Antineoplastic effects of 15(S)-hydroxyeicosatetraenoic acid and 13-S-hydroxyoctadecadienoic acid in non-small cell lung cancer.

BACKGROUND: Previous studies have shown that the levels of 15-lipoxygenase 1 (15-LOX-1) and 15-LOX-2 as well as their metabolites 13-S-hydroxyoctadecadienoic acid (13(S)-HODE) and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) are significantly reduced in smokers with non-small cell lung carcinoma (NSCLC). Furthermore, animal model experiments have indicated that the reduction of these molecules occurs before the establishment of cigarette smoking carcinogen-induced lung tumors, and this suggests roles in lung tumorigenesis. However, the functions of these molecules remain unknown in NSCLC. METHODS: NSCLC cells were treated with exogenous 13(S)-HODE and 15(S)-HETE, and then the ways in which they affected cell function were examined. 15-LOX-1 and 15-LOX-2 were also overexpressed in tumor cells to restore these 2 enzymes to generate endogenous 13(S)-HODE and 15(S)-HETE before cell function was assessed. RESULTS: The application of exogenous 13(S)-HODE and 15(S)-HETE significantly enhanced the activity of peroxisome proliferator-activated receptor γ (PPARγ), inhibited cell proliferation, induced apoptosis, and activated caspases 9 and 3. The overexpression of 15-LOX-1 and 15-LOX-2 obviously promoted the endogenous levels of 13(S)-HODE and 15(S)-HETE, which were demonstrated to be more effective in the inhibition of NSCLC. CONCLUSIONS: This study has demonstrated that exogenous or endogenous 13(S)-HODE and 15(S)-HETE can functionally inhibit NSCLC, likely by activating PPARγ. The restoration of 15-LOX activity to increase the production of endogenous 15(S)-HETE and 13(S)-HODE may offer a novel research direction for molecular targeting treatment of smoking-related NSCLC. This strategy can potentially avoid side effects associated with the application of synthetic PPARγ ligands.

Apolipoprotein A-I mimetic peptide 4F rescues pulmonary hypertension by inducing microRNA-193-3p.

BACKGROUND: Pulmonary arterial hypertension is a chronic lung disease associated with severe pulmonary vascular changes. A pathogenic role of oxidized lipids such as hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids is well established in vascular disease. Apolipoprotein A-I mimetic peptides, including 4F, have been reported to reduce levels of these oxidized lipids and improve vascular disease. However, the role of oxidized lipids in the progression of pulmonary arterial hypertension and the therapeutic action of 4F in pulmonary arterial hypertension are not well established. METHODS AND RESULTS: We studied 2 different rodent models of pulmonary hypertension (PH): a monocrotaline rat model and a hypoxia mouse model. Plasma levels of hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids were significantly elevated in PH. 4F treatment reduced these levels and rescued preexisting PH in both models. MicroRNA analysis revealed that microRNA-193-3p (miR193) was significantly downregulated in the lung tissue and serum from both patients with pulmonary arterial hypertension and rodents with PH. In vivo miR193 overexpression in the lungs rescued preexisting PH and resulted in downregulation of lipoxygenases and insulin-like growth factor-1 receptor. 4F restored PH-induced miR193 expression via transcription factor retinoid X receptor α. CONCLUSIONS: These studies establish the importance of microRNAs as downstream effectors of an apolipoprotein A-I mimetic peptide in the rescue of PH and suggest that treatment with apolipoprotein A-I mimetic peptides or miR193 may have therapeutic value.

5,14-HEDGE, a 20-HETE mimetic, reverses hypotension and improves survival in a rodent model of septic shock: contribution of soluble epoxide hydrolase, CYP2C23, MEK1/ERK1/2/IKKß/IκB-α/NF-κB pathway, and proinflammatory cytokine formation.

We have previously demonstrated that a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), restores vascular reactivity, blood pressure, and heart rate in endotoxemic rats. The aim of this study was to determine whether decreased renal expression and activity of soluble epoxide hydrolase (sEH), MEK1, ERK1/2, IKKß, IκB-α, and NF-κB as well as systemic and renal proinflammatory cytokine production associated with increased expression and activity of CYP2C23 contributes to the effect of 5,14-HEDGE to prevent hypotension, tachycardia, inflammation, and mortality in response to systemic administration of lipopolysaccharide (LPS). Blood pressure fell by 33 mmHg and heart rate rose by 57 beats/min in LPS (10 mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of sEH associated with a decrease in CYP2C23 mRNA and protein expression. Increased activity of sEH and p-MEK1, p-ERK1/2, p-IκB-α, NF-κB, and p-NF-κB protein levels as well as TNF-α and IL-8 production by LPS were also associated with a decreased activity of AA epoxygenases. These effects of LPS were prevented by 5,14-HEDGE (30 mg/kg, s.c.; 1 h after LPS). Treatment of endotoxemic mice with 5,14-HEDGE also raised the survival rate of animals from 84% to 98%. A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, 20-HEDE (30 mg/kg, s.c.; 1 h after LPS) prevented the effects of 5,14-HEDGE on blood pressure, heart rate, expression and/or activity of sEH, CYP2C23, and ERK1/2 as well as TNF-α and IL-8 levels in rats treated with LPS. These results suggest that decreased expression and/or activity of sEH and MEK1/ERK1/2/IKKß/IκB-α/NF-κB pathway as well as proinflammatory cytokine production associated with increased CYP2C23 expression and antiinflammatory mediator formation participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, inflammation, and mortality in the rodent model of septic shock.

Endogenously produced 20-HETE modulates myogenic and TGF response in microperfused afferent arterioles.

Previous studies have indicated that 20-hydroxyeicosatetraeonic acid (20-HETE) modulates vascular tone in large cerebral and renal arteries through inhibition of the large conductance, calcium sensitive potassium (BK) channel activity. However, the role of 20-HETE in modulating tubuloglomerular feedback (TGF) and the myogenic response in the afferent arteriole (Af-Art) is unknown. The present study examined the effects of inhibitors of the synthesis and action of 20-HETE on the myogenic and TGF responses of isolated rabbit and mouse Af-Arts. Luminal diameter decreased by 9.2±0.5% in mice and 8.9±1.3% in rabbit Af-Art when the perfusion pressure was increased from 60 to 120 mmHg. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 µM), or a selective 20-HETE antagonist, 6, 15-20-hydroxyeicosadienoic acid (6, 15-20-HEDE, 10 µM) completely blocked the myogenic response of both rabbit and mouse Af-Art, while addition of 5, 14-20-HEDE (10 µM), a 20-HETE agonist, restored the myogenic response in vessels treated with HET0016. Increases in NaCl concentration from 10 to 80 mM of the solution perfusing the macula densa constricted the Af-Art of rabbits by 6.0±1.4 µm (n=5). Addition of a 20-HETE agonist to the tubular perfusate potentiated the TGF-mediated vasoconstrictor response. This response was blocked by addition of a 20-HETE antagonist (6, 15-20-HEDE, 10 µM) to the vascular perfusate. These studies indicate that locally produced 20-HETE plays an important role in modulating the myogenic and TGF responsiveness of the Af-Art and may help explain how deficiencies in the renal formation of 20-HETE could promote the development of hypertension induced glomerular injury.

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that that contributes to infarct size following focal cerebral ischemia. However, little is known about the role of 20-HETE in global cerebral ischemia or neonatal hypoxia-ischemia (H-I). The present study examined the effects of blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-n-butyl-2-methylphenyl) formamidine (HET0016) in neonatal piglets after H-I to determine if it protects highly vulnerable striatal neurons. Administration of HET0016 after H-I improved early neurological recovery and protected neurons in putamen after 4 days of recovery. HET0016 had no significant effect on cerebral blood flow. cytochrome P450 4A immunoreactivity was detected in putamen neurons, and direct infusion of 20-HETE in the putamen increased phosphorylation of Na(+), K(+) -ATPase and NMDA receptor NR1 subunit selectively at protein kinase C-sensitive sites but not at protein kinase A-sensitive sites. HET0016 selectively inhibited the H-I induced phosphorylation at these same sites at 3 h of recovery and improved Na(+), K(+) -ATPase activity. At 3 h, HET0016 also suppressed H-I induced extracellular signal-regulated kinase 1/2 activation and protein markers of nitrosative and oxidative stress. Thus, 20-HETE can exert direct effects on key proteins involved in neuronal excitotoxicity in vivo and contributes to neurodegeneration after global cerebral ischemia in immature brain.

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation.

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BK(Ca) channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 microM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentrations. Together, these results indicate that 20-HETE-dependent activation of BK(Ca) channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle.

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.

Lipoxins and aspirin-triggered lipoxins in airway responses.

Here, we have demonstrated potent inhibition of allergen-mediated pulmonary inflammation and airway hyper-responsiveness by a novel dual-pronged action of LX's. Moreover, these results indicate that LX's play pivotal and previously unappreciated roles in regulating allergy and pulmonary inflammation.

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection.

The production of interleukin (IL)-12 is critical for the development of interferon (IFN)-gamma-dependent resistance to Toxoplasma gondii. Nevertheless, when this response is dysregulated, such as occurs in the absence of IL-10, the uncontrolled inflammation that results can have lethal consequences for the host. Recently, we demonstrated that lipoxin (LX)A(4), an eicosanoid mediator that depends on 5-lipoxygenase (LO) for its biosynthesis, exerts a regulatory role on dendritic cell IL-12 production triggered artificially by a T. gondii extract. We now formally establish the physiological relevance of this pathway in the systemic control of IL-12 production induced by live T. gondii infection and demonstrate its function to be distinct from that of IL-10. Thus, T. gondii-exposed wild-type, but not 5-LO-deficient animals, produced high levels of serum LXA(4) beginning at the onset of chronic infection. Moreover, 5-LO(-/-), in contrast to wild-type mice, succumbed during the same period displaying a marked encephalitis. The increased mortality of the 5-LO(-/-) animals was also associated with significant elevations of IL-12 and IFN-gamma and was completely prevented by the administration of a stable LXA(4) analogue. Together, these findings demonstrate a new pathway involving the induction of host LXs for the in vivo regulation of proinflammatory responses during microbial infection.

Suppression of proto-oncogene (AP-1) in a model of skin epidermal hyperproliferation is reversed by topical application of 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatrienoic acid.

The present study was conducted to delineate whether a possible mechanism for 13-(S)-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatrienoic acid (15-HETrE) reversal of experimentally-induced skin hyperproliferation in guinea pig is via the modulation of epidermal nuclear mitogen activator protein (AP-1), a nuclear transcription factor associated with tissue turnover. The data revealed that topical application of 13-HODE and/or 15-HETrE on the induced hyperproliferative skin reversed the hyperproliferation and up-regulated the suppressed AP-1 expression. A further analysis of the two major subunits of AP-1 (c-fos and c-jun) revealed a selective up-regulation of c-fos. These results underscore the modulatory role of lipoxygenase-derived hydroxy fatty acids on nuclear transcription factors and explains, at least in part, the antiproliferative effects of 13-HODE and 15-HETrE.

Cutting edge: lipoxin (LX) A4 and aspirin-triggered 15-epi-LXA4 block allergen-induced eosinophil trafficking.

Tissue eosinophilia prevention represents one of the primary targets to new anti-allergic therapies. As lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous "stop signals" produced in distinct pathologies including some eosinophil-related pulmonary disorders, we evaluated the impact of in situ LXA4/ATL metabolically stable analogues on allergen-induced eosinophilic pleurisy in sensitized rats. LXA4/ATL analogues dramatically blocked allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, inhibiting the earlier edema and neutrophilia associated with allergic reaction. The mechanisms underlying this LXA4/ATL-driven allergic eosinophilia blockade was independent of mast cell degranulation and involved LXA4/ATL inhibition of both IL-5 and eotaxin generation, as well as platelet activating factor action. These findings reveal LXA4/ATL as a novel class of endogenous anti-allergic mediators, capable of preventing local eosinophilia.

20-HETE requires increased vascular tone to constrict rabbit afferent arterioles.

Renal production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450-dependent arachidonate metabolite, increases during development of hypertension in spontaneously hypertensive rats, and inhibition of its production prevents hypertension. Since 20-HETE is a potent vasoconstrictor, these findings suggest that 20-HETE may contribute to the development of hypertension by elevating renal vascular resistance. In this study we examined the direct action of 20-HETE on the afferent arteriole, a vascular segment crucial to the control of renal vascular resistance. Rabbit afferent arterioles were microperfused at 60 mm Hg in vitro, and 20-HETE was added to the lumen. Although 20-HETE (10(-10) to 10(-6) mol/L) had no effect on the diameter of non-treated afferent arterioles (n=6), it caused dose-dependent constriction when vascular tone was increased with norepinephrine (0.3 micromol/L); 20-HETE at 10(-6) mol/L decreased diameter by 43 +/- 4% (n=6, P < .001). This constriction was abolished by disrupting the endothelium (n=5). Moreover, pretreatment with the cyclooxygenase inhibitor indomethacin (50 micromol/L) or the thromboxane/endoperoxide receptor antagonist SQ29548 (1 micromol/L) significantly (P < .03) attenuated 20-HETE-induced constriction: 20-HETE at 10(-6) mol/L constricted norepinephrine-treated afferent arterioles by 28 +/- 3% (n=6) and 25 +/- 4% (n=5), respectively. These results demonstrate that an increase in afferent arteriolar tone is required for the vasoconstrictor action of 20-HETE, which is partly mediated by the endothelial cyclooxygenase pathway. THus, increased production of 20-HETE in the kidney and increase in afferent arteriolar tone, both of which often precede the development of hypertension, may synergistically contribute to the development of hypertension by elevating renal vascular resistance.

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist, SC-53228: impact upon leukotriene B4- and 12(R)-HETE-mediated events.

Leukotriene B4 (LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig. LTB4 and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihy dro-8-propyl-2H - 1-benzopyran-2-carboxylic acid), a first-generation LTB4 receptor antagonist, inhibited the chemotactic actions of LTB4 when given orally with an ED50 value of 1.7 mg/kg. The second-generation LTB4 receptor antagonist, SC-53228 [(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4- [(methylamino)carbonyl]phenoxy)propoxy)-3,4-dihydro-8-propyl-2H-1- benzopyran-2-propanoic acid], inhibited LTB4-induced chemotaxis when given intragastrically with an ED50 value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED50 value of 5.8 mg/kg. When dosed orally over a range of 0.03-100 mg/kg, SC-53228 gave Cmax plasma concentrations of 0.015-41.1 micrograms/ml. SC-53228 inhibited LTB4-primed membrane depolarization of human neutrophils with an IC50 value of 34 nM. As a potent LTB4 receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB4 and/or 12(R)-HETE are implicated as inflammatory mediators.

Incorporation of 15-hydroxyeicosatrienoic acid in specific phospholipids of cultured human keratinocytes and psoriatic plaques.

15-hydroxyeicosatrienoic acid, 15-HETrE, the 15-lipoxygenase product of dihomogammalinolenic acid (DGLA), can inhibit the biosynthesis of the proinflammatory eicosanoids leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE). The purpose of the present study was to investigate the incorporation of [14C]15-HETrE in specific membrane phospholipids of cultured human keratinocytes in vitro. [14C]15-HETrE was rapidly incorporated into keratinocytes. When a plateau was reached after 3 hours, 15% of the added radioactivity was incorporated into lipids; 96.5% into phospholipids (PL) and 3.5% into neutral lipids (NL). Within the phospholipid classes, [14C]15-HETrE showed selectivity for incorporation into phosphatidylinositol (PI). The mean proportion of [14C]15-HETrE in the PI, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was 83.2%, 8.5% and 8.3%, respectively. We then investigated the incorporation of 15-HETrE in epidermal phospholipids of psoriatic skin intralesionally injected with 15-HETrE. Four patients took part in the study. In each patient four identical plaques were injected with 0.65 ml of 2.0 microM, 6.2 microM, 18.6 microM of 15-HETrE (0.4 micrograms, 1, 2 micrograms and 3.6 micrograms respectively) or 0.65 ml of 0.88% NaCl twice a week. After 3 wk keratome biopsies were obtained from the treated plaques. Phospholipids extracted from the skin biopsies were separated into major classes by two-dimensional thin layer chromatography. 15-HETrE was then released from specific phospholipids after treatment with phospholipase A2 and identified by reverse phase and straight phase high performance liquid chromatography. There was a dose-dependent incorporation of 15-HETrE into the specific phospholipids PI and PC. When expressed as ng 15-HETrE/micrograms phospholipid phosphate, 15-HETrE accumulated preferentially in PI.(ABSTRACT TRUNCATED AT 250 WORDS)