Suggestive evidence for the presence of an NADPH oxidase in platelets has been reported by us (10), as well as others (38, 39). None of these reports present rigorous evidence for a function- ing granulocyte-like NADPH oxidase system, however. Further- more, the activation of the NADPH oxidase and subsequent platelet fragmentation induced by Ab requires generation of the 12-LO product, 12(S)-HETE. This article presents such evidence based upon the following observations: (a) gel-filtered platelets are free of contaminating granulocytes/monocytes; (b) platelets from two different NADPH oxidase–deficient mice, p47 phox–/–
pathway has been implicated in tumor cell proliferation and motility, regulation of apoptosis, and in tumor angiogenesis (11–13). We have previously shown 12-LOX to be expressed in the prostate cancer cell lines DU-145 and PC3 (15); however, its exact role in these cells is not fully defined. The present study was aimed at investigating the effect of 12-LOX inhibition on prostate cancer cells and more impor- tantly, examining the mechanisms governing these effects. The results from this study indicate that the 12-LOX pathway is essential for prostate cancer growth and survival. Inhibition of 12-LOX by the specific inhibitors Baicalein or BHPP resulted in a dose-dependent decrease in proliferation of both DU-145 and PC3 cells. Inhibition of 5-LOX with Rev-5901 at similar concentrations had no effect on proliferation. These results are similar to those observed in pancreatic cancer cells after 5- or 12-LOX inhibition (13). However, 5-LOX does not appear to be as critical in the two prostate cancer lines studied
Cell culture and reagents. Liver macrophages were isolated from the liver of 7- to 10-week-old male C57BL/6 mice (Jackson Laboratory) by perfusion of liver using collagenase type IV (Sigma-Aldrich) as previ- ously described (25). For isolation of liver macrophages, nonparenchy- mal sufficient supernatant was centrifuged in 50%/25% Percoll (GE Healthcare). The layer containing liver macrophages was plated with RPMI-1640 (Hyclone) with 10% fetal bovine serum (FBS). The purity of liver macrophages exceeded 85% when estimated by either immu- nostaining or flow cytometry using anti-F4/80 (Santa Cruz Biotech- nology, sc-52664), anti–FITC-F4/80 (eBioscience, catalog 11-4801), anti–PE-CD11b (eBioscience, catalog 12-0112), or anti–APC-Ly6C (eBioscience, 17-5932) antibodies (3). Raw 264.7 and THP-1 cell lines were obtained from American Type Culture Collection (ATCC) and cul- tured in Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% FBS. Peritoneal macrophages were collected from the intra- peritoneal cavity filled with PBS and cultured in RPMI-1640 with 10% FBS. The cells were grown in an incubator with 5% CO 2 and 95% air at 37°C. Cells were incubated with DHA or MaR1 in free medium sup- plemented with 1% FA-free BSA or 0.1% FBS for 24 hours, respectively. DHA, IL-4, Wy-14,643, troglitazone, and 9-cis-retinoic acid were purchased from Sigma-Aldrich. SR1078 was purchased from Toc- ris Bioscience. T0901317 was purchased from Alexis Biomedicas. MaR1, MaR2, protectin D1, RvD1, RvD2, and RvE1 were purchased from Cayman Chemical.
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Computational selection of the best curcumin derivative Selectivity plays a major role in drug targeting process. It can help in identifying the most suitable ligand (key) for a specific enzyme (lock). Computational approaches are widely used nowadays to compute the free energy of binding, affinity and other parameters like LogP that have an indication of good fitting and predictive high selectivity. P-12-LOX is overexpressed in many tumor tissues . Arachidonic acid is metabolized by P-12- LOX to produce a hydroxyeicosatetraenoic acid that has been reported to be a main cause of cancer devel- opment [37, 38]. Thus, inhibition of P-12-LOX can decrease both cell proliferation and metastasis . Curcumin was reported to inhibit P-12-LOX (66 µmol/l) [28, 29]. Also, a number of synthetic curcuminoids were reported to have a promising P-12-LOX inhibitory activ- ity . Compound E26C, a curcumin derivative with benzofuran moiety, had P-12-LOX inhibitory activity IC50 = 17 µmol/l and showed the best fitting distance 3.3 Å among all the reported curcuminoids (Fig. 1). The discovery of a curcumin derivative that can possess high affinity toward P-12-LOX and can be radiolabeled for tumor imaging was the main objective of this study. Radiolabeling of a highly selective P-12-LOX inhibitor will also ensure high accuracy of tumor cells imaging, as this enzyme is overexpressed in tumor tissues as men- tioned before.
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Human neutrophils from peripheral blood may physically interact with platelets in several settings including hemostasis, inflammation, and a variety of vascular disorders. A role for lipoxygenase (LO)-derived products has been implicated in each of these events; therefore, we investigated the formation of lipoxins during coincubation of human neutrophils and platelets. Simultaneous addition of FMLP and thrombin to coincubations of these cells led to formation of both lipoxin A4 and lipoxin B4, which were monitored by reversed-phase high pressure liquid chromatography. Neither stimulus nor cell type alone induced the formation of these products. When leukotriene A4 (LTA4), a candidate for the transmitting signal, was added to platelets, lipoxins were formed. In cell-free 100,000 g supernatants of platelet lysates, which displayed 12-LO activity, LTA4 was also transformed to lipoxins. Platelet formation of lipoxins was inhibited by the LO inhibitor esculetin and partially sensitive to chelation of Ca2+, while neither acetylsalicylic acid nor indomethacin significantly inhibited their generation. In contrast, neutrophils did not transform LTA4 to lipoxins. Cell-free
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Generation of 12-LO KO mice. The generation of 12-LO KO mice has been described previously (14). The mice were backcrossed to the C57BL/6 genetic background 11 times (>99% C57BL/6). All animal studies were performed in accordance with guidelines set forth by the Research Animal Care Committees of City of Hope National Medical Center and University of Pennsylvania. Low dose STZ–induced diabetes. Male C57BL/6 and 12-LO KO mice aged 12–16 weeks were used in this study. Mice received 40 mg/kg of STZ intraperitoneally on 5 consecutive days between 0900 and 1000 hours. The mice were housed in a tem- perature-controlled room with 12-hour light/12-hour dark cycles. Mice were given free access to water and standard labo- ratory chow during these studies. Blood was obtained weekly from the retro-orbital plexus via capillary tube and used for glu- cose and insulin determinations. All specimens were obtained after an overnight fast. Mice were followed up for a total of 28 days, at which time they were sacrificed. We defined diabetes as fasting glucose >7.8 mM (∼140 mg/dL).
phosphate dibasic, monobasic potassium phosphate, hydrogen peroxide solution, thiobarbuturic acid, Xanthine oxidase from bovine milk (EC 220.127.116.11) and xanthine (2.6-dihydroxypurinol), allopurinol, hydrochloric acid, magnesium chloride, bovine serum albumine (BSA), 15-lipoxygenase (EC 18.104.22.168), linoleic acid and Boric acid were purchased from Sigma Aldrich chemie (Steinheim, Germany), ammonium ferric citrate, potassium persulfate, DPPH (2,2’-diphenyl-1- picrylhydrazyl) and trichoroacetic acid were supplied by Fluka chemie( Buchs, Switzerland); sulfuric acid, acetic anhydride, ferric trichlorure, hexane, chloroform, ethyl acetate, acetone, butanol, ethanol, methanol, sodium tetraborate, and potassium hexacyanoferrate [K 3 Fe(CN) 6 ] were sourced from Prolabo (Paris, France) ;
Oxidized LDL is present in human atherosclerotic lesions, but the mechanisms responsible for oxidation in vivo have not been definitively demonstrated. Circumstantial evidence has implicated the enzyme 15-lipoxygenase as a contributor to the formation of oxidized lipids in this disease. To assess whether oxidized lipids are indeed formed by the action of 15- lipoxygenase on polyunsaturated fatty acids (PUFAs) in vivo, we have used a sensitive and specific method (chiral phase HPLC) to analyze the lipid oxidation products present in human atherosclerotic lesions. Human 15-lipoxygenase is an omega-6 lipoxygenase that has previously been shown to oxidize esterified PUFA in a stereospecific manner, forming predominantly cholesteryl hydroperoxy-octadecadienoate (13(S)-HPODE) from cholesteryl linoleate substrate in LDL. This property allows its activity to be distinguished from
15-Lipoxygenase (15-LO) is a lipid-peroxidizing enzyme mainly expressed in airway epithelial cells, eosinophils, reticulocytes and macrophages. In humans, 15-LO exists as two different enzymes with different cell localizations and product profiles . 15-LO-1 converts AA to an unstable intermediate, 15- hydroperoxyeicosatetraenoic acid, which can be further con- verted to 15-hydroxyeicosatetraenoic acid (15-HETE). The 15- LO-1 enzyme has proinflammatory actions, with high levels of 15-HETE reported in sputum of asthmatic patients along with increased macrophage 15-LO-1 mRNA expression . 15- LO-1 expression is induced by IL-13 in human blood mono- cytes  and by IL-4 in monocytes, alveolar macrophages, dendritic cells, mast cells and rheumatoid arthritis synovial cells [12-18]. Only recently was it reported that 15-LO-1 can catalyze the metabolism of AA to the proinflammatory eoxins that can increase permeability of the endothelial cell monol- ayer in vitro, indicating that they can enhance vascular perme- ability . 15-LO-1 products, however, were also demonstrated to have protective roles in inflammatory disor- ders due to formation of anti-inflammatory lipoxins [20-22]. The 15-LO-1 mRNA was demonstrated to be present in RA synovial membranes  and its expression was stronger in RA compared with osteoarthritis (OA) biopsies .
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Various arachidonic acid (AA) metabolites are known to regulate immune cell function(s) and dictate the progression of both acute and chronic inflammatory reactions. Using a model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the in vivo effects of inhibitors of cyclooxygenase (CO) and lipoxygenase (LO) pathways on granuloma development and granuloma macrophage I-region-associated (Ia) antigen expression. In addition, by high performance liquid chromatography (HPLC) we have profiled the metabolism of AA by macrophages that are isolated from granulomatous foci, and have biochemically characterized the in vitro specificity and activity of selected CO and LO inhibitors. The development of hypersensitivity-type pulmonary granulomas in mice was dramatically suppressed by inhibitors with anti-LO activity (nordihydroguairetic acid (NDGA), nafazatrom, and BW755c) in a dose-dependent manner, while indomethacin, which is primarily CO-selective, had no significant effect. Furthermore, NDGA and nafazatrom profoundly arrested the normal progression of preformed granulomatous lesions. The inhibitors of the LO pathway also suppressed the in vivo kinetics of Ia antigen expression by granuloma macrophages. In contrast, indomethacin augmented Ia-antigen expression. The major AA metabolites that were synthesized by the granuloma
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EMT is a physiological process where epithelial cells are converted to mesenchymal cells by losing cellular polarity and cell – cell adhesion and acquiring invasive and migratory abil- ities. Recent studies indicate that EMT is a fundamental pro- cess in tumor progression and metastasis. 32 E-cadherin characterizes epithelial cells and plays a vital role in the main- tenance of intracellular binding structures. 33 The loss of E- cadherin is considered a critical event in the process of EMT. 34 Snail is a transcription factor involved in repressing the tran- scription of the E-cadherin gene by binding to the E-box of the E-cadherin promoter. 35 Dysregulation of Wnt/ β -catenin sig- naling genes has been reported in a variety of tumors including gastric, colorectal, breast and lung cancers. 8,9,36 – 39 Klamp ﬂ et al reported that in colorectal cancer, elevating the level of 12- LOX promoted Caco2 and SW480 cell migration by reducing the expression of E-cadherin and integrin- β 1. 40 Additionally, 12-LOX activity modulated by integrin β 4 is strongly corre- lated with the stage/grade of prostate cancer. 41 12-LOX and 12- HETE promote prostate cancer cell migration and invasion by enhancing matrix metallopeptidase 9 expression. 18 Therefore, we investigated whether the same molecular mechanism is operating in GC. In the present study, we found that over- expression of 12-LOX in GC cells resulted in a morphological
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granulocytes and monocytes. Within hematopoietic development, I concentrated on the mechanisms that underlie the defects in HSC function and monocyte development since these defects likely contribute to the myeloid leukemogenesis in Alox15 mice. Interestingly, I determined that 12/15-LOX promotes HSC self- renewal and quiescence, which is associated with the activation of canonical Wnt signaling. Moreover, my studies demonstrate that 12/15-LOX-mediated redox signaling of SHP-2 and the transcription factor ICSBP/ IRF-8 promotes monocyte development while inhibiting granulocyte development. This pathway is also conserved in IL-12p40 expression in macrophages. Therefore, I establish 12/15- LOX as a critical regulator of hematopoiesis and provide insight into novel mechanisms whereby HSC function and monocyte cell fate decisions are regulated. These findings have implications for leukemogenesis and immunity.
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In the present study, antioxidant activity, total phenolic and flavonoid content, and 15-lipoxygenase inhibitory activity of ethanol extract and its ethyl acetate, n-butanol and aqueous fractions of Eucalyptus citriodora kino were evaluated. The antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl and 2,2’-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) methods. The IC 50 values of the ethanol extract were 5.11±0.13, 2.72±0.08 and 25.86±1.81 μg/ml in the 2,2-diphenyl-1-picrylhydrazyl, 2,2’-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) methods and the 15-lipoxugenase assay, respectively. Total phenolic and flavonoid content of the ethanol extract were 490.77±1.95 mg catechin equivalents/g and 21.81±0.23 mg quercetin equivalents/g, respectively. Solvent partition of the ethanol extract yielded ethyl acetate, n-butanol and aqueous fractions. Among the three fractions, the ethyl acetate fraction showed the highest total phenolic and flavonoid contents, which were 575.87±3.92 mg catechin equivalents/g and 34.57±0.30 mg quercetin equivalents/g, respectively. This fraction also showed the strongest free radical scavenging activity in the two methods used as well as inhibitory activity against 15-lipoxygenase, with IC 50 values of 4.67±0.09, 2.57±0.06 and 14.67±0.93 μg/ml, respectively. These findings revealed that high antioxidant and lipoxygenase inhibitory activity of Eucalyptus citriodora kino might be due to high phenolic and flavonoid content. These results showed that Eucalyptus citriodora kino could be a potential source of natural antioxidants and lipoxygenase inhibitors which could be used to prevent free radical and lipoxygenase mediated diseases.
12 load in the lungs (5.19 ± 0.15 and 4.4 ± 0.16 cfu, respectively) compared to the n-3 treatment group (5.73 ±0.11 and 5.7 ± 0.25 cfu, respectively). T-cell function and proliferative abilities in the spleen were also examined after a mitogen-induced stimulation with concanavalin A (ConA). Guinea pigs fed the n-3 enriched diet displayed decreased T-cell proliferation (223 ± 27 SI, stimulation index) at 3 weeks post infection compared to the n-6 group (374 ± 41 SI) (McFarland et al., 2008). Previous work by Mayatepek et. al (Mayatepek et al., 1994) and Paul et. al (Paul et al., 1997) support these findings and suggest that n-3 supplementation can increase susceptibility to TB. In pigs, dietary supplementation of n-3 resulted in decreased immune cell function when challenged with LPS. Møller and colleagues (2006) evaluated the effects of n-6 and n-3 long- chain PUFA on ex vivo cytokine and eicosanoid production in PAM. Weaned pigs were fed diets containing 5% sunflower oil (n-6), fish oil (n-3), or a control diet containing animal fat for 28 days. Alveolar macrophages were cultured and stimulated with LPS for 18 hours. Cells from the n-3 treatment group had decreased concentration (mg/g) of ARA by 60% and 53% in the control group. Positive correlations between n-6 content, PGE 2 , TNF-α, and IL-8 production were
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Medicinal plants are rich source of secondary metabolites that could attribute to its medicinal property.These secondary metabolites are used as food additives, in pharmaceuticals industry and as herbicides 12, 13, 14 . Most of the secondary metabolites are present in the latex which helps its medicinal property. The presence of flavonoids may have an effect on anti-inflammatory mechanisms by means of their ability to inhibit reactive oxygen or nitrogen compounds. They have also been recommended to inhibit the pro-inflammatory activity of enzymes involved in free radical production, such as cyclooxygenase, lipoxygenase or inducible nitric oxide synthase, and to modify intracellular signaling pathways in immune cells, or in brain cells after a stroke. The presence of terpenoids, coumarins and phlobatannins have been reported for its wound healing properties, and also have anti-inflammatory and analgesic 15 and antitoxidant properties 16 . Plants having alkaloids are used in medicines for reducing headache and fever. These are attributed for antibacterial and analgesic properties 17 .These phytochemicals are known to promote anti-inflammatory activity in some plants such as Syzygium
Twenty micrograms of total proteins from chondrocyte lysates treated with HNE under the indicated conditions were loaded for discontinuous 4 to 12% SDS-PAGE. Protein transfer, immunodetection and semiquantitative measurements were performed as described previously . The primary antibodies were rabbit anti-COX-2 (Cayman Chemical, Hornby, ON, Canada), anti-mPGES- 1 (Cayman Chemical), anti-b-actin (Santa Cruz Biotech- nology, Santa Cruz, CA, USA) and anti-Egr-1 (Santa Cruz Biotechnology). After serial washes, primary anti- bodies were detected by goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Immunoreac- tive proteins were detected with SuperSignal blotting substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) and were exposed to clear-blue X-ray film (Pierce).
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ducing high PGE 2 levels . Moreover, chronic inhibition of COX-2 with a selective inhibitor such as NS-398 in OA oste- oblasts enhanced the production of leukotriene B 4 (LTB 4 ) , a situation also observed in other cell systems using selective COX-2 inhibitors . Hence, chronic inhibition of COX-2 may promote abnormal 5-LO activity. The exact mech- anism involved in this shunt toward the 5-LO pathway remains obscure. The production of LTs requires active 5-LO in the presence of calcium [25,26], yet arachidonic acid must be presented by the 5-LO-activating protein [25,27]. In macro- phages, the shunt from the COX to the 5-LO pathway is due to an increase in 5-LO expression [28,29], whereas in alveolar macrophages and in neutrophils it is due to altered 5-lipoxyge- nase-activating protein (FLAP) expression [30,31]. Whether
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L-12LO gene was apparently sufficient to yield similar lesion size in mice expressing 2 copies of the L-12LO gene on an apo E–deficient background, indicating no gene dosage effects. These lesion data are even more compelling because the total cholesterol and triglyceride levels, as well as the lipoprotein profiles, were similar among all groups of animals. Interestingly, although still statistically significant, the difference in atherosclerotic lesions in the aortic sinus region, as measured by section analysis, was less striking than that seen in the en face preparations of the distal aorta. In apo E–deficient mice, lesions in the aortic sinus area are among the most prominent early sites of predilection (51); thus, lesions there are usually the most advanced at any subsequent time they are measured. The reasons for this are not fully known, but the aortic sinus area may be subjected to higher turbulent flow forces than are distal regions. Thus, turbulent flow–related atherogenic mechanisms could diminish the early protection afforded by absence of macrophage L-12LO expression in the aortic sinus. These data suggest that lipoxygenases are involved in the early stages of atherogenesis. The initial lipid perox- ide resulting from the lipoxygenase reaction is stere- ospecific, but as nonenzymatic lipid peroxidation ensues, the hydroperoxides generated would be increas- ingly racemic. Indeed, consistent with this idea are the observations that predominantly stereospecific arachi-
The experiments were performed in accordance with The Guide for the Care and Handling of Laboratory Animals (NIH Publication No. 85-23) and Pennington Biomedical Research Center Protocol for Animal Studies. Mature male C57Bl6/J mice were purchased from Jack- son Laboratories. All the mice were fed standard mouse chow (PMI Nutrition International, Brentwood, MO, USA) and had ad libitum access to water. After a 7-day acclimation in a new environment, the mice were ran- domly divided into two groups. In one group, diabetes was induced by streptozotocin (STZ) as we described previously [24,25]. The mice with blood glucose ≥13.8 mM, three days post streptozotocin were considered dia- betic. The control and diabetic mice were kept for 12 weeks without treatment, and then divided into two sub- groups that were maintained with or without treatment with cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), 8 mg kg/d, s.c., for another 4 weeks. CDC, at the afore- mentioned dose, counteracted multiple manifestations of diabetic neuroapthy and oxidative-nitrosative stress in peripheral nerve and spinal cord in our previous study . Non-fasting blood glucose measurements were performed after induction of diabetes and at the end of the study period.
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DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell–driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase–meditated (12/15-LO–mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO–encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO–deficient DCs to 12/15-LO–derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO–deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune
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