Specific 5-lipoxygenase Inhibitor Research Articles

An Anti-Inflammatory Composition of Boswellia serrata Resin Extracts Alleviates Pain and Protects Cartilage in Monoiodoacetate-Induced Osteoarthritis in Rats.

The boswellic acids, the active compounds in Boswellia serrata gum resin extract, are potent anti-inflammatory agents and are specific nonredox inhibitors of 5-Lipoxygenase (5-LOX). Here, we present the anti-osteoarthritis (OA) efficacy of LI13019F1 (also known as Serratrin®), a unique composition containing the acidic and nonacidic fractions of B. serrata gum resin. This composition strongly inhibited 5-LOX activity with the half-maximal inhibitory concentration (IC50) of 43.35 ± 4.90 μg/mL. Also, LI13019F1 strongly inhibited the leukotriene B4 (IC50, 7.80 ± 2.40 μg/mL) and prostaglandin E2 (IC50, 6.19 ± 0.52 μg/mL) productions in human blood-derived cells. Besides, LI13019F1 reduced TNF-α production with the IC50 of 12.38 ± 0.423 μg/mL. On average, 1, 2.5, and 5 μg/mL doses of LI13019F1 protected 34.62, 47.66, and 62.29% SW1353 human chondrosarcoma cells from IL-1β induced SOX-9 depletion, respectively. Further, a 28-day preclinical proof-of-concept study evaluated the pain relief efficacy of LI13019F1 in monoiodoacetate- (MIA-) induced Sprague-Dawley rats. At the end of the study, 150 and 300 mg/kg doses of LI13019F1 supplemented rats showed significant improvements (55.17 ± 5.81 g (p < 0.05), and 66.22 ± 6.30 g (p < 0.05), respectively, vs. MIA: 31.22 ± 7.15 g) in body-weight-bearing capacities. Concurrently, LI13019F1-150 and LI13019F1-300 rats substantially (p < 0.05) increased the threshold of pain sensitivity to pressure (26.98 ± 2.36 and 28.06 ± 2.72-gram force, respectively; vs. 18.63 ± 5.82 in MIA) and increased (p < 0.05) the latent time to withdraw the paw after a thermal stimulus (23.61 ± 2.73 and 28.18 ± 1.90 sec, respectively; vs. 16.56 ± 1.22 sec. in MIA). Besides, the histological observations on Safranin-O green stained articular cartilage revealed that LI13019F1 also prevented the MIA-induced structural damage of the cartilage and reduced the loss of the extracellular matrix (ECM) components in the experimental rats. In conclusion, the present observations suggest that LI13019F1, a new composition of B. serrata gum resin extracts, reduces pain and protects articular cartilage from the damaging action of MIA in a rodent model.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

Detoxification of benzo[a]pyrene primarily depends on cytochrome P450, while bioactivation involves additional oxidoreductases including 5-lipoxygenase, cyclooxygenase, and aldo-keto reductase in the liver.

Cytochrome P450s are involved in detoxification and activation of benzo[a]pyrene (BaP) with unclear balance and unknown contribution of other oxidoreductases. Here, we investigated the BaP and BaP-induced mutagenicity in hepatic and extra-hepatic tissues using hepatic P450 reductase null (HRN) gpt mice. After 2-week treatment (50mg/kg, i.p. 4 days), BaP in the liver and lung of HRN-gpt mice were increased. BaP promoted gpt mutant frequency (MF) in HRN-gpt mice liver. MF of gpt in the lung and Pig-a in hematopoietic cells induced by BaP in HRN-gpt mice were increased than in gpt mice. BaP-7,8-diol-9,10-epoxide (BPDE)-DNA adducts in vitro was analyzed for enzymes detection in BaP bioactivation. Specific inhibitors of 5-lipoxygenase, cyclooxygenase-1&2, and aldo-keto reductase resulted in more than 80% inhibition rate in the DNA adduct formation, further confirmed by Macaca fascicularis hepatic S9 system. Our results suggested the detoxification of BaP primarily depends on cytochrome P450, while the bioactivation involves additional oxidoreductases.

Baicalein attenuates angiotensin II-induced cardiac remodeling via inhibition of AKT/mTOR, ERK1/2, NF-κB, and calcineurin signaling pathways in mice.

Baicalein, a specific lipoxygenase (LOX) inhibitor, has anti-inflammatory and antioxidant effects. However, the functional role of baicalein in angiotensin II (Ang II)-induced hypertension and cardiac remodeling remains unclear. Here we investigated the effect of baicalein on cardiac hypertrophy and fibrosis and the underlying mechanism. Wild-type (WT) mice were injected with Ang II (1,200ng/kg/min) alone or together with 12/15-LOX inhibitor baicalein (25mg/kg) for 14 days. Histological examinations were performed on heart sections with hematoxylin and eosin, Masson's trichrome, wheat germ agglutinin staining, and immunohistochemistry. The messenger RNA (mRNA) expression of cytokines and protein levels were detected by real-time polymerase chain reaction (PCR) and western blot analysis respectively. Ang II infusion significantly increased blood pressure but decreased cardiac contractile function reflected by fractional shortening% and ejection fraction% compared with saline-treated mice. Moreover, Ang II infusion resulted in marked cardiac hypertrophy and fibrosis, promoted accumulation of macrophages and T cells, the expression of proinflammatory cytokines and malondialdehyde (MDA) production. However, these actions were markedly reversed by administration of baicalein in mice. Mechanistically, the protective effects of baicalein were associated with the inhibition of inflammation, oxidative stress, and multiple signaling pathways (AKT/mTOR, ERK1/2, nuclear factor-κB (NF-κB), and calcineurin) in the Ang II-treated mice. This study demonstrates that baicalein can significantly ameliorate Ang II-induced hypertension and cardiac remodeling, and may be a novel therapeutic drug for prevention of hypertensive heart diseases.

Methyl jasmonate-induced cell death in grapevine requires both lipoxygenase activity and functional octadecanoid biosynthetic pathway

Grapevine (Vitis vinifera L., cv. Limberger) leaf tissues and suspension-cultured cells were induced to undergo programmed cell death (PCD) by exogenously added methyl jasmonate (MeJA). The elicitor signaling pathway involved in MeJA-induced PCD was further investigated using pharmacological, biochemical and histological approaches. Pharmacological dissection of the early events preceding the execution of MeJA-triggered PCD indicated that this process strongly depends on both, de novo protein and mRNA synthesis. Treatment of leaf discs and cell suspensions with lipase inhibitor Ebelactone B and specific lipoxygenase inhibitor Phenidone blocked MeJA-induced PCD. These results suggest that some chloroplast membrane-derived compound(s) is required for MeJA-induced PCD in grapevine. The progression of MeJAtriggered PCD may be further inhibited by the use of metabolic inhibitors of key enzymes of octadecanoid biosynthesis including AOS, AOC, and OPR indicating that the functional jasmonate biosynthetic pathway is an integral part of the MeJA-induced signal transduction cascade that results in the coordinate expression of events leading to PCD. Finally, the activation of the octadecanoid pathway, as a critical point in MeJA-induced PCD, was independently demonstrated with cellulysin, a macromolecular elicitor acting via octadecanoid signaling. The cellulysin was shown to be a very potent enhancer of MeJA-triggered PCD in grapevine cells.

Abstract 5615: Combination therapy for chronic myelogenous leukemia: Targeting the BCR-ABL coiled-coil domain and other pathways for synthetic lethality

Abstract Single-agent tyrosine kinase inhibitor (TKI) therapy for chronic myeloid leukemia (CML) has been largely effective in keeping the disease at bay. However, resistance to therapy and persistence of a subset of leukemic cells demonstrate the necessity for multiple-agent therapy. We have developed a unique interfering peptide, CCmut2 (Dixon et al., JBC 2011) able to disrupt BCR-ABL dimers. This disruption of trans-auto phosphorylation exacts its activity via the coiled-coil domain in BCR, but leads to an overall similar effect as TKIs - induction of apoptosis and reduction in proliferation. Here we detail the results of this peptide (delivered as a gene and transcribed in vitro) in combination with small molecule or biologic agents. The secondary agents include: chloroquine which is used to inhibit lysosomal acidification and block cellular macroautophagy; zileuton, a specific inhibitor of arachidonate 5-lipoxygenase (Alox5) known to enhance the proliferative capacity of CML cells; and GO-201, an interfering peptide targeting MUC-1, a known oncogene expressed in CML cells. These agents in combination with CCmut2 were hypothesized to improve therapeutic potency and enhance leukemia-specific cytotoxicity. All assays were performed in BCR-ABL expressing K562 cells in vitro. Western blots confirming knockdown of the autophagy gene ATG7, Alox5, and MUC-1 using shRNA were carried out. The autophagy pathway was further validated by blotting for autophagic microtubule-associated protein light chain 3 (LC3) conversion from LC3-I to LC3-II following combination therapy with CCmut2 and chloroquine. Transformative ability of K562 cells was evaluated using a methylcellulose colony forming assay following co-transfection with CCmut2 and a shRNA. Next, the specific drugs targeting these pathways were also used in combination with the CCmut2 and assessed for apoptotic potential using caspase 3/7 activity and DNA segmentation. Proliferation was investigated using trypan blue exclusion and transformative ability was further assessed with multi-drug combinations. MUC-1 inhibition in combination with CCmut2 resulted in a potent combination in K562 cells. Other combinations hinted at an additive or synergistic enhancement of apoptosis and reduction in proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5615. doi:1538-7445.AM2012-5615

Synthesis of Volatile Compounds of Virgin Olive Oil Is Limited by the Lipoxygenase Activity Load during the Oil Extraction Process

The aim of this work was to determine whether the lipoxygenase (LOX) activity is a limiting factor for the biosynthesis of virgin olive oil (VOO) volatile compounds during the oil extraction process. For this purpose, LOX activity load was modified during this process using exogenous LOX activity and specific LOX inhibitors on olive cultivars producing oils with different volatile profiles (Arbequina and Picual). Experimental data suggest that LOX activity is a limiting factor for the synthesis of the oil volatile fraction, this limitation being significantly higher in Picual cultivar than in Arbequina, in line with the lowest content of volatile compounds in the oils obtained from the former. Moreover, there is evidence that this limitation of LOX activity takes place mostly during the milling step in the process of olive oil extraction.

Novel insights into the mechanism of decreased expression of type X collagen in human mesenchymal stem cells from patients with osteoarthritis cultured on nitrogen‐rich plasma polymers: Implication of cyclooxygenase‐1

Recent evidence indicates that a major drawback of current cartilage- and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from patients with osteoarthritis rapidly express type X collagen (COL10A1), a marker of late stage chondrocyte hypertrophy associated with endochondral ossification. We recently demonstrated that COL10A1 expression was inhibited in MSCs from patients with osteoarthritis cultured on nitrogen-rich plasma polymerized (PPE:N) coatings. Here, we sought to understand the mechanisms of action of this effect by culturing MSCs on PPE:N surfaces in the presence of different inhibitors of kinases and cyclooxygenases. The effect of PPE:N surfaces on COL10A1 expression was found to be mimicked by the cyclooxygenase inhibitor NPPB, but not by daphnetin (an inhibitor of protein kinases) nor by genistein (an inhibitor of tyrosine kinases). COL10A1 expression was also suppressed by the specific cyclooxygenase-1 (COX-1: SC-560) and 5-lipoxygenase (5-LOX: MK-866) inhibitors, but not by COX-2 (COX-2 inhibitor 2) and 12-LOX (baicalein) inhibitors. Finally, the incubation of MSCs on PPE:N surfaces inhibited the expression of COX-1 while 5-LOX was not expressed in these cells. Taken together, these results indicate that PPE:N surfaces inhibit COL10A1 expression via the suppression of COX-1.

A mutant of hepatitis B virus X protein (HBxΔ127) enhances hepatoma cell migration via osteopontin involving 5-lipoxygenase

To explore a novel function of a mutant of the hepatitis B virus X protein (HBx Delta 127) in the promotion of hepatoma cell migration. The effect of HBx Delta 127 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 HBx Delta 127-transfected hepatoblastoma HepG2 (HepG2-X Delta 127) and hepatocellular carcinoma H7402 (H7402-X Delta 127) 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-X Delta 127 and H7402-X Delta 127 cells were examined using luciferase reporter gene assays and RT-PCR, respectively. Finally, the migration ability of HepG2-X Delta 127 was observed after treatment with siRNA targeting OPN mRNA and HBx mRNA using wound healing assays. HepG2-X Delta 127 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-X Delta 127 and H7402-X Delta 127 cells. Moreover, MK886 abolished the HBx Delta 127-mediated upregulation of OPN. Wound healing assays demonstrated that the migration ability of HepG2-X Delta 127 cells can be suppressed by treatment with siRNA targeting OPN mRNA and siRNA targeting HBx mRNA. HBx Delta 127 strongly promotes hepatoma cell migration via activation of OPN involving 5-LOX.

Protein expression of 5-lipoxygenase and activation and cytotoxicity of Benzidine in human bronchial epithelial cells

To investigate the effect of intracellular 5-lipoxygenase on oxidation of benzidine in HBE cells and to provide further evidence that lipoxygenase is an alternative pathway for the oxidation of xenobiotics mediated by cytochrome P450. Enzyme system test: Soybean lipoxygenase (SLO), substrate (benzidine) and other components reacted in the enzyme system, followed by detection of the reaction products by spectrophotometry. In vitro test: After HBE cells were exposed to benzidine, the protein levels of 5-lipoxygenase in HBE cells were assessed by Western-blot, and the DNA damage by the single cell gel electrophoresis. At last, the effect of the specific inhibitor of 5-lipoxygenase (AA861) on 5-lipoxygenase protein expression and DNA damage in HBE cells were detected. SLO could catalyze the co-oxidation of benzidine to generate benzidine diimine in the presence of hydrogen peroxide. Under optimal condition, numax value of the oxidation of benzidine catalyzed by SLO was 1.42 nmol*min(-1) SLO, and the Km value of benzidine was 1.48 mmol/L. EGCG could inhibit the oxidation of benzidine by SLO. Benzidine could induce 5-lipoxygenase protein expression in HBE cells, but AA861 was invalid. Benzidine caused DNA damage in HBE cells, which could be significantly inhibited by AA861. 5-LOX protein expression in HBE cells can be regulated by benzidine, which suggests that the co-oxidation of benzidine by 5-LOX could produce into electrophile that could covalently bind to DNA and induce DNA damage, which could be one of the mechanisms for carcinogenesis of BZD. 5-LOX inhibitor AA861 can inhibit this effect.

Mechanisms involved in the cell cycle and apoptosis of HT-29 cells pre-treated with MK-886 prior to photodynamic therapy with hypericin

In our previous study we have proved that colon cancer cells HT-29 pre-treated with specific 5-lipoxygenase inhibitor MK-886 became more susceptible to photodynamic therapy (PDT) with hypericin and we also found that this mutual combination induced cell cycle arrest and stimulated onset of apoptosis (Kleban et al., 2007. J. Photochem. Photobiol. B 84, 2). To further explain events associated with MK-886 mediated sensitization of tumor cells toward PDT with hypericin, more detailed study of signaling pathways leading to increase in apoptosis as well as cell cycle perturbations was performed and is presented herein. Intensive accumulation of HT-29 cells in G 0/ G 1 phase of cell cycle led to expression analyses of several G 0/ G 1 checkpoint molecules (cyclin A, cyclin E, cdk-2, pRb). Similarly, accumulation of apoptotic cells invoked analyses of key molecules involved in apoptotic signaling (caspase-3, -8, -9; PARP; Lamin B; Mcl-1; Bax) by Western blotting and caspase activity assay. Long term survival of cells was examined by clonogenicity test. As the effect of PDT is mediated by ROS production, levels of hydrogen peroxides and superoxide anion were monitored by flow cytometric analyses. In addition, an impact of MK-886 on LTB 4 production and expression of 5-LOX was monitored. Massive G 0/ G 1 arrest in the cell cycle accompanied by increase in cyclin E level and decrease/absention of cyclin A, cdk-2 and pRb expression indicated incapability for G 1/ S transition. Minimal changes in cleavage of procaspases observed in cells treated with non-toxic concentrations of either agent alone or their mutual combination were not quite in line with their activity (caspase-3, -8, -9) which was significantly increased mainly in combinations. Treatment with non-toxic concentration of MK-886 had minimal influence over ROS production compared to control cells. In contrast, hypericin alone markedly increased the level of ROS, but no additional effect of MK-886 pre-treatment was detected. Further analyses of particular ROS groups unveiled an impact of increasing MK-886 concentration on superoxide accumulation accompanied with depletion of hydrogen peroxide level within the cells. The clonogenicity test revealed disruption of colony formation after mutual combination of both agents as compared to MK-886 or PDT alone. In conclusion, we presume that stimulation of apoptosis in our experimental model was accomplished preferentially through the mitochondrial pathway, although caspase-8 activation was also noticed. Interestingly, pre-treatment with MK-886 modulated distribution of ROS production in mutual combination with PDT.

Protection of mouse brain from aluminum-induced damage by caffeic acid.

The natural product caffeic acid is a specific inhibitor of 5-lipoxygenase (5-LOX); it also possesses antioxidant and antiinflammatory properties. The current study was designed to determine whether the neuroprotective properties of caffeic acid are due to inhibition of 5-LOX. Cerebral damage was induced in mice by intracerebroventricular microinjection of aluminum (5.0 microg aluminum in 2.0 microL, once a day, for 5 days). Caffeic acid was administered intragastrically at 30 min prior to aluminum and repeated daily for an additional 10 days. The brain injury was determined by observation of behavioral changes in mice, as well as by measuring biochemical and pathological changes in the cerebral tissue. The levels of 5-LOX proteins and 5-LOX mRNA expression were measured in brain tissue. Aluminum impaired learning and memory in mice produced neuronal death in hippocampi, elevated brain malondialdehyde levels, increased protein expression of amyloid precursor protein (APP), amyloid beta, and 5-LOX. It also increased 5-LOX mRNA expression and decreased choline acetyl transferase (ChAT) protein expression in the brain tissue of mice. Caffeic acid prevented brain damage as well as behavioral and biochemical changes caused by aluminum overload. The results of this study suggest that overexpression of 5-LOX accompanies the cerebral injury induced by aluminum overload in mice, and that selective inhibitors of 5-LOX may have potential value in the treatment of aluminum neurotoxicity and conceivably of diseases associated with neuronal injury.

Interleukin-4 inhibition of interleukin-1-induced expression of matrix metalloproteinase-3 (MMP-3) is independent of lipoxygenase and PPARγ activation in human gingival fibroblasts

BackgroundInterleukin 4 (IL-4) has been shown to suppress interleukin-1 (IL-1) induced expression of matrix metalloproteinase-3 (MMP-3) in human synovial and gingival fibroblasts, but the mechanism of suppression has not been determined. Activators of peroxisome proliferator-activated receptor-γ (PPARγ) have been shown to inhibit cytokine induced expression of MMPs in other cell types, and IL-4 has been shown to activate PPARγ by stimulating production of ligands through the lipoxygenase pathway. It has been suggested that PPARγ may inhibit expression of MMPs by competing with transcription factor AP-1 for binding to a putative composite binding element in the promoters. The objective of this study was to determine whether the suppressive effects of IL-4 on the IL-1 induced expression of MMP-3 involve activation of lipoxygenase and/or PPARγ.ResultsWestern blotting revealed the presence of PPARγ in nuclear extract of HGF. IL-1 induced binding of nuclear extract to the putative composite PPRE/AP-1 site was diminished in the presence of pioglitazone, but there was no evidence of any change in the composition of the retarded complexes, and no evidence of PPARγ binding to this site. Nordihydroguaiaretic acid (NDGA), a non-selective lipoxygenase inhibitor, and MK886, a specific inhibitor of 5-lipoxygenase, induced MMP-3 expression synergistically with IL-1. However IL-4 was still able to inhibit MMP-3 expression in the presence of NDGA or MK886 and IL-1. Activation of PPARγ with pioglitazone not only failed to inhibit IL-1 induced expression of MMP-3 mRNA, but rather super-induced MMP-3 in the presence of IL-1. PPARγ antagonist GW9662 failed to abolish the suppressive effects of IL-4. Another PPARγ activator, 15-deoxy-Delta12,14prostaglandin J2 (15dPGJ2), also super-induced MMP-3 mRNA, and this was due at least in part to increased transcription.ConclusionIL-4 suppression of IL-1-induced MMP-3 expression in HGF is independent of lipoxygenase activity and activation of PPARγ. Super-induction of MMP-3 by pioglitazone may have important implications for patients using pioglitazone to treat type II diabetes in the presence of chronic inflammation.

5‐LOX Inhibitor Modulates the Inflammatory Responses Provoked by Helicobacter pylori Infection

Arachidonic acid metabolites have been considered as pivotal mediators in Helicobacter pylori-induced inflammatory response, which are mainly metabolized by two distinct enzymes: cyclooxygenase (COX) and lipoxygenase (LOX). While COX has become well known to play a key role in either carcinogenesis or inflammation related to H. pylori infection, little is known regarding the implication of LOX in H. pylori infection. In this study, we evaluated the roles of 5-LOX and its metabolites in H. pylori-induced host responses and further a potential beneficial action of specific LOX inhibitors against H. pylori infection. Expressions of cytosolic phospholipase A(2) (cPLA(2)), COX-2, and 5-LOX after H. pylori infection were evaluated by immunofluorescence staining and Western blotting. Synthesis of LOX metabolites was measured with reversed-phase high-performance liquid chromatography. For analyzing the influence of 5-LOX inhibitors, nordihydroguaiaretic acid (NDGA) and geraniin, on H. pylori-induced inflammatory responses, RNase protection assay and RT-PCR were performed. H. pylori stimulated the translocation of cPLA(2) from cytoplasm to nucleus and increased the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) as a predominant form of 5S-HETE in gastric epithelium. NDGA exerted a strong suppression activity of H. pylori-induced 5-LOX signaling. The administration of LOX inhibitors was related with down-expression of proinflammatory mediators such as interleukin-8 and tumor necrosis factor-alpha in both H. pylori-infected gastric epithelial cells and macrophage cells. LOX modulation with its specific inhibitors could impose significant anti-inflammatory responses after H. pylori infection, based on the fact that H. pylori infection provoked gastric inflammation through metabolizing arachidonic acid by the 5-LOX pathway.

Co-Administration of Acetyl-11-Keto-β-Boswellic Acid, a Specific 5-Lipoxygenase Inhibitor, Potentiates the Protective Effect of COX-2 Inhibitors in Kainic Acid-Induced Neurotoxicity in Mice

Cyclooxygenase (COX) and lipoxygenase (LOX) are responsible for the metabolism of arachidonic acid into inflammatory metabolites, prostaglandins and leukotrienes, respectively. The upregulation of these enzymes in the central nervous system has been demonstrated to be responsible for the increased neuronal vulnerability to degeneration. Kainic acid, a glutamate receptor agonist and responsible for neuronal excitotoxicity and oxidative damage via different mechanisms, is capable of stimulating mRNA of both COX-2 and 5-LOX in the brain. The present study was designed to study the effects of COX inhibitors (indomethacin, nimesulide, rofecoxib) and a 5-LOX inhibitor (acetyl-11-keto-β-boswellic acid; AKBA) and the combination of these inhibitors (dual inhibition) on kainic acid induced excitotoxicity and oxidative and nitrosative damage in mice. The results from the present study indicated that AKBA, indomethacin, and nimesulide per se did not produce any change in the behavioural parameters after kainic acid administration; however, rofecoxib per seproduced a significant increase in the latency of clonic (seizure-like) movement and a decrease in mortality rate as compared with kainic acid treated animals. In combination studies AKBA, rofecoxib, and nimesulide produced a more pronounced effect than either of these drugs alone. Further, the effect of AKBA combined with rofecoxib was significantly more marked when compared with AKBA combined with nimesulide. Besides this, identical results were found for the effect of these agents and their combination against oxidative damage induced by kainic acid. These findings indicate the potential role of COX-2 inhibitors and also their combination with the 5-LOX inhibitor in kainic acid induced excitotoxicity and oxidative damage by virtue of their antioxidant effect and suggest the need for the development of dual inhibitors for the treatment of neuronal excitotoxicity.

Cancer Cells Cause Vascular Endothelial Cell (vEC) Retraction via 12(S)HETE Secretion; The Possible Role of Cancer Cell Derived Microparticle

Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7. In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM. These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.

Effect of arachidonic acid supplementation and cyclooxygenase/lipoxygenase inhibition on the development of early bovine embryos

The effect of arachidonic acid (AA) cascade on bovine embryo development in a granulosa cell co-culture system was studied. Arachidonic acid (100 µM) was supplemented from 1-cell to 8-16 cell block stage (first three days of co-culture) and from 1-cell to hatching. Specific cyclooxygenase (indomethacin, 28 µM) and lipoxygenase (nordihydroguaiaretic acid - NDGA, 28 µM) inhibitors were used from 1-cell to 8-16 cell block stage with AA. Embryo development was assessed by cleavage, day 7-day 8 and hatched embryo rates and by measuring growth rates through development stages found in days 7-10 of culture (day 0 = insemination day). Embryo quality was scored at day 8. A 6.5-10.4% increase on cleavage rate after AA supplementation was found. This AA supplementation from 1-cell to hatching delayed embryo growth rate beyond day 7 and a reduction on hatching rate was detected. When AA supplementation was restricted to the first three days of co-culture those negative effects were overcome. Also, indomethacin and NDGA prevented the positive effect of AA and induced a significant reduction on cleavage, respectively. NDGA further decreased day 7 embryo rate and quality. Results suggest that AA has a two-phase action on bovine embryos, promoting early development and impairing embryo growth from day 7 onwards and hatching rates. Both cyclooxygenase and lipoxygenase were found to be important pathways to promote cleavage.

Stimulation of Suicidal Erythrocyte Death by Lipoxygenase Inhibitor Bay-Y5884

The prostaglandin PGE₂, a metabolite of the cyclooxygenase pathway, activates Ca²⁺-permeable cation channels in erythrocyte cell membranes leading to entry of Ca²⁺ with subsequent eryptosis, i.e. cell shrinkage, breakdown of phosphatidylserine (PS) asymmetry and membrane blebbing, all features typical for apoptosis in nucleated cells. PS exposing cells are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether the specific lipoxygenase inhibitor Bay-Y5884 influences eryptosis. As determined by competitive ELISA, Bay-Y5884 (20 µM) enhanced the release of PGE₂ from human erythrocytes. According to whole-cell patch-clamp, Bay-Y5884 (20 µM) activated nonselective cation channels. The effect of Bay-Y5884 on cation channels was abolished by the cyclooxygenase inhibitor diclophenac (10 µM). Bay-Y5884 (30-40 µM) significantly increased erythrocyte free Ca²⁺ concentration and PS exposure as analyzed in flow cytometry by Fluo3 fluorescence and annexin-V binding, respectively. PS exposure triggered by 20 µM (but not by 40 µM) Bay-Y5884 was blunted by cyclooxygenase inhibitors acetylsalicylic acid (50 µM) and diclophenac (10 µM). In conclusion, the lipoxygenase inhibitor Bay-Y5884 enhances erythrocyte PGE₂ formation with subsequent activation of non-selective cation channels, Ca²⁺ entry and phospholipid scrambling.

Leukotriene B4 plays a pivotal role in CD40-dependent activation of chronic B lymphocytic leukemia cells

AbstractBiosynthesis of leukotrienes (LTs) occurs in human myeloid cells and B lymphocytes. However, the function of leukotrienes in B lymphocytes is unclear. Here, we report that B-cell chronic lymphocytic leukemia (B-CLL) cells produce leukotriene B4, and that specific leukotriene biosynthesis inhibitors counteracted CD40-dependent activation of B-CLL cells. Studies on the expression of the high-affinity receptor for LTB4 (BLT1) by flow cytometry analysis showed that the receptor was expressed, to a varying degree, in all investigated B-CLL clones. At a concentration of 100 nM, the drugs BWA4C (a specific 5-lipoxygenase inhibitor) and MK-886 (a specific 5-lipoxygenase activating protein inhibitor) markedly inhibited CD40-induced DNA synthesis (45% and 38%, respectively) and CD40-induced expression of CD23, CD54, and CD150. Addition of exogenous LTB4 (150 nM) almost completely reversed the effect of the inhibitors on DNA synthesis and antigen expression. Taken together, the results of the present study suggest that leukotriene biosynthesis inhibitors may have a therapeutic role in B-CLL.

Leukotriene B4 Plays a Pivotal Role in CD40 Dependent Activation of Chronic B Lymphocytic Leukemia Cells.

Biosynthesis of leukotrienes occurs in human myeloid cells and B lymphocytes. However, the function of leukotrienes in B lymphocytes is unclear. Here we report that B-cell chronic lymphocytic leukemia (B-CLL) cells produce leukotriene (LT) B4 and that specific leukotriene biosynthesis inhibitors counteracted CD40-dependent activation of B-CLL cells. Studies on the expression of the high affinity receptor for LTB4 (BLT1) by flow cytometry analysis showed that the receptor was expressed, to a varying degree, in all investigated B-CLL clones. The drugs BWA4C (a specific 5-lipoxygenase inhibitor) and MK-886 (a specific 5-lipoxygenase activating protein inhibitor), at a concentration of 100 nM, markedly inhibited CD40-induced DNA synthesis (45% and 38%, respectively) and CD40-induced expression of CD23, CD54 and CD150. Addition of exogenous LTB4 (150 nM) almost completely reversed the effect of the inhibitors on DNA synthesis and antigen expression.Taken together, the results of the present study suggest that leukotriene biosynthesis inhibitors may have a therapeutic role in B-CLL.