ABSTRACT
We previously reported that the soy isoflavone daidzein (Dz) suppresses the intracellular replication of influenza virus and that arachidonic acid-derived oxidation product via lipid oxidase 5-lipoxygenase (5-LOX) is involved in its antiviral effect. The activation of 5-LOX by Dz triggers anti-influenza activity; however, the mechanism of activation of 5-LOX remains unclear. Therefore, in this study, we aimed to clarify the activation mechanism using human monocyte-derived THP-1 cells differentiated using phorbol 12-myristate 13-acetate. THP-1 cells expressed 5-LOX endogenously and Dz did not induce 5-LOX expression. However, 8 h after treatment with Dz, the amount of 5-hydroxyeicosatetraenoic acid (5-HETE), an arachidonic acid oxidation product via 5-LOX, increased significantly suggesting that the enzyme is activated regardless of changes in 5-LOX protein levels. Intracellular Ca2+ content, ATP concentration, 5-LOX protein phosphorylation, and 5-LOX intracellular localization are known 5-LOX activation factors. The intracellular Ca2+ and ATP concentrations were not affected by Dz treatment. The enzymatic activity of 5-LOX is regulated by the phosphorylation of three serine residues and four tyrosine residues. Pretreatment with inhibitors of each kinase revealed that Dz-induced 5-HETE production was suppressed by the MEK/ERK inhibitor. 5-LOX in which the Ser663 residue was phosphorylated was found to be increased in the nuclear fraction of Dz-treated THP-1 cells. Furthermore, immunocytochemistry showed that 5-LOX translocates to the nuclear envelope following Dz treatment. These results indicate that Dz activates 5-LOX by phosphorylating Ser663 via the MEK/ERK pathway. Thus, these results demonstrate that Dz exerts anti-influenza virus activity via the MEK/ERK signal transduction pathway.
Subject(s)
Arachidonate 5-Lipoxygenase , Isoflavones , MAP Kinase Signaling System , Humans , Adenosine Triphosphate/metabolism , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 5-Lipoxygenase/drug effects , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Isoflavones/pharmacology , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Influenza, Human/metabolismABSTRACT
BACKGROUND: Non-allergic angioedema is a potentially life-threatening condition caused by accumulation of bradykinin and subsequent activation of bradykinin type 2 receptors (B2). Since COX activity plays a pivotal role in B2 signaling, the aim of this study was to determine which prostaglandins are the key mediators and which COX, COX-1 or COX-2, is predominantly involved. METHODS: We used Miles assays to assess the effects of inhibitors of COX, 5-lipoxygenase, epoxyeicosatrienoic acid generation, cytosolic phospholipase A2α and a variety of prostaglandin receptor antagonists on bradykinin-induced dermal extravasation in C57BL/6 and COX-1-deficient mice (COX-1-/-). In addition, the prostacyclin metabolite 6-keto-PGF1α was quantified by ELISA in subcutaneous tissue from C57BL/6 and human dermal microvascular endothelial cells. In the latter, 6-keto-PGF1α was also quantified and identified by LC-MS/MS. RESULTS: Unspecific COX inhibition by ibuprofen and diclofenac significantly reduced B2-mediated dermal extravasation in C57BL/6 but not COX-1-/-. Likewise, inhibition of cytosolic phospholipase A2α showed similar effects. Furthermore, extravasation in COX-1-/- was generally lower than in C57BL/6. Of the prostaglandin antagonists used, only the prostacyclin receptor antagonist RO1138452 showed a significant reduction of dermal extravasation. Moreover, 6-keto-PGF1α concentrations were increased after bradykinin treatment in subcutaneous tissue from C57BL/6 as well as in human dermal microvascular endothelial cells and this increase was abolished by diclofenac. CONCLUSION: Our findings suggest that COX-1-dependent prostacyclin production is critically involved in dermal extravasation after activation of B2 in small dermal blood vessels. Targeting prostacyclin production and/or signaling appears to be a suitable option for acute treatment of non-allergic angioedema.
Subject(s)
Angioedema/pathology , Cyclooxygenase 1/metabolism , Epoprostenol/metabolism , Angioedema/chemically induced , Animals , Arachidonate 5-Lipoxygenase/drug effects , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Bradykinin/pharmacology , Diclofenac/pharmacology , Endothelial Cells/drug effects , Group IV Phospholipases A2/drug effects , Group IV Phospholipases A2/metabolism , Ibuprofen/pharmacology , Male , Mice , Mice, Inbred C57BL , Oxygenases/drug effects , Oxygenases/metabolism , Prostaglandin-Endoperoxide Synthases/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Receptors, Prostaglandin/antagonists & inhibitorsABSTRACT
BACKGROUND: Recent studies highlight the regulatory role of arachidonate lipoxygenase5 (Alox5) and its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in cancer tumorigenesis and progression. In this study, we analyzed the expression, biological function and the downstream signaling of Alox5 in gastric cancer. METHODS: Alox5 protein levels were measured using IHC and ELISA. Growth, migration and survival assays were performed. Phosphorylation of molecules involved in growth and survival signaling were analyzed by WB. Analysis of variance and t-test were used for statistic analysis. RESULTS: Alox5 and 5-HETE levels were upregulated in gastric cancer patients. ALOX5 overexpression or 5-HETE addition activates gastric cancer cells and reduces chemotherapy's efficacy. In contrast, ALOX5 inhibition via genetic and pharmacological approaches suppresses gastric cancer cells and enhances chemotherapy's efficacy. In addition, Alox5 inhibition led to suppression of ERK-mediated signaling pathways whereas ALOX5-5-HETE activates ERK-mediated signaling in gastric cancer cells. CONCLUSIONS: Our work demonstrates the critical role of ALOX5-5-HETE in gastric cancer and provides pre-clinical evidence to initialize clinical trial using zileuton in combination with chemotherapy for treating gastric cancer.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Cell Proliferation , Hydroxyeicosatetraenoic Acids/metabolism , MAP Kinase Signaling System/physiology , Stomach Neoplasms/metabolism , Analysis of Variance , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Arachidonate 5-Lipoxygenase/drug effects , Cell Survival , Disease Progression , Humans , Hydroxyurea/analogs & derivatives , Hydroxyurea/therapeutic use , Lipoxygenase Inhibitors/therapeutic use , MAP Kinase Signaling System/drug effects , Phosphorylation , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Up-RegulationABSTRACT
Five new compounds viz kaempferol 3-O-(4â³-galloyl)-ß-d-glucopyranosyl-(1â´â6â³)-O-ß-d-glucopyranoside (1), kaempferol 3-O-ß-d-mannuronopyranoside (2), kaempferol 3-O-ß-d-mannopyranoside (3), quercetin 3-O-ß-d-mannuronopyranoside (4), 2, 3 (S)- hexahydroxydiphenoyl]-d-glucose (5) along with fifteen known compounds were isolated from 80% aqueous methanol extract (AME) of C. viminalis. AME and compounds exerted similar or better antioxidant activity to ascorbic acid using DPPH, O2-, and NO inhibition methods. In addition, compounds 16, 4, and 7 showed cytotoxic activity against MCF-7 cell lines while 3, 7 and 16 exhibited strong activity against HepG2. An in silico analysis using molecular docking for polyphenolic compounds 2, 3, 7, 16 and 17 against human stable 5-LOX was performed and compared to that of ascorbic acid and quercetin. The binding mode as well as the enzyme-inhibitor interactions were evaluated. All compounds occupied the 5-LOX active site and showed binding affinity greater than ascorbic acid or quercetin. The data herein suggest that AME, a source of polyphenols, could be used against oxidative-stress-related disorders.
Subject(s)
Antineoplastic Agents/pharmacology , Antioxidants/chemistry , Arachidonate 5-Lipoxygenase/drug effects , Myrtaceae/chemistry , Antineoplastic Agents/chemistry , Antioxidants/pharmacology , Arachidonate 5-Lipoxygenase/genetics , Humans , MCF-7 Cells , Oxidative Stress/drug effects , Plant Components, Aerial/chemistry , Polyphenols/chemistry , Polyphenols/pharmacologyABSTRACT
Commonly used to treat skin injuries in Asia, several Homalium spp. have been found to promote skin regeneration and wound healing. While ethnobotanical surveys report the use of H. bhamoense trunk bark as a wound salve, there are no studies covering bioactive properties. As impaired cutaneous healing is characterized by excessive inflammation, a series of inflammatory mediators involved in wound healing were targeted with a methanol extract obtained from H. bhamoense trunk bark. Results showed concentration-dependent inhibition of hyaluronidase and 5-lipoxygenase upon exposure to the extract, with IC50 values of 396.9 ± 25.7 and 29.0 ± 2.3 µg mL-1, respectively. H. bhamoense trunk bark extract also exerted anti-inflammatory activity by significantly suppressing the overproduction of interleukin 6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages at concentrations ranging from 125 to 1000 µg mL-1, while leading to a biphasic effect on nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) levels. The phenolic profile was elucidated by HPLC-DAD, being characterized by the occurrence of ellagic acid as the main constituent, in addition to a series of methylated derivatives, which might underlie the observed anti-inflammatory effects. Our findings provide in vitro data on anti-inflammatory ability of H. bhamoense trunk bark, disclosing also potential cutaneous toxicity as assessed in HaCaT keratinocytes.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Interleukin-6/adverse effects , Lipopolysaccharides/adverse effects , Macrophages/drug effects , Medicine, Traditional/methods , Nephropidae/chemistry , Plant Extracts/pharmacology , Animals , Arachidonate 5-Lipoxygenase/drug effects , Cell Survival/drug effects , Cytokines/metabolism , Herbal Medicine , Hyaluronoglucosaminidase/drug effects , Hydroxybenzoates , Inflammation Mediators/pharmacology , Inhibitory Concentration 50 , Interleukin-6/metabolism , Keratinocytes , Lipopolysaccharides/metabolism , Mice , Nitric Oxide/metabolism , RAW 264.7 Cells , Tumor Necrosis Factor-alphaABSTRACT
The obligatory testing of drug molecules and their impurities to protect users against toxic compounds seems to provide interesting opportunities for new drug discovery. Impurities, which proved to be non-toxic, may be explored for their own therapeutic potential and thus be a part of future drug discovery. The essential role of pharmaceutical analysis can thus be extended to achieve this purpose. The present study examined these objectives by characterizing the major degradation products of zileuton (ZLT), a 5-lipoxygenase (5-LOX) inhibitor being prevalently used to treat asthma. The drug sample was exposed to forced degradation and found susceptible to hydrolysis and oxidative stress. The obtained Forced Degradation Products (FDP's) were resolved using an earlier developed and validated Ultra-High-Pressure Liquid Chromatography Photo-Diode-Array (UHPLC-PDA) protocol. ZLT, along with acid-and alkali-stressed samples, were subjected to Liquid-chromatography Mass-spectrometry Quadrupole Time-of-flight (LC/MS-QTOF) studies. Major degradation products were isolated using Preparative TLC and characterized using Q-TOF and/or Proton nuclear magnetic resonance (1HNMR) studies. The information obtained was assembled for structural conformation. Toxicity Prediction using Komputer Assisted Technology (TOPKAT) toxicity analyses indicated some FDP's as non-toxic when compared to ZLT. Hence, these non-toxic impurities may have bio-affinity and can be explored to interact with other therapeutic targets, to assist in drug discovery. The drug molecule and the characterized FDP's were subjected to 3-Dimensional Extra Precision (3D-XP)-molecular docking to explore changes in bio-affinity for the 5-LOX enzyme (PDB Id: 3V99). One FDP was found to have a higher binding affinity than the drug itself, indicating it may be a suitable antiasthmatic. The possibility of being active at other sites cannot be neglected and this is evaluated to a reasonable extent by Prediction of Activity Spectra for Substances (PASS). Besides being antiasthmatic, some FDP's were predicted antineoplastic, antiallergic and inhibitors of Complement Factor-D.
Subject(s)
Drug Contamination , Hydroxyurea/analogs & derivatives , Arachidonate 5-Lipoxygenase/drug effects , Chromatography, Liquid/methods , Computer Simulation , Drug Discovery/methods , Hydrolysis , Hydroxyurea/chemistry , Hydroxyurea/therapeutic use , Hydroxyurea/toxicity , Magnetic Resonance Spectroscopy/methods , Molecular Docking Simulation , Molecular Structure , Oxidative Stress , Software , Tandem Mass Spectrometry/methodsABSTRACT
Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Arachidonate 5-Lipoxygenase/drug effects , Lysophospholipids/pharmacology , Receptors, Lysosphingolipid/physiology , Sphingosine/analogs & derivatives , Animals , Arachidonate 5-Lipoxygenase/biosynthesis , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Calcium/metabolism , Cell Line , Female , Humans , Lysophospholipids/metabolism , Mice , Mice, Inbred C57BL , Neutrophils/enzymology , Neutrophils/metabolism , Pneumonia/metabolism , Reactive Oxygen Species/metabolism , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Sphingosine/metabolism , Sphingosine/pharmacology , Substrate SpecificityABSTRACT
OBJECTIVES: This work describes the synthesis, the bioactivity and the structure-activity relationship of new derivatives from a natural coumarin. METHODS: (-)-Deltoin 1 and the corresponding isoxazolines and aziridines were characterized by spectroscopic means. The cytotoxic (HTC-116, IGROV-1 and OVCAR-3 cancer cell lines) and 5-lipoxygenase activity of (-)-deltoin 1 and its structural analogues have been evaluated. KEY FINDINGS: The phytochemical investigation of the ethyl acetate extract of the flowers of Ferula lutea (Poir.) Maire has led to the isolation of (-)-deltoin 1. A series of new isoxazoline 2a,a'-2f,f' and aziridine 3a,a'-3e,e' derivatives have been prepared by 1,3-dipolar cycloaddition. It has been found that the derivatives 2a (IC50 = 3.3 ± 0.1 µm), 3a,a' (IC50 = 5.9 ± 0.1 µm), 3b,b' (IC50 = 6.1 ± 0.7 µm) and 3c,c' (IC50 = 7.3 ± 0.9 µm) bearing a phenyl isoxazoline, a phenylaziridine, a 4-methlphenylaziridine and a 4-methoxyphenylaziridine, respectively, are more cytotoxic than (-)-deltoin 1 (IC50 = 14.3 ± 0.2 µm). The diastereoisomers in mixture (2f,f') with a 6-chloropyridin-2-yl system have shown the best anti-5-lipoxygenase activity (% inhibition = 53.1 ± 4.8% at 200 µm). CONCLUSIONS: Some analogues have been found more bioactive than deltoin 1. Their activity has been related to the nature of the added heterocycles. It would be interesting to evaluate their in-vivo activity.
Subject(s)
Antineoplastic Agents/pharmacology , Aziridines/pharmacology , Furocoumarins/chemistry , Isoxazoles/pharmacology , Plant Extracts/pharmacology , Antineoplastic Agents/chemistry , Arachidonate 5-Lipoxygenase/drug effects , Aziridines/chemistry , Cell Line, Tumor , Drug Screening Assays, Antitumor , Flowers , Furocoumarins/pharmacology , Humans , Isoxazoles/chemistry , Plant Extracts/chemistry , Structure-Activity RelationshipABSTRACT
A new series of 4-((5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)methyl)-benzoic acids 4a-o and 2-(5-fluoro-6-(substituted)-1H-benzo[d]imidazol-2-ylthio)-2-methylpropanoic acids 8a-e were synthesized, and their inhibitory potencies against soluble epoxide hydrolase (sEH) and 5-lipoxygenase (5-LOX) were investigated. These molecules were designed based on the combination of 5-LOX and sEH pharmacophores, resulting in hybrid analogs with potent sEH and 5-LOX inhibitory activity. Compound 4g showed remarkable activity with IC50 values of less than 1 µM (0.9 µM) against 5-LOX, while compound 4k displayed promising activity against sEH with IC50 ≤ 1 µM (0.7 µM). These compounds were evaluated for their in vivo potential using the carrageenan-induced rat paw edema assay. Based on the obtained results, the structure-activity relationship was established and a correlation between the activities was observed. Compounds 4f, 4g, 4k, 4n, and 8e showed potent anti-inflammatory activity and significant inhibition of edema (64.13, 67.39, 66.30, 65.21, and 58.69%, respectively) at a dose of 100 mg/kg, comparable to the standard drug ibuprofen (70.65%) at 3 h.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Benzimidazoles/pharmacology , Edema/drug therapy , Inflammation/drug therapy , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Arachidonate 5-Lipoxygenase/drug effects , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Carrageenan , Disease Models, Animal , Dose-Response Relationship, Drug , Edema/enzymology , Epoxide Hydrolases/antagonists & inhibitors , Female , Humans , Ibuprofen/pharmacology , Inflammation/enzymology , Inhibitory Concentration 50 , Lipoxygenase Inhibitors/chemical synthesis , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/pharmacology , Male , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
Neurodegenerative processes involve numerous and closely related events that ultimately culminate in neuronal cell injury. The aim of this study was (i) to assess, for the first time, the neuroprotective potential of acetone extracts of six edible species of Ochrophyta, by evaluating their cholinesterase and lipoxygenase inhibitory activity in cell-free assays, as well as their capacity to attenuate glutamate-induced toxicity in neuronal (SH-SY5Y) cells, and (ii) to try to relate the chemical composition of the extracts with their biological activity, evaluating also the effect of the main compounds thereof. In spite of a modest cholinesterase inhibition, a dose-dependent response towards lipoxygenase was found for all macroalgae extracts. At non-cytotoxic concentrations, the extracts from Fucus serratus Linnaeus and Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders were able to improve the viability of glutamate-insulted SH-SY5Y cells. These results encourage further studies for a more detailed understanding of the mechanisms beyond the documented biological activities, and point to the potential interest of the selected seaweed species and their extracts as promising candidates for in vivo studies.
Subject(s)
Glutamic Acid/toxicity , Neurons/drug effects , Neuroprotective Agents/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plants, Edible/chemistry , Seaweed/chemistry , Arachidonate 5-Lipoxygenase/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cell-Free System , Cholinesterases/drug effects , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Gas Chromatography-Mass Spectrometry , Humans , Neurons/enzymology , Oleic Acid/pharmacology , Palmitic Acid/pharmacology , Xanthophylls/pharmacologyABSTRACT
BACKGROUND: Anti-inflammatory drug development efforts for lung disease have been hampered in part by the lack of noninvasive inflammation biomarkers and the limited ability of animal models to predict efficacy in humans. We used 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in a human model of lung inflammation to assess whether pioglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, and zileuton, a 5-lipoxygenase inhibitor, reduce lung inflammation. METHODS: For this single center, single-blind, placebo-controlled cohort study, we enrolled healthy volunteers sequentially into the following treatment cohorts (N = 6 per cohort): pioglitazone plus placebo, zileuton plus placebo, or dual placebo prior to bronchoscopic endotoxin instillation. 18F-FDG uptake pre- and post-endotoxin was quantified as the Patlak graphical analysis-determined Ki (primary outcome measure). Secondary outcome measures included the mean standard uptake value (SUVmean), post-endotoxin bronchoalveolar lavage (BAL) cell counts and differentials and blood adiponectin and urinary leukotriene E4 (LTE4) levels, determined by enzyme-linked immunosorbent assay, to verify treatment compliance. One- or two-way analysis of variance assessed for differences among cohorts in the outcome measures (expressed as mean ± standard deviation). RESULTS: Ten females and eight males (29±6 years of age) completed all study procedures except for one volunteer who did not complete the post-endotoxin BAL. Ki and SUVmean increased in all cohorts after endotoxin instillation (Ki increased by 0.0021±0.0019, 0.0023±0.0017, and 0.0024±0.0020 and SUVmean by 0.47±0.14, 0.55±0.15, and 0.54±0.38 in placebo, pioglitazone, and zileuton cohorts, respectively, p<0.001) with no differences among treatment cohorts (p = 0.933). Adiponectin levels increased as expected with pioglitazone treatment but not urinary LTE4 levels as expected with zileuton treatment. BAL cell counts (p = 0.442) and neutrophil percentage (p = 0.773) were similar among the treatment cohorts. CONCLUSIONS: Endotoxin-induced lung inflammation in humans is not responsive to pioglitazone or zileuton, highlighting the challenge in translating anti-inflammatory drug efficacy results from murine models to humans. TRIAL REGISTRATION: ClinicalTrials.gov NCT01174056.
Subject(s)
Arachidonate 5-Lipoxygenase/drug effects , Hydroxyurea/analogs & derivatives , Peroxisome Proliferator-Activated Receptors/agonists , Thiazolidinediones/therapeutic use , Adult , Female , Healthy Volunteers , Humans , Hydroxyurea/therapeutic use , Male , Pioglitazone , Placebos , Positron-Emission Tomography , Single-Blind Method , Young AdultABSTRACT
AIMS: Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. RESULTS: In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE2) in plasma when compared with either HIV or alcohol alone. INNOVATION: This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. CONCLUSION: These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.
Subject(s)
Alcohols/toxicity , Arachidonate 5-Lipoxygenase/drug effects , Gene Expression Regulation/drug effects , HIV Infections/metabolism , Adult , Alcohols/immunology , Alcohols/metabolism , Arachidonate 5-Lipoxygenase/genetics , Arachidonic Acid/genetics , Arachidonic Acid/metabolism , Blood Donors , Cyclooxygenase 2/genetics , Disease Progression , Female , Gene Expression Regulation/immunology , Glutathione Peroxidase/genetics , Glutathione Synthase/genetics , HIV/drug effects , HIV/immunology , HIV/pathogenicity , HIV Infections/immunology , HIV Infections/pathology , HIV Infections/virology , Humans , Male , Membrane Microdomains/drug effects , Membrane Microdomains/immunology , Membrane Microdomains/virology , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , Sterol Regulatory Element Binding Proteins/genetics , Superoxide Dismutase/geneticsABSTRACT
Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.
Subject(s)
Acetophenones/isolation & purification , Acetophenones/pharmacology , Anti-Inflammatory Agents/pharmacology , Arachidonate 5-Lipoxygenase/metabolism , Lipoxygenase Inhibitors/pharmacology , Prostaglandin-E Synthases/antagonists & inhibitors , Rutaceae/chemistry , Acetophenones/chemistry , Animals , Arachidonate 5-Lipoxygenase/drug effects , Disease Models, Animal , Humans , Hydroxyurea/analogs & derivatives , Hydroxyurea/pharmacology , Inhibitory Concentration 50 , Intramolecular Oxidoreductases/antagonists & inhibitors , Mice , Molecular Structure , Prenylation , Structure-Activity RelationshipABSTRACT
OBJECTIVE: The goal of the present study was to investigate the effects of 5-lipoxygenase (5-LOX) inhibition, alone and with cyclooxygenase (COX) inhibitors, on inflammatory parameters and apoptosis in ischemia/reperfusion (I/R)-induced myocardial damage in rats. For this purpose, zileuton, a selective and potent inhibitor of 5-LOX, resulting in suppression leukotriene production, was used. METHODS: Male Wistar rats (200-250 g; n=12 per group) were used in the study. I/R was performed by occluding the left coronary artery for 30 minutes and 2 hours of reperfusion of the heart. Experimental groups were I/R group, sham I/R group, zileuton (5 mg/kg orally, twice daily)+I/R group, zileuton+indomethacin (5 mg/kg intraperitoneally)+I/R group, zileuton+ketorolac (10 mg/kg subcutaneously)+I/R group, and zileuton+nimesulide (5 mg/kg subcutaneously)+I/R group. Following I/R, blood samples were collected to measure tumor necrosis factor alpha (TNF-α), and left ventricles were excised for evaluation of microscopic damage; malondialdehyde (MDA), glutathione, nuclear factor (NF)-κB assays; and evaluation of apoptosis. RESULTS: Left ventricle MDA in I/R group was higher compared to sham group; however, it did not show significant change with zileuton. Although tissue injury in I/R group was less severe in all treatment groups, it was not statistically significant. NF-κB H-score and apoptotic index, which were higher in I/R group compared to sham I/R, were decreased with application of zileuton (H-score: p<0.01; apoptotic index: p<0.001). Zileuton had no significant effect on increased serum TNF-α levels in I/R group. CONCLUSION: 5-LOX inhibition in rat myocardial infarction model attenuated increased left ventricle NF-κB expression and apoptosis and these actions were not modulated by COX inhibitors.
Subject(s)
Arachidonate 5-Lipoxygenase/drug effects , Hydroxyurea/analogs & derivatives , Leukotriene Antagonists/therapeutic use , Myocardial Infarction/drug therapy , Myocardial Reperfusion Injury/prevention & control , Animals , Arachidonate 5-Lipoxygenase/blood , Disease Models, Animal , Hydroxyurea/pharmacology , Hydroxyurea/therapeutic use , Leukotriene Antagonists/pharmacology , Male , Myocardial Infarction/blood , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/bloodABSTRACT
PURPOSE: 5-Lipoxygenase (5-LOX) oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). The RPE and retina express the PNPLA2 gene for pigment epithelium-derived factor receptor (PEDF-R), a lipase involved in cell survival. The purpose here was to investigate the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE. METHODS: Lipoxygenase activity assays were performed with soybean and potato lipoxygenase. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-R-derived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with indicated concentrations of H2O2/TNF-α. Reverse transcription-PCR of ALOX5 and PNPLA2 genes was performed. Cell viability and death rates were determined using respective biomarkers. Leukotriene B4 levels were measured by ELISA. RESULTS: Among five peptides spanning between positions Leu159 and Met325 of human PEDF-R polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited lipoxygenase activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. The two inhibitor peptides E5b and P1 promoted cell viability and decreased cell death of RPE cells undergoing oxidative stress. Oxidative stress decreased the levels of PNPLA2 transcripts with no effect on ALOX5 expression. Exogenous additions of P1 peptide or overexpression of the PNPLA2 gene decreased both LTB4 levels and death of RPE cells undergoing oxidative stress. CONCLUSIONS: A novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Cell Death/drug effects , Gene Expression Regulation , Lipase/genetics , Lipoxygenase Inhibitors/pharmacology , Oxidative Stress/genetics , Retinal Pigment Epithelium/metabolism , Arachidonate 5-Lipoxygenase/biosynthesis , Arachidonate 5-Lipoxygenase/drug effects , Blotting, Western , Cell Survival , Cells, Cultured , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Humans , Lipase/biosynthesis , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/pathology , Oxidative Stress/drug effects , RNA/genetics , Receptors, Neuropeptide/metabolism , Retinal Pigment Epithelium/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Sclerotiorin, isolated from the fermented broth of Penicillium frequentans, exhibited potent inhibition against human polymorphonuclear leukocytes 5-lipoxygenase and human platelet aggregation with a half maximal value 36 µM and 250 µM, respectively. Further, the Ames test has demonstrated the sclerotiorin to be non-mutagenic.
Subject(s)
Arachidonate 5-Lipoxygenase/drug effects , Benzopyrans/pharmacology , Lipoxygenase Inhibitors/pharmacology , Neutrophils/enzymology , Platelet Aggregation Inhibitors/pharmacology , Fermentation , Humans , Mutagenicity Tests , Penicillium/metabolism , Salmonella typhimurium/geneticsABSTRACT
We present the design, synthesis and biological evaluation of compounds containing a 2-(benzylidene)hexanoic acid scaffold as multi-target directed γ-secretase-modulators. Broad structural variations were undertaken to elucidate the structure-activity-relationships at the 5-position of the aromatic core. Compound 13 showed the most potent activity profile with IC50 values of 0.79µM (Aß42), 0.3µM (5-lipoxygenase) and an EC50 value of 4.64µM for PPARγ-activation. This derivative is the first compound exhibiting low micromolar to nanomolar activities for these three targets. Combining γ-secretase-modulation, PPARγ-agonism and inhibition of 5-lipoxygenase in one compound could be a novel disease-modifying multi-target-strategy for Alzheimer's disease to concurrently address the causative amyloid pathology and secondary pathologies like chronic brain inflammation.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/drug effects , Arachidonate 5-Lipoxygenase/drug effects , Caproates/therapeutic use , Lipoxygenase Inhibitors/pharmacology , PPAR gamma/agonists , Caproates/chemistry , Caproates/pharmacology , Humans , Lipoxygenase Inhibitors/therapeutic use , Structure-Activity RelationshipABSTRACT
BACKGROUND: The effectiveness of intermittent montelukast for wheeze in young children is unclear. We aimed to assess whether intermittent montelukast is better than placebo for treatment of wheeze in this age group. Because copy numbers of the Sp1-binding motif in the arachidonate 5-lipoxygenase (ALOX5) gene promoter (either 5/5, 5/x, or x/x, where x does not equal 5) modifies response to montelukast in adults, we stratified by this genotype. METHODS: We did this multicentre, parallel-group, randomised, placebo-controlled trial between Oct 1, 2010, and Dec 20, 2013, at 21 primary care sites and 41 secondary care sites in England and Scotland. Children aged 10 months to 5 years with two or more wheeze episodes were allocated to either a 5/5 or 5/x+x/x ALOX5 promoter genotype stratum, then randomly assigned (1:1) via a permuted block schedule (size ten), to receive intermittent montelukast or placebo given by parents at each wheeze episode over a 12 month period. Clinical investigators and parents were masked to treatment group and genotype strata. The primary outcome was number of unscheduled medical attendances for wheezing episodes. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01142505. FINDINGS: We randomly assigned 1358 children to receive montelukast (n=669) or placebo (n=677). Consent was withdrawn for 12 (1%) children. Primary outcome data were available for 1308 (96%) children. There was no difference in unscheduled medical attendances for wheezing episodes between children in the montelukast and placebo groups (mean 2·0 [SD 2·6] vs 2·3 [2·7]; incidence rate ratio [IRR] 0·88, 95% CI: 0·77-1·01; p=0·06). Compared with placebo, unscheduled medical attendances for wheezing episodes were reduced in children given montelukast in the 5/5 stratum (2·0 [2·7] vs 2·4 [3·0]; IRR 0·80, 95% CI 0·68-0·95; p=0·01), but not in those in the 5/x+x/x stratum (2·0 [2·5] vs 2·0 [2·3]; 1·03, 0·83-1·29; p=0·79, pinteraction=0·08). We recorded one serious adverse event, which was a skin reaction in a child allocated to placebo. INTERPRETATION: Our findings show no clear benefit of intermittent montelukast in young children with wheeze. However, the 5/5 ALOX5 promoter genotype might identify a montelukast-responsive subgroup. FUNDING: Medical Research Council (UK) and National Institute for Health Research.
Subject(s)
Acetates/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Quinolines/therapeutic use , Respiratory Sounds/drug effects , Acetates/administration & dosage , Appointments and Schedules , Arachidonate 5-Lipoxygenase/drug effects , Child, Preschool , Cyclopropanes , Cysteine/urine , Drug Administration Schedule , Female , Genotype , Humans , Infant , Leukotrienes/urine , Male , Quinolines/administration & dosage , Sulfides , Treatment OutcomeABSTRACT
A phytochemical investigation on the 5-lipoxygenase (5-LOX) inhibitory methanolic extract of Rudbeckia hirta L. flowers yielded 10 phenolic metabolites, including three phenolic acids, two phenolic acid esters, four flavonol glycosides and a trimethylated flavonol. The structures of the isolated metabolites were determined on the basis of spectroscopic analyses and by comparison with the literature data. Seven of these metabolites were isolated for the first time from the genus Rudbeckia. The in vitro 5-LOX inhibitory, immunomodulatory and antioxidant (oxygen radical absorbance capacity) activities of the isolated compounds were evaluated, and the results provided a new scientific evidence for the ethnopharmacological use of the herb in inflammatory conditions.
Subject(s)
Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Antioxidants/isolation & purification , Antioxidants/pharmacology , Arachidonate 5-Lipoxygenase/drug effects , Flavonols/isolation & purification , Flavonols/pharmacology , Flowers/chemistry , Glycosides/isolation & purification , Glycosides/pharmacology , Immunologic Factors/isolation & purification , Immunologic Factors/pharmacology , Lipoxygenase Inhibitors/isolation & purification , Lipoxygenase Inhibitors/pharmacology , Phenols/isolation & purification , Phenols/pharmacology , Plants, Medicinal/chemistry , Rudbeckia/chemistry , Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , Flavonols/chemistry , Glycosides/chemistry , Immunologic Factors/chemistry , Lipoxygenase Inhibitors/chemistry , Phenols/chemistry , Plant Extracts/chemistryABSTRACT
BACKGROUND/AIMS: The major compounds of Cochinchina momordica seed extract (SK-MS10) include momordica saponins. We report that the gastroprotective effect of SK-MS10 in an ethanol-induced gastric damage rat model is mediated by suppressing proinflammatory cytokines and downregulating cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and the activation of calcitonin gene-related peptide. In this study, we evaluated the gastroprotective effects of SK-MS10 in the nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage rat model. METHODS: The pretreatment effect of SK-MS10 was evaluated in the NSAID-induced gastric damage rat model using aspirin, indomethacin, and diclofenac in 7-week-old rats. Gastric damage was evaluated based on the gross ulcer index by gastroenterologists, and the damage area (%) was measured using the MetaMorph 7.0 video image analysis system. Myeloperoxidase (MPO) was measured by enzyme-linked immunosorbent assay, and Western blotting was used to analyze the levels of cyclooxygenase (COX)-1, COX-2, cPLA2, and 5-LOX. RESULTS: All NSAIDs induced gastric damage based on the gross ulcer index and damage area (p<0.05). Gastric damage was significantly attenuated by SK-MS10 pretreatment compared with NSAID treatment alone (p<0.05). The SK-MS10 pretreatment group exhibited lower MPO levels than the diclofenac group. The expression of cPLA2 and 5-LOX was decreased by SK-MS10 pretreatment in each of the three NSAID treatment groups. CONCLUSIONS: SK-MS10 exhibited a gastroprotective effect against NSAID-induced acute gastric damage in rats. However, its protective mechanism may be different across the three types of NSAID-induced gastric damage models in rats.