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The 5-lipoxygenase/leukotriene system has been implicated in both physiological and pathological states within the central nervous system. Understanding how this system interacts with the dopaminergic system could provide valuable insights into dopamine-related pathologies. This study focused on examining both motor and non-motor dopamine-related responses in 5-lipoxygenase/leukotriene-deficient mice. We used pharmacological agents such as amphetamine, apomorphine, and reserpine to challenge the dopaminergic system, evaluating their effects on prepulse inhibition reaction (PPI), general motor activity, and oral involuntary movements. Additionally, we analyzed striatal glial marker expression (GFAP and Iba-1) in reserpine-treated mice. The 5-lipoxygenase/leukotriene-deficient mice exhibited increased spontaneous locomotor activity, including both horizontal and vertical exploration, along with stereotyped behavior compared to wild-type mice. This hyperactivity was reduced by acute apomorphine treatment. Although basal PPI responses were unchanged, 5-lipoxygenase/leukotriene-deficient mice displayed a significant reduction in susceptibility to amphetamine-induced PPI disruption. Conversely, these mice were more vulnerable to reserpine-induced involuntary movements. There were no significant differences in the basal expression of striatal GFAP and Iba-1 positive cells between 5-lipoxygenase/leukotriene-deficient and wild-type mice. However, reserpine treatment significantly increased GFAP immunoreactivity in wild-type mice, an effect not observed in 5-lipoxygenase-deficient mice. Additionally, the percentage of activated microglia was significantly higher in reserpine-treated wild-type mice, an effect absents in 5-lipoxygenase/leukotriene-deficient mice. Our findings suggest that 5-lipoxygenase/leukotriene deficiency leads to a distinctive dopaminergic phenotype, indicating that leukotrienes may influence the modulation of dopamine-mediated responses. [ABSTRACT FROM AUTHOR]

Objective: To explore the potential mechanism of lysionotin in treating glioma. Methods: First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. Results: Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. Conclusion: Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway. [ABSTRACT FROM AUTHOR]

Leukotrienes (LTs) are a group of substances that cause inflammation. They are produced by the enzyme 5-lipoxygenase (5-LOX) from arachidonic acid. Cysteinyl LTs are a group of lipid molecules that have a prominent role in inflammatory signaling in the allergic diseases. Although they are traditionally known for their role in allergic disease, current advancements in bio-medical research have shed light on the involvement of these inflammatory mediators in diseases such as in the inflammation related to central nervous system (CNS) disorders. Among the CNS diseases, LTs, along with 5-LOX and their receptors, have been shown to be associated with multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease (PD). Through a comprehensive review of current research and experimentation, this investigation provides an insight on the biosynthesis, receptors, and biological effects of LTs in the body. Furthermore, implications of leukotriene signaling in CNS and its intricate role in neurodegeneration are also studied. Through the revelation of these insights, our aim is to establish a foundation for the development of enhanced and focused therapeutic approaches in the continuous endeavor to combat neurodegeneration. Furthermore, the pharmacological inhibition of leukotriene signaling with selective inhibitors offers promising prospects for future interventions and treatments for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, mediators of the innate immune system that also play an important role in inflammatory diseases and cancer. In this study, we present compounds, containing a Michael-reactive cyanoacrylate moiety as potent inhibitors of 5-LO. Representatives of the tyrosine kinase inhibitor family called tyrphostins, structurally related to known 5-LO inhibitors, were screened for their 5-LO inhibitory properties using recombinant human 5-LO, intact human PMNL (polymorphonuclear leukocytes), and PMNL homogenates. Their mode of action was characterized by the addition of glutathione, using a fourfold cysteine 5-LO mutant and mass spectrometry analysis. SAR studies revealed several members of the tyrphostin family containing a Michael-reactive cyanoacrylate to efficiently inhibit 5-LO. We identified degrasyn (IC50 0.11 µM), tyrphostin A9 (IC50 0.8 µM), AG879 (IC50 78 nM), and AG556 (IC50 64 nM) as potent 5-LO inhibitors. Mass spectrometry analysis revealed that degrasyn and AG556 covalently bound to up to four cysteines, including C416 and/or C418 which surround the substrate entry site. Furthermore, the 5-LO inhibitory effect of degrasyn was remarkably impaired by the addition of glutathione or by the mutation of cysteines to serines at the surface of 5-LO. We successfully identified several tyrphostins as potent inhibitors of human 5-LO. Degrasyn and AG556 were able to covalently bind to 5-LO via their cyanoacrylate moiety. This provides a promising mechanism for targeting 5-LO by Michael acceptors, leading to new therapeutic opportunities in the field of inflammation and cancer. [ABSTRACT FROM AUTHOR]

As innate immune cells of the central nervous system (CNS), microglia are involved in the physiological processes of the CNS, including neural development and maintenance of homeostasis, and in the occurrence and development of most CNS diseases. Lipoxygenases (LOXs) are a family of non-heme, iron-containing enzymes that generate lipid mediators that regulate cellular inflammation by catalyzing the oxidation of polyunsaturated fatty acids. Many previous studies have demonstrated the indispensable role of the LOX pathway in microglia-mediated neuroinflammation, especially the 5-LOX and 12/15-LOX pathways. Emerging evidence indicates that the LOX pathway is also implicated in physiological processes, such as synaptic pruning and synaptic phagocytosis mediated by microglia, and that deficiency can contribute to neurodevelopmental disorders. The present review summarizes the impact of the LOX pathway on microglia-related physiological and pathological processes in the CNS and describes the potential for inhibition of the LOX pathway as a future strategy for the treatment of CNS diseases. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, known as coronavirus disease 2019 (COVID-19) causes cytokine release syndrome (CRS), leading to acute respiratory distress syndrome (ARDS), acute kidney and cardiac injury, liver dysfunction, and multiorgan failure. Although several studies have discussed the role of 5-lipoxygenase (5-LOX) in viral infections, such as influenzae and SARS, it remains unexplored in the pathophysiology of COVID-19. 5-LOX acts on free arachidonic acid (AA) to form proinflammatory leukotrienes (LTs). Of note, numerous cells involved with COVID-19 (e.g., inflammatory and smooth muscle cells, platelets, and vascular endothelium) widely express leukotriene receptors. Moreover, 5-LOX metabolites induce the release of cytokines (e.g., tumour necrosis factor-α [TNF-α], interleukin-1α [IL-1α], and interleukin-1β [IL-1β]) and express tissue factor on cell membranes and activate plasmin. Since macrophages, monocytes, neutrophils, and eosinophils can express lipoxygenases, activation of 5-LOX and the subsequent release of LTs may contribute to the severity of COVID-19. This review sheds light on the potential implications of 5-LOX in SARS-CoV-2-mediated infection and the anticipated therapeutic role of 5-LOX inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: The leukotriene pathway may be implicated in the induction of virus-induced inflammation. Respiratory epithelial cells may express low levels of 5-lipoxygenase (5-LO) and release leukotrienes (LTs) C4, D4, and E4, upon exposure to viruses or other stimuli. Enhanced expression of 5-LO pathway proteins after rhinovirus (RV) infection has previously been described. We hypothesized that anti-leukotriene treatment of epithelial cells, with or without exposure to RV-infected peripheral blood mononuclear cells (PBMCs)-conditioned media, may inhibit RV-induced up-regulation of inflammatory cytokines. Methods: PBMCs from a healthy donor were exposed to RV1B and supernatants were harvested at 48 h post infection. BEAS-2B cells were infected with RV, with or without conditioning with the PBMC supernatant. Treatment with anti-LT agents was performed either on both PBMCs and BEAS-2B or at the bronchial epithelial level only, with varying concentrations of montelukast (CysLT receptor antagonist) or MK-886 [FLAP(5-lipoxygenase-activating-protein) inhibitor]. Evaluation of the inflammatory cytokines IL-8, RANTES, IL-11, IL-6, and IP-10 was performed using ELISA. Results: Our results show that anti-LT treatment of RV-infected bronchial epithelial cells suppresses epithelial RV-mediated cytokine production, independent of conditioning. Conclusions: This observation may represent an indirect mode of action of the anti-leukotrienes in virus-induced asthma. [ABSTRACT FROM AUTHOR]

It is well established that cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) play a vital role in the initiation and progression of inflammatory reactions. Hence, thiazole and thiazolidene-based pharmacophore molecules were synthesized to obtain dual COX-2 and 5-LOX inhibitory activity. The synthesis of target compounds has been achieved by a novel green strategy. In vitro COX-1, COX-2, and 5-LOX evaluation of these molecules have shown the potential for an improved anti-inflammatory profile. Most promising compound among the series (2-(diphenylamino)-4-(4-nitrophenyl)thiazol-5-yl)(naphthalen-1-yl)methanone 7h (IC50 = 0.07 ± 0.02 μM) showed equivalent COX-2 inhibitory potency as that of positive control etoricoxib (IC50 = 0.07 ± 0.01 μM) and an enhanced selectivity index of 115.14. Compound 7h exhibited 5-LOX IC50 of 0.29 ± 0.09 μM and reference drug zileuton showed IC50 of 0.15 ± 0.05 μM. In vivo studies of 7h including carrageenan-induced paw edema assay (63% inhibition of paw edema), antiulcer studies, biochemical assays, qRT-PCR analysis, and anticancer studies indicated that the present study has identified a good lead compound for the development of a potential anti-inflammatory drug having improved gastric safety profile. [ABSTRACT FROM AUTHOR]

Inflammation secondary to tissue injuries serves as a double-edged sword that determines the prognosis of tissue repair. As one of the most important enzymes controlling the inflammation process by producing leukotrienes, 5-lipoxygenase (5-LOX, also called 5-LO) has been one of the therapeutic targets in regulating inflammation for a long time. Although a large number of 5-LOX inhibitors have been explored, only a few of them can be applied clinically. Surprisingly, phosphorylation of 5-LOX reveals great significance in regulating the subcellular localization of 5-LOX, which has proven to be an important mechanism underlying the enzymatic activities of 5-LOX. There are at least three phosphorylation sites in 5-LOX jointly to determine the final inflammatory outcomes, and adjustment of phosphorylation of 5-LOX at different phosphorylation sites brings hope to provide an unrecognized means to regulate inflammation. The present review intends to shed more lights into the set-point-like mechanisms of phosphorylation of 5-LOX and its possible clinical application by summarizing the biological properties of 5-LOX, the relationship of 5-LOX with neurodegenerative diseases and brain injuries, the phosphorylation of 5-LOX at different sites, the regulatory effects and mechanisms of phosphorylated 5-LOX upon inflammation, as well as the potential anti-inflammatory application through balancing the phosphorylation-depended set-point. [ABSTRACT FROM AUTHOR]

Massive neutrophil infiltration is an early key event in infectious inflammation, accompanied by chemotactic leukotriene (LT)B4 generation. LTB4 biosynthesis is mediated by 5-lipoxygenase (5-LOX), but which pathogenic factors cause 5-LOX activation during bacterial infections is elusive. Here, we reveal staphylococcal exotoxins as 5-LOX activators. Conditioned medium of wild-type Staphylococcus aureus but not of exotoxin-deficient strains induced 5-LOX activation in transfected HEK293 cells. Two different staphylococcal exotoxins mimicked the effects of S. aureus-conditioned medium: (1) the pore-forming toxin α-hemolysin and (2) amphipathic α-helical phenol-soluble modulin (PSM) peptides. Interestingly, in human neutrophils, 5-LOX activation was exclusively evoked by PSMs, which was prevented by the selective FPR2/ALX receptor antagonist WRW4. 5-LOX activation by PSMs was confirmed in vivo as LT formation in infected paws of mice was impaired in response to PSM-deficient S. aureus. Conclusively, exotoxins from S. aureus are potent pathogenic factors that activate 5-LOX and induce LT formation in neutrophils. [ABSTRACT FROM AUTHOR]

Brain accumulation of increasing amount of phosphorylated microtubule associated tau protein is one the major hallmark lesions of Alzheimer’s disease (AD) and related tauopathies. Consistent evidence from clinical and animal studies has shown that neuroinflammation characterizes these diseases. The 5-lipoxygenase (5LO) is an enzyme protein whose metabolic products are lipids with potent inflammatory actions. Previously, we showed that blockade of 5LO activation ameliorates the phenotype of the htau transgenic mice. Here, by employing a vector system to overexpress 5LO in the brain of the same mouse model, we investigated its role and contribution to their behavioral deficits and development of tau neuropathology. Compared with controls, 5LO gene targeted mice manifested significant impairments in their memory and learning ability. On the other hand, brain tissues of the same mice had higher 5LO protein level and activity which resulted in intense neuroinflammation and synaptic pathology. Further, the same mice had a significant elevation of tau phosphorylation, which associated with the activation of the cdk5 kinase and an accumulation of insoluble tau. The functional involvement of this kinase in the 5LO-dependent tau phosphorylation was confirmed in neuronal cells. Taken together, our findings demonstrate that neuronal 5LO is directly involved in tau phosphorylation and tau neuropathology, and for this reason, it should be considered a good therapeutic target for tauopathies. [ABSTRACT FROM AUTHOR]

Previously undescribed chromenyl derivatives, characterized as 7-(2′-ethyl-1′-hydroxynonan-2′-yl)-6,7,8,8a-tetrahydro-3 H-isochromen-1-(5 H)-one ( 1) and 6-(3-(( E)-3-(furan-2′-yl)-prop-3-en-3-yl)-4a,5,6,8a-tetrahydro-8-methyl-2 H-chromen-6-yl)-ethyl-5′′-methyl-hexanoate ( 2) were isolated from ethyl acetate-methanol extract of yellow-foot bivalve clam, Paphia malabarica. Their structures have skeletons composed of isochromen-(5 H)-one and furanyl-2 H-chromenyl moieties, which are reported for the first time in marine organism. These were characterized by extensive one and two-dimensional nuclear magnetic resonance, infrared, and high-resolution mass spectroscopic experiments. The title compounds were evaluated for therapeutic potentials with regard to anti-inflammatory and antioxidant properties compared to known standards. These compounds exhibited comparable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities with α-tocopherol (IC ~0.6-0.7 mg/mL). The significantly higher anti-5-lipoxygenase activity of the title compounds (IC 0.76-0.82 mg/mL) than ibuprofen (IC 0.93 mg/mL) indicated their potential anti-inflammatory properties. The selectivity indices (IC anti-cyclooxygenase-1/ICanti-cyclooxygenase-2) of compounds 1 (1.19) and 2 (1.31) well established its safety profiles as an anti-inflammatory when compared to known drug, ibuprofen (0.44). The target bioactivities of chromenyl derivatives were guided by hydrophobic and electronic parameters. These compounds can be used as potential bioactive leads in functional food and medicinal applications. [ABSTRACT FROM AUTHOR]

Previous studies from our laboratories showed that an anti-inflammatory drug, 5-lipoxygenase inhibitor zileuton, attenuates ischemic brain damage via inhibiting inflammatory reaction. However, it was elusive whether zileuton attenuates inflammatory reaction and ischemic brain damage through the modulation of PI3K/Akt signaling pathway. In the present study, we, for the first time, investigated whether PI3K/Akt pathway was involved in zileuton's anti-inflammatory and neuroprotective properties against brain damage following experimental ischemic stroke. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), then received treatment with zileuton or vehicle after the onset of ischemia. LY294002 was injected intracerebroventricularly to inhibit the activation of PI3K/Akt signaling pathway selectively. Neurological deficit scores, cerebral infarct volume, morphological characteristic and cerebral water content were assessed 24 h after cerebral ischemia. The enzymatic activity of myeloperoxidase (MPO) was measured 24 h after cerebral ischemia. Expression of p-Akt, t-Akt and COX-2 in ischemic brain were determined by western blot. NF-κB p65 immuno-positive cells in ischemic brain were detected 24 h after cerebral ischemia. The content of TNF-α in blood was examined by ELISA. 5-LOX inhibitor zileuton significantly reduces neurological deficit scores, cerebral infarct volume, cerebral water content, ischemic neuronal injury and the enzymatic activity of MPO, all of which were abolished by LY294002 administration. Zileuton significantly up-regulates the expression of p-Akt, which was inhibited by LY294002 administration. Zileuton significantly down-regulates the over-expression of NF-κB p65 and COX-2, and attenuates the release of TNF-α, all of which were disminished by LY294002 administration. These results suggest that zileuton attenuates ischemic brain damage by inhibiting inflammatory reaction through the activation of PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Cocoa is a rich source of polyphenols that has been traditionally used as the treatment of several types of inflammation related disease. The response to inflammation comprises the consecutive release of mediators and the enlistment of circulating leukocytes, such as macrophages. Currently, Cocoa-derived polyphenolics have shown anti-inflammatory effects in vivo, but the therapeutic benefits in vitro remain unclear. Therefore, in this study, the effect of cocoa polyphenolic extract (CPE) on RAW 264.7 macrophage cells sensitized by lipopolysaccharide as in vitro inflammatory model was investigated. The anti-inflammatory activity of CPE was assessed by measuring its ability to inhibit the pro-inflammatory enzyme 5-lipoxygenase (5-LOX) and the pro-inflammatory mediators prostaglandin E (PGE), reactive oxygen species (ROS), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α). The results show that CPE significantly inhibits 5-LOX activity ( p < 0.01). In addition, CPE dose-dependently suppressed the production of PGE, ROS, NO and TNF-α in RAW 264.7 cells. These data suggest that CPE may be used for the treatment of inflammation and it's related-diseases. [ABSTRACT FROM AUTHOR]

Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity. [ABSTRACT FROM AUTHOR]

Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism and are markers and mediators of pulmonary inflammation. Research over the past two decades has established that LTs modulate inflammation in pulmonary arterial hypertension (PAH). The purpose of this review was to summarize the current knowledge of LTs in the pathophysiology of PAH and to highlight a recent study that advances our understanding of how leukotriene B (LTB) specifically contributes to pulmonary vascular remodeling. The results of these studies suggest that pharmacological inhibition of LT pathways, especially LTB, has high potential for the treatment of PAH. [ABSTRACT FROM AUTHOR]

New potential inhibitors of 5-lipoxygenase (5-LOX) based on the structure of 2-(3-benzoylphenyl)propanoic acid (an active component of the nonsteroidal antiinflammatory drug Ketoprofen) were designed using the SARD-21 program. The docking of these compounds in the active site of 5-LOX suggested that seven compounds can interact with this enzyme. Two of them can also be dual inhibitors of 5-LOX and two isoforms of cyclooxygenase. [ABSTRACT FROM AUTHOR]

A fifty-four compound series of 5-lipoxygenase and cyclooxygenase inhibitory activity of substituted 3,4-dihydroxychalcones was subjected to the development of a robust quantitative structure-activity relationship (QSAR) and pharmacophore model and the investigation of structure-activity relationship analysis using Molecular Design Suite software version 3.5. The requirements for the 5-lipoxygenase and cyclooxygenase activity are explored with 2D, group based and k-Nearest Neighbor studies. Simulated annealing is applied as variable selection methods for an effective comparison and model development. Several statistical expressions were developed using partial least square (PLS) analysis. The best QSAR models were further validated by leave-one-out method of cross-validation. The statistically significant best 2D-QSAR model was selected, having correlation coefficient r = 0.9338, and cross-validated squared correlation coefficient q = 0.7832 with external predictive ability of pred_ r = 0.8169 was developed by simulated annealing PLS with the descriptors like Average −ve potential, SsCHE-index, SsClE-index, SsOH count, and HUMO Energy. Group based QSAR model indicates that molar refractivity and methoxy, ethoxy, carboxylic groups in R1 positions can enhance activity. The obtained 3D-QSAR ( k-Nearest Neighbor) model using simulated annealing as a variable selection method has an excellent correlation coefficient value ( r = 0.8537) along with good statistical significance as shown by high Fisher's ratio ( F = 73.86). The model also exhibits good predictive power confirmed by the high value of cross-validated correlation coefficient ( q = 0.7841). The k-Nearest Neighbor contour maps suggest some important structural features like electronegative substituents which are essential for the activity exhibited by these compounds, and inclusion of electron-donating substituents will enhance the 5-lipoxygenase and cyclooxygenase inhibition activity. The pharmacophore analysis of the molecules demonstrated that the aromatic/aliphatic and hydrogen bond donor features are important pharmacophore contours favorable for these activities. The information rendered by 2D-QSAR, group based and 3D-QSAR models may lead to a better understanding of structural requirements of chalcone derivatives and also aid in designing novel potent 5-lipoxygenase and cyclooxygenase molecules. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharide ascophyllan was isolated from the brown algae Padina tetrastromatica and purified by ion-exchange chromatography. Anti-inflammatory effect of ascophyllan fraction against carrageenan-induced paw edema in rats was studied. Paw edema in rats was induced by injecting 0.1 ml, 1 % carrageenan suspension in 0.9 % NaCl solution into the sub-plantar tissue of the right hind paw. Carrageenan caused a significant increase in the activity of inflammatory marker enzymes like lipoxygenases and cyclooxygenase in peripheral blood mononuclear cells and paw tissue and also increased the concentration of prostaglandin E2 (PGE2) and myeloperoxidase (MPO) in paw tissue. When compared to the reference drug diclofenac, ascophyllan fraction-3 ( AF3) treatment significantly reduced the activities of anti-inflammatory enzymes, concentration of PGE2 and MPO. AF3 treatment decreased the mRNA level expression of TNF-α and IL-6. Concentration of thiobarbituric acid reactive substances was decreased. Activities of antioxidant enzymes and reduced glutathione level were increased on treatment with AF3. Histopathology of paw tissue showed decreased edema formation and cellular infiltration on supplementation with AF3. Thus the results demonstrated the potential beneficiary effect of ascophyllan fraction on carrageenan-treated rats. [ABSTRACT FROM AUTHOR]

Previous studies from our laboratory demonstrated that zileuton, a selective 5-lipoxygenase (5-LOX) inhibitor, attenuates ischemic brain damage in rats of focal cerebral ischemia. Enormous evidences showed that inflammatory reaction and neuronal apoptosis are the two important pathophysiological events in ischemia-induced brain damage. Our previous studies demonstrate that zileuton attenuates ischemic brain damage via inhibiting inflammatory reaction. The present study was performed to explore whether 5-LOX inhibitor zileuton attenuates neuronal apoptosis following focal cerebral ischemia and further investigate the potent mechanisms underlying its neuroprotection. Adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO) for 72 h, then received intragastric gavage with zileuton or vehicle as a bolus after the onset of MCAO. Neurological deficit scores, cerebral infarct volume, and neuronal damage were measured 72 h after MCAO. TUNEL staining was performed to measure the extent of neuronal apoptosis. Reverse transcription-polymerase chain reaction was performed to determine the expression of caspase-1 mRNA. Western blot was performed to determine the expression of procaspase-3 and cleaved caspase-3 in rat brain. Neurological deficit scores, infarct volume, and neuronal damage were significantly attenuated by administration of zileuton. MCAO caused the elevation of neuronal apoptosis, which was significantly inhibited by the administration of zileuton. MCAO caused the over-expression of caspase-1 and cleaved caspase-3, both of which were significantly inhibited by the administration of zileuton. Expression of procaspase-3 was reduced after MCAO, which was significantly up-regulated by administration of zileuton. Our studies suggested that 5-LOX inhibitor zileuton reduces MCAO-induced brain damage and neuronal apoptosis, which might be associated with the inhibition of caspase-1 and the regulation of caspase-3. [ABSTRACT FROM AUTHOR]

Molecular modeling and pharmacophore study has been performed on a series of 2-Substituted-1-naphthols derivatives with potent 5-lipoxygenase inhibitory activity. Structural features responsible for the activity of the compounds were characterized by using physicochemical, topological, and electrotopological descriptors, calculated from the Molecular Design Suite Software (V-life MDS™ 3.5). The relationship developed between biological activity and electrostatic, steric, hydrophobicity parameters was explored to generate combinatorial library with various substitution. This library can be explored for the synthesis and study of future potential anti-inflammatory agent. The requirements for the 5-lipoxygenase inhibitory activity are explored with 2D-QSAR, group based QSAR (G-QSAR), and k- nearest neighbour studies. The statistically significant 2D-QSAR model having r = 0.8502 and q = 0.8144 with pred_ r = 0.8072 and the best group based QSAR (G-QSAR) model having r = 0.9137 and q = 0.7808 with pred_ r = 0.8490 was developed by simulated annealing based partial least squares method. Further analysis using k- nearest neighbour 3D-QSAR technique identifies two models obtained by simulated annealing (SA) and genetic algorithm (GA)-based partial least squares methods leading to 5-lipoxygenase activity prediction. The best simulated annealing QSAR model showed q = 0.8547, r = 0.8963, and standard error = 0.3677. It was observed that steric properties predicted by k- nearest neighbour MFA contours can be related to 5-lipoxygenase activity. The predictive ability of the resultant model was evaluated using a test set molecules and the predicted r = 0.8162. The best pharmacophore model with hydrophobic, hydrogen bond donor (HBD), and aromatic features has root mean square deviation (RMSD) of 0.5746. The QSAR model suggests that electron-withdrawing character is favorable for the 5-lipoxygenase inhibitory activity. This suggests that electron-withdrawing groups like a chlorine or fluorine atoms at naphthol moiety are good for anti-inflammatory activity. In addition to the electron-withdrawing character, electron-donating groups, hydrophobic and hydrogen bond donor groups positively contribute to the 5-lipoxygenase inhibition. The results of 2D-QSAR, Group based QSAR, and k- nearest neighbour 3D-QSAR studies give detailed structural insights as well as highlights important binding features of these 2-Substituted-1-naphthols derivatives as anti-inflammatory agents which can provide guidance for the rational design of novel potent 5-lipoxygenase inhibitors. [ABSTRACT FROM AUTHOR]

A cyclodextrin-modified microemulsion electrokinetic chromatography method employing head column field-amplified sample stacking was developed for the analysis of arachidonic acid metabolites of the lipoxygenase pathways. The influence of the concentration of boric acid, the surfactant sodium dodecyl sulfate, the co-surfactant 1-butanol and the oil phase octane as well as the pH of the background electrolyte, the separation voltage and the separation temperature was studied. The optimized microemulsion consisting of 20 mM boric acid buffer, pH 9.0, 3.0 % (m/v) sodium dodecyl sulfate, 0.5 % (v/v) octane, 5.0 % (v/v) 1-butanol and 15 mM α-cyclodextrin enabled the separation of 20-hydroxy-leukotriene B, leukotriene B, 6- trans-leukotriene B, 6- trans-12- epi-leukotriene B, 5( S)-hydroxy-6- trans-8,11,14- cis-eicosatetraenoic acid, 12( S)-hydroxy-5,8,14- cis-10- trans-eicosatetraenoic acid, 15( S)-hydroxy-5,8,11- cis-13- trans-eicosatetraenoic acid as well as the internal standard prostaglandin B in <10 min employing a separation voltage of 17.5 kV at a temperature of 23 °C. A matrix peak from solid-phase extraction sample workup co-migrated with 5( S)-hydroxy-6- trans-8,11,14- cis-eicosatetraenoic acid affecting peak integration. The addition of 5 % (v/v) 2-propanol to the microemulsion resulted in the separation of this eicosatetraenoic acid and the matrix components at the expense of analysis time and peak resolution between the diastereomers 6- trans-leukotriene B and 6- trans-12- epi-leukotriene B. In summary, the MEEKC method appeared to be especially suitable for the more polar arachidonic acid metabolites. [ABSTRACT FROM AUTHOR]

Adventitia is the outer part of the arterial wall where the inflammatory response often occurs. Urotensin II (UII) is a potent vasoconstrictive peptide that also promotes the inflammatory process in patients with cardiovascular disease. Leukotriene C (LTC), a lipid mediator, was recently found to play a role in the inflammatory process in the artery. We hypothesized that the adventitia is one of the resources of LTC and that UII may promote LTC production through the 5-LO (5-lipoxygenase) pathway in adventitial fibroblasts. Rat adventitial fibroblasts were isolated and incubated in serum-free medium with either UII alone or in combination with inhibitors of p38 MAPK, ERK, and UII receptors. The expression of 5-LO was detected using real-time polymerase chain reaction and Western blot. The translocation and binding activity of nuclear factor (NF)-κB were measured using immunofluorescence and electrophoretic mobility shift assay, respectively. The production of LTC was measured by enzyme-linked immunosorbent assay. The results indicated that: (1) adventitial fibroblasts were a source of LTC production; (2) UII increased the expression of the 5-LO mRNA and the protein by NF-κB activation through p38 MAPK and ERK pathways; and (3) UII promoted the LTC release in fibroblasts through the 5-LO pathway by p38 MAPK and ERK activations. The 5-LO pathway mediates LTC production, which may be a new mechanism in the pathogenesis of the vascular adventitial inflammation caused by UII. [ABSTRACT FROM AUTHOR]

Structural features characteristic of high-, medium-, and low-efficiency inhibitors of 5-lipoxygenase (5-LOG) catalytic activity were established. The degree of their influence on the inhibition efficiency was assessed. Two models (M1 and M2) were constructed for the prediction and recognition of the efficiency intervals of 5-LOG inhibitors. The confidence level of activity prediction for M1 and M2 reached 90 and 70%, respectively. The revealed structural features could be used for constructing highly selective inhibitors of 5-LOG catalytic activity. [ABSTRACT FROM AUTHOR]

Although some anti-allergic activities of the rhizome of Atractylodes japonica have been previously reported, the active principle(s) for anti-allergic action is not fully elucidated and the effect of this plant material on atopic dermatitis (AD) is not known. In this study, the 70% ethanol extract of the rhizome of A. japonica was found to significantly inhibit 5-lipoxygenase (5-LOX)-catalyzed leukotrienes (LT) production from rat basophilic leukemia (RBL)-1 cells. From the extract of A. japonica, three major sesquiterpene derivatives including atractylenolide I, atractylenolide III and eudesma-4,7-dien-8-one were successfully isolated. Among these compounds, only atractylenolide I was shown to strongly inhibit 5-LOX from RBL-1 cells (IC = 18.6 μM). To evaluate the effects of experimental AD, the ethanol extract of A. japonica (200 mg/day) was administered orally to hapten-treated NC/Nga mice which is an animal model of AD. It was firstly found that the extract significantly inhibited AD-like symptoms in mice, as judged by severity score and scratching behavior. Taken together, it is concluded that A. japonica possesses the inhibitory activity on 5-LOX and an animal model of AD, and atractylenolide I may contribute, at least in part, to these anti-allergic actions of A. japonica. [ABSTRACT FROM AUTHOR]

Lipoxin A (LXA), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA inhibits the upregulation of both leukotriene B (LTB) and leukotriene C (LTC) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati ( P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C (LTC) generation in mast cells. Cit suppresses both degranulation and the generation of LTC in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca, phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A (PLA) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation. [ABSTRACT FROM AUTHOR]

Background: It has recently been found that 5-lipoxygenase (5-LO) and cytochrome P450-2J2 (CYP2J2), molecules capable of arachidonic acid (AA) metabolism, might promote cancer cell viability through several mechanisms similar to those of cyclooxygenase-2 (COX-2). We found that not only COX-2 expression, but also the expression of 5-LO and CYP2J2 is up-regulated in head and neck squamous cell carcinoma (HNSCC) cell lines. From these observations, we hypothesized that AA metabolism by 5-LO and/or CYP2J2 may lower the efficacy of anti-cancer effect by COX-2 inhibition. Methods and Results: Although COX-2 was highly expressed in all cell lines tested, COX-2-specific inhibition showed little growth-inhibitory effect in some cell lines. Inhibition of COX-2 resulted in increased production of LTB and 14-15-DHET/EET, metabolites of 5-LO and CYP2J2, respectively. Combined knock-down of COX-2 and 5-LO or CYP2J2 by siRNA results in a decrease in cell proliferation and VEGF production. Furthermore, these results are dependent on 5-LO and CYP2J2 expression in cells. Conclusion: Therefore, combined inhibition of COX-2 and 5-LO or CYP2J2 may be one way to overcome low efficacy of single inhibition of COX-2 in cancer cells. In addition, combined therapies should be chosen based on the expression of members of other AA metabolism pathways. [ABSTRACT FROM AUTHOR]

Cyclooxygenase (COX)-1, COX-2, and 5-lipoxygenase (5-LOX) enzymes produce effectors of pain and inflammation in osteoarthritis (OA) and many other diseases. All three enzymes play a key role in the metabolism of arachidonic acid (AA) to inflammatory fatty acids, which contribute to the deterioration of cartilage. AA is derived from both phospholipase A (PLA) conversion of cell membrane phospholipids and dietary consumption of omega-6 fatty acids. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the COX enzymes, but show no anti-5-LOX activity to prevent the formation of leukotrienes (LTs). Cysteinyl LTs, such as LTC, LTD, LTE, and leukoattractive LTB accumulate in several organs of mammals in response to NSAID consumption. Elevated 5-LOX-mediated AA metabolism may contribute to the side-effect profile observed for NSAIDs in OA. Current therapeutics under development, so-called 'dual inhibitors' of COX and 5-LOX, show improved side-effect profiles and may represent a new option in the management of OA. [ABSTRACT FROM AUTHOR]

Purpose: Pancreatic cancer has one of the highest fatality rates of all cancers, and new strategies or reagents to tackle this disease are needed. Triptolide (TL) is able to potently inhibit the growth of pancreatic tumor cells in vitro. On the other hand, blockage of 5-LOX pathway might be useful for treatment of pancreatic cancer. In the current study, we tested the effects of 5-LOX RNA interference and TL individually or in combination in suppressing human pancreatic tumor growth in xenograft mouse model. Methods: 5-LOX short hairpin RNA (shRNA) vectors were developed and screened out for their efficacy in human pancreatic cancer cell line SW1990 in vitro. Their antitumor effects were also evaluated by measuring cell proliferation and apoptosis. An effective 5-LOX shRNA was given alone or in combination with TL to treat pancreatic tumor xenograft. Expression levels of 5-LOX and VEGF were measured with Western blotting and immunohistology. Results: Knocking down 5-LOX gene suppressed cancer cell growth in vitro and intra-tumoral delivering of 5-LOX shRNA inhibited growth of transplanted tumor in vivo. TL treatment induced tumor suppression and greatly enhanced antitumor effects of 5-LOX shRNA in the mouse model. 5-LOX RNA interference or TL treatment suppresses VEGF expression in tumor tissue, and combined treatment further reduces its expression. Conclusions: Both treatments exerted antitumor effects in vivo, and combined use of the two approaches produced more powerful antitumor effects. Synergistic effects of combined treatment in VEGF expression may contribute to the mechanisms of the strong antitumor effects. [ABSTRACT FROM AUTHOR]

Cerumen, or propolis, is a mixture of plant resins enriched with bee secretions. In Australia, stingless bees are important pollinators that use cerumen for nest construction and possibly for colony's health. While extensive research attests to the therapeutic properties of honeybee ( Apis mellifera) propolis, the biological and medicinal properties of Australian stingless bee cerumen are largely unknown. In this study, the chemical and biological properties of polar extracts of cerumen from Tetragonula carbonaria in South East Queensland, Australia were investigated using gas chromatography-mass spectrometry (GC-MS) analyses and in vitro 5-lipoxygenase (5-LOX) cell-free assays. Extracts were tested against comparative (commercial tincture of A. mellifera propolis) and positive controls (Trolox and gallic acid). Distinct GC-MS fingerprints of a mixed diterpenic profile typical of native bee cerumen were obtained with pimaric acid (6.31 ± 0.97%, w/w), isopimaric acid (12.23 ± 3.03%, w/w), and gallic acid (5.79 ± 0.81%, w/w) tentatively identified as useful chemical markers. Characteristic flavonoids and prenylated phenolics found in honeybee propolis were absent. Cerumen extracts from T. carbonaria inhibited activity of 5-LOX, an enzyme known to catalyse production of proinflammatory mediators (IC 19.97 ± 2.67 μg/ml, mean ± SEM, n = 4). Extracts had similar potency to Trolox (IC 12.78 ± 1.82 μg/ml), but were less potent than honeybee propolis (IC 5.90 ± 0.62 μg/ml) or gallic acid (IC 5.62 ± 0.35 μg/ml, P < 0.001). These findings warrant further investigation of the ecological and medicinal properties of this stingless bee cerumen, which may herald a commercial potential for the Australian beekeeping industry. [ABSTRACT FROM AUTHOR]

From the 70% ethanol extract of the rhizomes of Cyperus rotundus (CRE), several major constituents including the sesquiterpene derivatives (valencene, nootkatone, and caryophyllene α-oxide), monoterpenes (β-pinene, 1,8-cineole, and limonene) and 4-cymene were isolated and examined for their anti-allergic activity in vitro and in vivo. In rat basophilic leukemia (RBL)-1 cells, the sesquiterpenes strongly inhibited 5-lipoxygenase-catalyzed leukotrienes production. In addition, they inhibited β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, with valencene having the highest inhibitory effect. CRE inhibited leukotrienes production and β-hexosaminidase release at 300 μg/mL. It was also found that the most active sesquiterpene (valencene) and CRE inhibited β-hexosaminidase degranulation by inhibiting the initial activation reaction, Lyn phosphorylation, in IgE-stimulated RBL-2H3 cells. Moreover, CRE, valencene and nootkatone significantly inhibited the delayed-type hypersensitivity reaction in mice when administered orally at 50-300 mg/kg. In conclusion, C. rotundus and its constituents, valencene, nootkatone, and caryophyllene α-oxide, exert anti-allergic activity in vitro and in vivo. These sesquiterpenes, but not monoterpenes, certainly contribute to the anti-allergic activity of the rhizomes of C. rotundus. [ABSTRACT FROM AUTHOR]

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300-400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5-10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2-10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed. [ABSTRACT FROM AUTHOR]

Objective: Twice-daily flavocoxid, a cyclooxygenase and 5-lipoxygenase inhibitor with potent antioxidant activity of botanical origin, was evaluated for 12 weeks in a randomized, double-blind, active-comparator study against naproxen in 220 subjects with moderate-severe osteoarthritis (OA) of the knee. As previously reported, both groups noted a significant reduction in the signs and symptoms of OA with no detectable differences in efficacy between the groups when the entire intent-to-treat population was considered. This post-hoc analysis compares the efficacy of flavocoxid to naproxen in different subsets of patients, specifically those related to age, gender, and disease severity as reported at baseline for individual response parameters. Methods: In the original randomized, double-blind study, 220 subjects were assigned to receive either flavocoxid (500 mg twice daily) or naproxen (500 mg twice daily) for 12 weeks. In this subgroup analysis, primary outcome measures including the Western Ontario and McMaster Universities OA index and subscales, timed walk, and secondary efficacy variables, including investigator global assessment for disease and global response to treatment, subject visual analog scale for discomfort, overall disease activity, global response to treatment, index joint tenderness and mobility, were evaluated for differing trends between the study groups. Results: Subset analyses revealed some statistically significant differences and some notable trends in favor of the flavocoxid group. These trends became stronger the longer the subjects continued on therapy. These observations were specifically noted in older subjects (>60 years), males and in subjects with milder disease, particularly those with lower subject global assessment of disease activity and investigator global assessment for disease and faster walking times at baseline. Conclusions: Initial analysis of the entire intent-to-treat population revealed that flavocoxid was as effective as naproxen in managing the signs and symptoms of OA of the knee. Detailed analyses of subject subsets demonstrated distinct trends in favor of flavocoxid for specific groups of subjects. [ABSTRACT FROM AUTHOR]

5-Lipoxygenase inhibitor zileuton has been demonstrated to attenuate ischemic brain damage in rats of permanent focal cerebral ischemia in previous work. To further investigate the mechanism underlying zileuton's neuroprotection, adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO), then received treatment with zileuton or vehicle after the onset of ischemia. Neurological deficit, cerebral infarction, and morphological characteristic were measured 6 and 24 h after MCAO. The enzymatic activity of myeloperoxidase (MPO) was assessed 6 and 24 h after MCAO and the lipid peroxidation levels were evaluated by malondialdehyde assay. Expression of nuclear factor-kappa B (NF-κB) p65 in rat brain was detected by immunohistochemistry and Western blot. Expression of inducible nitric oxide synthase (iNOS) in rat brain was determined by RT-PCR and Western blot. Nitric oxide production in rat brain was also measured 24 h after MCAO. The concentration of TNF-α and IL-1β in serum were detected by ELISA. Zileuton significantly reduced neurological deficit scores, cerebral infarct volume, MPO activity, and the lipid peroxidation levels. It also inhibited the expression of NF-κB and decreased the expression and activity of iNOS in rat brain. In addition, zileuton attenuated the release of TNF-α and IL-1β in serum. Our results suggest that zileuton reduces inflammatory reaction and brain damage in a rat model of permanent focal cerebral ischemia. The neuroprotective effect of zileuton in cerebral ischemia might be associated with the inhibition of inflammatory reaction. [ABSTRACT FROM AUTHOR]

Introduction: Flavocoxid is a novel flavonoid-based 'dual inhibitor' of the 5-lipoxygenase (5-LOX) enzyme and the cyclooxygenase (COX) enzymes. This study was designed to compare the effectiveness and safety of flavocoxid to naproxen in subjects with moderate to severe osteoarthritis (OA) of the knee. Methods: In this randomized, multicenter, double-blind study, 220 subjects were assigned to receive either flavocoxid (500 mg twice daily) or naproxen (500 mg twice daily) for 12 weeks. The trial was structured to show noninferiority of flavocoxid to naproxen. Primary outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and subscales and a timed walk. Results: More than 90% of the subjects in both groups noted significant reduction in the signs and symptoms of knee OA. There were no statistically significant differences in efficacy between the flavocoxid and naproxen groups when the entire intent-to-treat population was analyzed. The flavocoxid group had significantly fewer upper gastrointestinal (UGI) and renal (edema) adverse events (AEs) as well as a strong trend toward fewer respiratory AEs. Conclusion: Flavocoxid, a first-in-class flavonoid-based therapeutic that inhibits COX-1 and COX-2 as well as 5-LOX, was as effective as naproxen in managing the signs and symptoms of OA of the knee. Flavocoxid demonstrated better UGI, renal (edema), and respiratory safety profiles than naproxen. [ABSTRACT FROM AUTHOR]

We aimed to study the intracellular expression of 5-lipoxygenase (5-LOX), the primary competitor with cyclooxygenase-2 in arachidonic acid metabolism, as inflammatory enzymes may be involved in blocking apoptosis and promoting cancer growth by changing arachidonic acid metabolism within cells. Our purpose was to investigate the possible connection between 5-LOX expression and colon carcinogenesis by characterizing 5-LOX expression in histologically different colonic adenomas, determining the relationship between high expression of 5-LOX and various conventional clinicopathological features of adenomas, and finally characterizing the histological localization of cells with 5-LOX overexpression. A total of 111 patients were examined and 120 histologically different colonic adenomas analyzed (including four cases of intramucosal adenocarcinoma in a polyp). Immunohistochemical staining with polyclonal anti-5-LOX antibodies was performed. There was a significant correlation between high 5-LOX expression and patient age, increased polyp size, high grade of intraepithelial neoplasia, villous and tubulovillous adenoma, and histological epithelial localization. We observed a strong positive correlation between 5-LOX overexpression and the appearance of typical high-risk factors for malignant transformation in adenomatous polyps. The results support the role of 5-LOX in early stages of colon carcinogenesis. [ABSTRACT FROM AUTHOR]

AbstractAim:To explore a novel function of a mutant of the hepatitis B virus X protein (HBxΔ127) in the promotion of hepatoma cell migration.Methods:The effect of HBxΔ127 and wild type HBx on the migration ability of hepatoblastoma HepG2 cells were examined using wound healing assays in stable transfection systems. The full-length osteopontin(OPN) promoter sequence was cloned into the pGL3-Basic plasmid. The promoter activities of OPN in stably HBxΔ127-transfected hepatoblastoma HepG2 (HepG2-XΔ127) and hepatocellular carcinoma H7402 (H7402-XΔ127) cells were determined using luciferase reporter gene assays. The mRNA expression levels of OPN were detected by RT-PCR. And the effect of MK886, a specific inhibitor of 5-lipoxygenase (5-LOX), on OPN promoter activity and mRNA expression in HepG2-XΔ127 and H7402-XΔ127 cells were examined using luciferase reporter gene assays and RT-PCR, respectively. Finally, the migration ability of HepG2-XΔ127 was observed after treatment with siRNA targeting OPN mRNA and HBx mRNA using wound healing assays.Results:HepG2-XΔ127 cells exhibited a greater capacity for wound repair compared to HepG2-X cells. The promoter activity and mRNA expression levels of OPN were also increased in HepG2-XΔ127 and H7402-XΔ127 cells. Moreover, MK886 abolished the HBxΔ127-mediated upregulation of OPN. Wound healing assays demonstrated that the migration ability of HepG2-XΔ127 cells can be suppressed by treatment with siRNA targeting OPN mRNA and siRNA targeting HBx mRNA.Conclusion:HBxΔ127 strongly promotes hepatoma cell migration via activation of OPN involving 5-LOX. [ABSTRACT FROM AUTHOR]

Mounting evidence suggests that lipoxygenase (LO)-catalyzed products may play a key role in the development and progression of human cancers. In this study, we analyzed the effects of a 5-LO inhibitor, which inhibits the conversion of arachidonic acid to leukotrienes, on cell proliferation and apoptosis in human malignant glioma cells, including 5-LO-expressing cells U-87MG, A172 and 5-LO non-expressing cell U373. Growth of U-87MG and A172 cells, but not that of U373 cells, was inhibited in a dose-dependent manner by treatment with MK886. Similarly, specific 5-LO silencing by small interfering RNA reduced the growth of U-87MG and A172 cells. MK886 treatment reduced 5-LO activity independently of 5-LO-activating protein (FLAP) in human malignant glioma cells. MK886 treatment also induced cell apoptosis, measured by DNA fragmentation and nuclear condensation, in U-87MG and A172 cells but there were no signs in U373 cells. Moreover, this treatment reduced ERKs phosphorylation and anti-apoptotic molecule Bcl-2 expression, and increased Bax expression in U-87MG and A172 cells. In summary, our results show there is a link between the 5-LO expression status and the extent of MK886-inhibited cell proliferation and apoptosis. Taken together, this study suggest that 5-LO is a possible target for treating patients with gliomas, and 5-LO inhibition might be potent therapy for patients with 5-LO-expressing malignant gliomas. [ABSTRACT FROM AUTHOR]

AbstractAim:Previously, we identified a natural mutant of hepatitis B virus X gene (HBx) with a deletion from 382 to 401 base pairs (termed HBxΔ127), which could potently enhance growth of hepatoma cells. In this study, we further investigated the mechanism of increased hepatoma cell growth that was mediated by HBxΔ127.Methods:We examined the effect of HBxΔ127 on the transcription factor sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS) in a model of HepG2-XΔ127 (or H7402-XΔ127) cells, which was stably transfected with HBxΔ127 gene in a human hepatoma HepG2 (or H7402) cell line.Results:Relative to wild type HBx, HBxΔ127 was able to potently activate SREBP-1c at the levels of promoter activity, mRNA and protein by a luciferase reporter gene assay, RT-PCR and Western blot analysis. Then, using the treatment with MK886, a specific 5-lipoxygenases (5-LOX) inhibitor, (or 5-LOX siRNA) we identified that 5-LOX was responsible for the upregulation of SREBP-1c by luciferase reporter gene assay, RT-PCR and Western blot analysis. Because FAS was a target gene of SREBP-1c, we further showed that HBxΔ127 was able to strongly activate the promoter activity of FAS and upregulated the mRNA expression level of FAS as well, by luciferase reporter gene assay and RT-PCR. In function, flow cytometry analysis revealed that FAS contributed to the growth of hepatoma cells that was mediated by HBxΔ127, using cerulenin (a FAS inhibitor).Conclusion:HBxΔ127 promotes hepatoma cell growth through activating SREBP-1c involving 5-LOX. [ABSTRACT FROM AUTHOR]

Ruta graveolens L. (Rutaceae) are traditionally used for the treatment of rheumatism, arthritis and other inflammatory conditions in the traditional medicine of India, were evaluated for their protective effect in acute and chronic models of inflammation. Carrageenan induced rat paw edema and adjuvant induced arthritis were employed as the experimental models of acute and chronic inflammation respectively. Isolated polyphenolic and alkaloid fraction ( AFR) from Ruta graveolens and evaluated its anti inflammatory activity in carrageenan induced acute model. AFR with a dose 10 mg/kg showed higher anti inflammatory effect than polyphenols and standard drug diclofenec. AFR significantly decreased the paw edema in arthritic rats. TBARS, COX-2, 5-LOX and MPO level were decreased and the levels of antioxidant enzymes and GSH level were increased on treatment with AFR. The increment in CRP level and ceruloplasmin level observed in arthritic animals were also found to be significantly restored in AFR treated rats. The results demonstrated the potential beneficiary effect of isolated polyphenolic and alkaloid fraction of Ruta graveolens L. on acute and chronic models of inflammation in rats. [ABSTRACT FROM AUTHOR]

AbstractAim:To determine whether the flavonoid baicalin attenuates oxygen-glucose deprivation (OGD)-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase (5-LOX) activation in PC12 cells.Methods:The effects of baicalin and the 5-LOX inhibitor zileuton on the changes induced by OGD/recovery or H2O2 (an exogenous reactive oxygen species [ROS]) in green fluorescent protein-5-LOX-transfected PC12 cells were compared.Results:Both baicalin and zileuton attenuated OGD/recovery- and H2O2-induced injury and inhibited OGD/recovery-induced production of 5-LOX metabolites (cysteinyl leukotrienes) in a concentration-dependent manner. However, baicalin did not reduce baseline cysteinyl leukotriene levels. Baicalin also reduced OGD/recovery-induced ROS production and inhibited 5-LOX translocation to the nuclear envelope and p38 phosphorylation induced by OGD/recovery and H2O2. In contrast, zileuton did not show these effects.Conclusion:Baicalin can inhibit 5-LOX activation after ischemic injury, which may partly result from inhibition of the ROS/p38 mitogen-activated protein kinase pathway. [ABSTRACT FROM AUTHOR]

We previously showed that, in vitro, hyperforin from St. John’s wort ( Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo . Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B4 formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo. [ABSTRACT FROM AUTHOR]

Background and objective: Umbelliprenin, the natural prenylated coumarin distributed in the plants of apiaceae family, has shown various biological activities, especially as a cancer chemopreventive agent. In the present study, umbelliprenin, was examined for in vitro antioxidant activity, in vitro inhibitory activity against lipoxygenase, and in vivo anti-inflammatory activity. Methods: The applied tests were interaction with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, inhibition of lipid peroxidation, inhibition of soybean lipoxygenase and in vivo inhibition of the carrageenin-induced rat paw edema. Results: Umbelliprenin did not show any significant antioxidant activity but exhibited a remarkable and potent inhibition against soybean lipoxygenase (IC50 = 0.0725 µM). This compound, in the in vivo anti-inflammatory test, could also inhibit the carrageenin induced paw edema significantly (39 %). Conclusion: The observed inhibition of lipoxygenase may be a plausible mechanism for the potent cancer chemopreventive activity of umbelliprenin and may pose this compound as a valuable agent for the treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

We previously used an integrative genetics approach to demonstrate that 5-lipoxygenase (5-LO) deficiency in mice ( Alox5 −/−) protects against atherosclerosis despite increasing lipid levels and fat mass. In the present study, we sought to further examine the role of 5-LO in adiposity and pancreatic function. Alox5 −/− and wild-type (WT) mice were characterised with respect to adiposity and glucose/insulin metabolism using in vivo and in vitro approaches. The role of ALOX5 in pancreatic function in human islets was assessed through short interfering RNA (siRNA) knockdown experiments. Beginning at 12 weeks of age, Alox5 −/− mice had significantly increased fat mass, plasma leptin levels and fasting glucose levels, but lower fasting insulin levels ( p < 0.05). Although Alox5 −/− mice did not exhibit insulin resistance, they had impaired insulin secretion in response to a bolus glucose injection. Histological analyses revealed that Alox5 −/− mice had increased islet area, beta cell nuclear size, and numbers of beta cells/mm2 islet ( p < 0.05), indicative of both hyperplasia and hypertrophy. Basal and stimulated insulin secretion in isolated Alox5 −/− islets were significantly lower than in WT islets ( p < 0.05) and accompanied by a three- to fivefold decrease in the expression of the genes encoding insulin and pancreatic duodenal homeobox 1 ( Pdx1). Direct perturbation of ALOX5 in isolated human islets with siRNA decreased insulin and PDX1 gene expression by 50% and insulin secretion by threefold ( p < 0.05). These results provide strong evidence for pleiotropic metabolic effects of 5-LO on adiposity and pancreatic function and may have important implications for therapeutic strategies targeting this pathway for the treatment of cardiovascular disease. [ABSTRACT FROM AUTHOR]

5-Methoxy-8-(2-hydroxy-3-buthoxy-3-methylbutyloxy)-psoralen (MP) is a medicinal herbal product isolated from Angelica dahurica that inhibits the cyclooxygenase-2 (COX-2)-dependent phase of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (IC50, 23.5 μM). Western blotting with specific anti-COX-2 antibodies showed that the decrease in PGD2 production was accompanied by a decrease in COX-2 protein levels. In addition, this compound consistently inhibited the production of leukotriene C4 in a dose dependent manner, (IC50, 2.5 μM). These results demonstrate that MP inhibits both cyclooxygenase-2 and 5-lipoxygenase activity. Furthermore, this compound also inhibited the degranulation reaction (IC50, 4.1 μM). Therefore, this compound might provide a basis for novel anti-inflammatory drug development. [ABSTRACT FROM AUTHOR]

Meso-dihydroguaiaretic acid (MDGA) is a medicinal herbal product isolated from the aerial parts of Saururus chinensis that inhibits the cyclooxygenase-2 (COX-2)-dependent phase of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) (IC50 9.8 μM). However, this compound did not inhibit COX-2 protein expression in BMMC at concentrations up to 30 μM, indicating that MDGA directly inhibits COX-2 activity. In addition, this compound consistently inhibited the production of leukotriene C4 (IC50 1.3 μM). These results demonstrate that MDGA inhibits both COX-2 and 5-lipoxygenase. Furthermore, this compound strongly inhibited the degranulation reaction in BMMC (IC50 11.4 μM). Therefore, this compound might provide a basis for novel anti-inflammatory drug development. [ABSTRACT FROM AUTHOR]

5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine’s actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine’s actions. [ABSTRACT FROM AUTHOR]

In our ongoing search for anti-inflammatory agents originating from Korean medicinal plants, we found that the hexane and BuOH fractions of the MeOH extract from the whole plants of Melandrium firmum Rohrbach inhibited 5-lipoxygenase (5-LOX) activity. By activity-guided fractionation, eleven compounds, α-spinaterol ( 1), ursolic acid ( 2), ergosterol peroxide ( 3), α-spinaterol glucoside ( 4), 2-methoxy-9-β-D-ribofuranosyl purine ( 5), aristeromycin ( 6), ecdysteron ( 7), polypodoaurein ( 8), (-)-bornesitol ( 9), mannitol ( 10) and cytisoside ( 11) were isolated from the hexane and BuOH fractions using column chromatography. Compounds 2, 5, 6, 8, 9, 10 and 11 were isolated for the first time from this plant. Compounds 1, 3, 4 and 7 inhibited 5-LOX activity with IC50 values of 21.04 μM, 42.30 μM, 32.82 μM, and 17.18 μM, respectively. [ABSTRACT FROM AUTHOR]