receptor antagonist, 1–10 μM). OGD-induced 5-LOX translocation to the nuclear envelope as detected by immunoblotting, immunocytochemistry and 5-LOX transfection; this translocation was inhibited by baicalin (5 μM) and MK-801 (5 μM) but not by caffeic acid (5 μM). During 0.5- to 2-h recovery after 1.5-h OGD, the production of 5-LOX metabolites, cysteinyl leukotrienes, was increased; this increased production was inhibited by baicalin and MK-801, while both the increased and baseline production were inhibited by caffeic acid. In addition baicalin and MK-801, not caffeic acid, inhibited glutamate-induced elevation of intracellular calcium. These results indicate that baicalin attenuates ischemic-like injury in the neurons, and this effect partly relates to the inhibition of NMDA receptor-mediated 5-LOX activation.

Licofelone, a Balanced Inhibitor of Cyclooxygenase and 5-Lipoxygenase, Reduces Inflammation in a Rabbit Model of Atherosclerosis

Licofelone, a dual anti-inflammatory drug that inhibits 5-lipoxygenase (LOX) and cyclooxygenase (COX) enzymes, may have a better cardiovascular profile that cycloxygenase-2 inhibitors due to cycloxygenase-1 blockade-mediated antithrombotic effect and a better gastrointestinal tolerability. We examined the anti-inflammatory effect of licofelone on atherosclerotic lesions as well as in isolated neutrophils from whole blood of rabbits compared with a selective inhibitor of COX-2, rofecoxib. We also assessed the antithrombotic effect of licofelone in rabbit platelet-rich plasma. For this purpose, 30 rabbits underwent injury of femoral arteries, and they were randomized to receive 10 mg/kg/day licofelone or 5 mg/kg/day rofecoxib or no treatment during 4 weeks with atherogenic diet in all cases. Ten healthy rabbits were used as controls. Neutrophils and platelets were isolated from peripheral blood of rabbits for ex vivo studies. Licofelone reduced intima/media ratio in injured arteries, the macrophages infiltration in the neointimal area, monocyte chemoattractant protein-1 (MCP-1) gene expression, and the activation of nuclear factor-kappaB in rabbit atheroma. Moreover, licofelone inhibited COX-2 and 5-LOX protein expression in vascular lesions. Rofecoxib only diminished COX-2 protein expression and MCP-1 gene expression in vascular atheroma. Prostaglandin E(2) in rabbit plasma was attenuated by both drugs. Licofelone almost abolished 5-LOX activity by inhibiting leukotriene B4 generation in rabbit neutrophils and prevented platelet thromboxane B2 production from whole blood. Licofelone reduces neointimal formation and inflammation in an atherosclerotic rabbit model more markedly than rofecoxib. This effect, together with the antiplatelet activity of licofelone, suggests that this drug may have a favorable cardiovascular profile.

Design, synthesis, and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-(aryl)acrylic acids as dual inhibitors of cyclooxygenases and lipoxygenases

A group of (E)-3-(4-methanesulfonylphenyl)acrylic acids possessing a substituted-phenyl ring (4-H, 4-Br, 3-Br, 4-F, 4-OH, 4-OMe, 4-OAc, and 4-NHAc) attached to the acrylic acid C-2 position were prepared using a stereospecific Perkin condensation reaction. A related group of compounds having 4- and 3-(4-isopropyloxyphenyl)phenyl, 4- and 3-(2,4-difluorophenyl)phenyl and 4- and 3-(4-methanesulfonylphenyl)phenyl substituents attached to the acrylic acid C-2 position were also synthesized, using a palladium-catalyzed Suzuki cross-coupling reaction, for evaluation as dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors. (E)-2-(3-Bromophenyl)-3-(4-methanesulfonylphenyl)acrylic acid (9h), and compounds having 4-(4-isopropyloxyphenyl-, 2,4-difluorophenyl-, or 4-methylsulfonylphenyl)phenyl moieties at the acrylic acid C-2 position (11a,b,d), were particularly potent COX-2 inhibitors with a high COX-2 selectivity index (COX-2 IC50≈0.32μM, SI>316) similar to the reference drug rofecoxib (COX-2 IC50=0.5μM, SI>200). Acrylic acid analogs with a C-2 4-hydoxyphenyl (9d, IC50=0.56μM), or 4-acetamidophenyl (9g, IC50=0.11μM), substituent were particularly potent 5-LOX inhibitors that may participate in an additional specific hydrogen-bonding interaction. A number of compounds possessing a C-2 substituted-phenyl moiety (4-Br, 4-F, and 4-OH), or a 4- or 3-(2,4-difluorophenyl)phenyl moiety, showed potent 15-LOX inhibitory activity (IC50 values in the 0.31–0.49μM range) relative to the reference drug luteolin (IC50=3.2μM). Compounds having a C-2 4-acetylaminophenyl, or 4-(2,4-difluorophenyl)phenyl, moiety exhibited anti-inflammatory activities that were equipotent to aspirin, but less than that of celecoxib. The structure–activity data acquired indicate the acrylic acid moiety constitutes a suitable scaffold (template) to design novel acyclic dual inhibitors of the COX and LOX isozymes.

Molecular modeling of the non-covalent binding of the dietary tomato carotenoids lycopene and lycophyll, and selected oxidative metabolites with 5-lipoxygenase

Numerous studies on human prostate cancer cell lines indicate a role for arachidonic acid (AA) and its oxidative metabolites in prostate cancer proliferation. The metabolism of AA by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression, and metastasis. In particular, products of the 5-LOX pathway (including 5-HETE and 5-oxo-EET) have been implicated as potential ‘survival factors’ that may confer escape after androgen withdrawal therapy through fatty-acid (i.e., AA) drive. Potent natural dietary antioxidant compounds such as lycopene and lycophyll, with tissue tropism for human prostate, have been shown to be effective in ameliorating generalized oxidative stress at the DNA level. Suppressing the 5-LOX axis pharmacologically is also a promising avenue for intervention in human patients. The recently recognized direct interaction of the astaxanthin-based soft-drug Cardax™ to human 5-LOX with molecular modeling, and the downregulation of both 5-HETE and 5-oxo-EET in vivo in a murine peritonitis model, suggest that other important dietary carotenoids may share this enzyme regulatory feature. In the current study, the acyclic tomato carotene lycopene (in all- trans and 5- cis isomeric configurations) and its natural dihydroxy analog lycophyll (also present in tomato fruit) were subjected to molecular modeling calculations in order to investigate their predicted binding interaction(s) with human 5-LOX. Two bioactive oxidative metabolites of lycopene (4-methyl-8-oxo-2,4,6-nonatrienal and 2,7,11-trimethyl-tetradecahexaene-1,14-dial) were also investigated. A homology model of 5-LOX was constructed using 8-LOX and 15-LOX structures as templates. The model was validated by calculating the binding energy of Cardax™ to 5-LOX, which was demonstrated to be in good agreement with the published experimental data. Blind docking calculations were carried out in order to explore the possible binding sites of the carotenoids on 5-LOX, followed by focused docking to more accurately calculate the predicted energy of binding. Lycopene and lycophyll were predicted to bind with high affinity in the superficial cleft at the interface of the β-barrel and the catalytic domain of 5-LOX (the ‘cleavage site’). Carotenoid binding at this cleavage site provides the structural rationale by which polyenic compounds could modify the 5-LOX enzymatic function via an allosteric mechanism, or by radical scavenging in proximity to the active center. In addition, the two bioactive metabolites of lycopene were predicted to bind to the catalytic site with high affinity—therefore suggesting potential direct competitive inhibition of 5-LOX activity that should be shared by both lycopene and lycophyll after in vivo supplementation, particularly in the case of the dial metabolite.

N-3 PUFAs reduce oxidative stress in ESRD patients on maintenance HD by inhibiting 5-lipoxygenase activity

Reactive oxygen species formation and release of pro-inflammatory/pro-atherogenic cytokines, that is, interleukin 1-beta and tumor necrosis factor-alpha, need the activation of the arachidonic acid cascade via the enzyme 5-lipoxygenase (5-Lox). 5-Lox activity and expression are significantly increased in peripheral blood mononuclear cells (PBMCs) of end-stage renal disease (ESRD) patients on maintenance hemodialysis (HD). Diets enriched with n-3 polyunsaturated fatty acids (PUFAs) (omega-3) have been associated to a lower incidence of coronary heart disease (CHD) and a reduction in atherosclerotic lesions. Omega-3 may interfere with the arachidonic acid cascade by inhibiting 5-Lox. Lipid peroxidation, leukotriene B(4) (LTB(4)) production, 5-Lox activity and expression were investigated in PBMC isolated from ESRD patients under maintenance HD before and after a 3-month oral supplementation with omega-3 at a daily dose of 2700 mg of n-3 PUFAs at the average eicosapentaenoic acid/docosaesaenoic acid ratio of 1.2 and finally after a further 3-month washout with no omega-3 supplementation. PBMCs from non-uremic volunteers were also investigated for comparison to normal parameters. Administration of omega-3 reduced significantly lipid peroxidation (P < 0.0001), LTB(4) synthesis (P < 0.0001) and 5-Lox activity (P < 0.0001), with no effect on 5-Lox protein expression. After the 3-month washout, all parameters were comparable to those observed before treatment. Our results resemble those obtained after oral administration of vitamin E and are consistent with a reversible, dose-dependent inhibition of 5-Lox by omega-3. Upregulation of 5-Lox may also be related to the increased mitochondrial damage and apoptosis of PBMCs observed in ESRD patients compared to non-uremic controls. Omega-3 may thus protect PBMCs of ESRD patients against oxidative stress.

Minocycline protects PC12 cells against NMDA-induced injury via inhibiting 5-lipoxygenase activation

Recently, we have reported that minocycline, a semi-synthetic tetracycline with neuroprotective effects, inhibits the in vitro ischemic-like injury and 5-lipoxygenase (5-LOX) activation in PC12 cells. In the present study, we further determined whether minocycline protects PC12 cells from excitotoxicity via inhibiting 5-LOX activation. We used N-methyl- d-aspartate (NMDA, 200 μM) to induce early (exposure for 6 h) and delayed (exposure for 6 h followed by 24 h recovery) injuries. We found that NMDA receptor antagonist ketamine, 5-LOX inhibitor caffeic acid and minocycline concentration dependently attenuated NMDA-induced early and delayed cell injuries (viability reduction and cell death). However, only ketamine (1 μM) inhibited NMDA-evoked elevation of intracellular calcium. In addition, immunohistochemical analysis showed that NMDA induced 5-LOX translocation to the nuclear membrane after 1- to 6-h exposure which was confirmed by Western blotting, indicating that 5-LOX was activated. Ketamine, caffeic acid and minocycline (each at 1 μM) inhibited 5-LOX translocation after early injury. After delayed injury, PC12 cells were shrunk, and 5-LOX was translocated to the nuclei and nuclear membrane; ketamine, caffeic acid and minocycline inhibited both cell shrinking and 5-LOX translocation. As a control, 12-LOX inhibitor baicalein showed a weak effect on cell viability and death, but no effect on 5-LOX translocation. Therefore, we conclude that the protective effect of minocycline on NMDA-induced injury is partly mediated by inhibiting 5-LOX activation.

Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase

Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A (2) inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy- a -cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1 - 2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis.

Involvement of arachidonic acid metabolism and EGF receptor in neurotensin-induced prostate cancer PC3 cell growth

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [ 3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca 2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.

Identification and characterization of an arachidonate 11 R-lipoxygenase

11 R-Lipoxygenase (11 R-LOX) activity has been detected in several marine invertebrates, and here we report the first cloning and expression of the enzyme. The cDNA encoding a protein of 77 kDa was isolated by RT-PCR from the soft coral Gersemia fruticosa and expressed in Escherichia coli. Incubations of recombinant enzyme with arachidonic acid yielded a single product, identified by RP-HPLC, GC–MS, and chiral phase-HPLC as 11 R-hydroperoxyeicosatetraenoic acid. Other C18, C20, and C22 substrates are also oxygenated, preferentially at the ω10 position. Significantly, both Ca 2+-ions and a membrane fraction are required for catalytic activity. Calcium effects translocation of the soluble 11 R-LOX to the membrane and this association is reversible by Ca 2+ chelation. The enzyme sequence contains some conserved amino acids implicated in calcium activation of mammalian 5-LOX, and with its obligate requirement for membrane interaction the 11 R-LOX may thus provide a new model for further analysis of this aspect of lipoxygenase activation.

Inhibition of COXs and 5-LOX and activation of PPARs by Australian Clematis species (Ranunculaceae)

The species of Clematis (Ranunculaceae) have been traditionally used for inflammatory conditions by indigenous Australians. We have previously reported that the ethanol extract of Clematis pickeringii inhibited COX-1. In this study, we examined the ethanol extracts and fractions of three Clematis species, Clematis pickeringii, Clematis glycinoides and Clematis microphylla, on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). We further examined the activating effects on the protein expression of peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) in HepG2 cells. The ethanol extracts of three Clematis species inhibited the activities of COX-1, COX-2 and 5-LOX in the different extents. The stem extract of Clematis pickeringii showed the highest inhibitory activities among the three species on COX-1, COX-2 and 5-LOX with the IC50 values of 73.5, 101.2 and 29.3μg/mL. One of its fractions also significantly elevated PPARα expression by 173, 280 and 435% and PPARγ expression by 140, 228 and 296% at 4, 8 and 16μg/mL, respectively.

NF-κB, Nrf2, and HO-1 Interplay in Redox-Regulated VCAM-1 Expression

Oxidative processes are considered to play a crucial role in the induction of cell adhesion molecules, a key event in inflammatory processes. We recently reported on an unexpected unidirectional effect of an overexpressed antioxidant [phospholipid hydroperoxide glutathione peroxidase (PHGPx)] and an oxidant [15-lipoxygenase (15-LOX)] enzyme on the basal and interleukin-1 induced vascular cell adhesion molecule-1 (VCAM-1) expression in vascular smooth muscle cells (SMC). Both enzymes inhibited VCAM-1 expression and reduced the cellular protein thiol content, thus, both exerting an oxidant effect. We now investigated whether transcription factors known to be regulated by oxidation, i.e., the nuclear factor-kappaB and the Keap1/Nrf2 system, were affected in our set of cells: SMC, SMC(PHGPx), and SMC(LOX), as well as ECV and ECV(PHGPx). PHGPx and 15-LOX inhibited nuclear factor-kappaB activation most efficiently at a step downstream of DNA binding, which explains their inhibitory effect on VCAM-1 expression. Both enzymes up-regulated endogenous heme oxygenase-1 most probably via activation of Nrf2. Transfected Nrf2 strongly inhibited VCAM-1 promoter activity, which could be reversed by cotransfection with Keap1. The key player in this complex cross-talk obviously is heme oxygenase-1, which is known to be induced by oxidant-activated Nrf2. The moderate oxidative stress initiated by enhanced PHGPx or 15-LOX activity appears to induce a defense system that diminishes the response to further proinflammatory stimuli.

Contributory role of 5-lipoxygenase and its association with angiogenesis in the promotion of inflammation-associated colonic tumorigenesis by cigarette smoking.

Our previous study shows that cigarette smoking can promote inflammation-associated adenoma formation in the mouse colon, but the underlying mechanism remains unknown. Several studies suggest that there is a link between 5-lipoxygenase (5-LOX) and carcinogenesis in humans and animals. In the present study, we aims to investigate whether the promoting action of cigarette smoke on inflammation-associated colon cancer formation is associated with 5-LOX activation in mice. Results showed that exposure to the mainstream smoke of unfiltered cigarettes enhanced the 5-LOX protein expression in the inflammation-associated colonic adenomas. It was accompanied with an up-regulation of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF). Both are the key angiogenic factors for tumorigenesis. 5-LOX inhibitors decreased the incidence of colonic adenoma formation and reduced angiogenesis, MMP-2 activity and VEGF protein expression in the colons of these animals. Taken together, these results strongly suggest that cigarette smoke can induce 5-LOX expression which plays an important role in activation of MMP-2 and VEGF to induce angiogenic process and promotion of inflammation-associated adenoma formation in mice.