5-LOX Activity Research Articles

Ethyl acetate fraction of Terminalia bellirica (Gaertn.) Roxb. fruits inhibits proinflammatory mediators via down regulating nuclear factor-κB in LPS stimulated Raw 264.7 cells

Inflammation has been considered as a major risk factor for various kinds of human diseases. Macrophages play substantial roles in host defense against infection. It can be activated by lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria. The current study aims to investigate the anti-inflammatory effects of ethyl acetate fraction isolated from T. bellerica (EFTB) in LPS stimulated RAW 264.7 macrophage cell lines. The inhibitory effects of EFTB on total cyclooxygenase (COX), 5-lipoxygenase (5-LOX) activity, nitrate and inducible nitric oxide synthase (iNOS) level, reactive oxygen species (ROS) production were studied. The gene level expression of COX-2, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and Nuclear factor-κB (NF-κB) were also studied in LPS stimulated RAW 264.7 cells. EFTB (100μg/mL) inhibited all inflammatory markers in dose dependent manner. Moreover, EFTB down regulated the mRNA expression of TNF-α, IL-6, COX-2 and NF-κB against LPS stimulation. Our results demonstrated that EFTB is able to attenuate inflammatory response possibly via suppression of ROS and NO species, inhibiting the production of arachidonic acid metabolites, proinflammatory mediators and cytokines release.

Targeted lipidomics reveals activation of resolution pathways in knee osteoarthritis in humans.

To investigate the presence of inflammation and resolution pathways in osteoarthritis (OA). Tissues were obtained from knee OA patients and control rheumatoid arthritis (RA) patients. Cells in synovial fluid (SF) were visualized by flow cytometry. Cytokines and chemokines were measured by multiplex assay. Lipid mediators (LMs) were determined by targeted lipidomics using liquid-chromatography mass spectrometry. SF of OA patients contained less cells, especially neutrophils, less cytokines and comparable levels of chemokines compared to RA controls. Thirty-seven lipids were detected in the soluble fraction of SF, including polyunsaturated fatty acids (PUFAs) and their pro-inflammatory and pro-resolving lipoxygenase (LOX) and cyclooxygenase (COX) pathway markers in both OA and RA patients. Among these, pro-inflammatory LM such as prostaglandin E2 (PGE2) and thromboxane B2, as well as precursors and pathway markers of resolution such as 17-HDHA and 18-HEPE were detected. Interestingly, the pro-resolving lipid RvD2 could also be detected, but only in the insoluble fraction (cells and undigested matrix). Ratios of metabolites to their precursors indicated a lower activity of 5-LOX and 15-LOX in OA compared to RA, with no apparent differences in COX-derived products. Interestingly, synovial tissue and SF cells could produce 5-LOX and 15-LOX metabolites, indicating these cells as possible source of LM. By using a state-of-the-art technique, we show for the first time that resolution pathways are present in OA patients. A better understanding of these pathways could guide us to more effective therapeutic approaches to inhibit inflammation and further structural damage in OA and RA.

Dietary chitosan affects metabolism of arachidonic acid in weaned piglets

The effects of chitosan on immune function via arachidonic acid (AA) pathway in weaned piglets were investigated. A total of 180 piglets (Duroc × Yorkshire × Landrace) were randomly assigned to 5 dietary treatments and fed a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. Results showed that serum AA, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) contents in piglets were increased in a linear or quadratic dose-dependent manner with increasing chitosan on day 28 (P < 0.05). Chitosan increased serum cytosolic-phospholipase A2 (cPLA2) activity in a linear or quadratic dose-dependent manner on day 14 or 28, and improved 5-lipoxygenase (5-LOX) activity in a linear manner and cyclooxygenase-2 (COX-2) activity quadratically on day 28 (P < 0.05). Moreover, chitosan elevated gene expression of cPLA2 mRNA quadratically in the small intestine on days 14 and 28, increased the COX-2 mRNA expression in the duodenum or jejunum in a linear or quadratic manner on day 28, and improved the 5-LOX mRNA expression quadratically in the small intestine (P < 0.05). These results implied that the metabolism of AA was regulated by chitosan in a dose-dependent relationship, which may be one reason why chitosan affected immune function via AA pathway in weaned piglets.

Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Propofol is an intravenous anesthetic that produces its anesthetic effect, largely via the GABAA receptor in the CNS, and also reduces the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil respiratory burst. Because fMLP-stimulated neutrophils produce leukotriene (LT)B4, we examined the effect of propofol on LTB4 production in vivo and in vitro Cecal ligation and puncture surgery was performed in mice, with or without exposure to propofol. Propofol attenuated the production of 5-lipoxygenase (5-LOX)-related arachidonic acid (AA) derivatives in the peritoneal fluid. Also, in the in vitro experiments on fMLP-stimulated neutrophils and 5-LOX-transfected human embryonic kidney cells, propofol attenuated the production of 5-LOX-related AA derivatives. Based on these results, we hypothesized that propofol would directly affect 5-LOX function. Using meta-azi-propofol (AziPm), we photolabeled stable 5-LOX protein, which had been used to solve the X-ray crystallographic structure of 5-LOX, and examined the binding site(s) of propofol on 5-LOX. Two propofol binding pockets were identified near the active site of 5-LOX. Alanine scanning mutagenesis was performed for the residues of 5-LOX in the vicinity of propofol, and we evaluated the functional role of these pockets in LTB4 production. We demonstrated that these pockets were functionally important for 5-LOX activity. These two pockets can be used to explore a novel 5-LOX inhibitor in the future.-Okuno, T., Koutsogiannaki, S., Ohba, M., Chamberlain, M., Bu, W., Lin, F.-Y., Eckenhoff, R. G., Yokomizo T., Yuki, K. Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Investigation on silymarin impact on lipopolysaccharide induced inflammation model based on arachidonic acid metabolism pathway

The objective of this research is to investigate the suppressive effect of silymarin on vitro cell culture model of inflammatory macrophage RAW264.7 induced by Kdo2-Lipid A, and explore its mechanism based on cell metabonomics. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was used in the cell metabonomic assay to quantitative analysis of metabolites related to eicosanoids pathway. Then chemometric approaches such as principal component analysis were used to process the metabolic data. Within the established method, a total of 59 eicosanoids standards (containing 15 deuterated internal standards) were simultaneously separated in a single 5 min run, and the analytical method is proved to be rapid, sensitive and accurate. Whereafter, the metabolites with VIP> 1 and P value< 0.05 were considered as biomarkers. 12-OxoLeukotriene B4 (12-OxoLTB4) was eventually identified as metabolic biomarkers of silymarin treatment group in this research, and according to the related inflammatory pathways, we speculated silymarin has anti-inflammatory activities by inhibiting the 5-lipoxygenase (5-LOX) activity and blocking lipid peroxidation in 5-LOX metabolic pathways to reduce the formation of peroxides and oxygen free radicals. This study provide a novel approach to the mechanism research on the silymarin treatment on RAW264.7 cells based on cell metabonomics.

Celastrol modulates inflammation through inhibition of the catalytic activity of mediators of arachidonic acid pathway: Secretory phospholipase A2 group IIA, 5-lipoxygenase and cyclooxygenase-2

Elevated production of arachidonic acid (AA)-derived pro-inflammatory eicosanoids due to the concerted action of secretory phospholipase A2 group IIA (sPLA2IIA), 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) is a common feature of many inflammatory disorders. Hence, modulation of the bioactivity of these 3 enzymes is an important strategy to control inflammation. However, the failure of drugs specific for an individual enzyme (sPLA2IIA-, 5-LOX- or COX-2) and the success of 5-LOX/COX-2 dual inhibitors in effectively controlling inflammation in clinical trials prompted us to evaluate a common inhibitor for sPLA2IIA, 5-LOX and COX-2 enzymes. Celastrol, a quinone methide triterpene, was selected in this regard through molecular docking studies. We provide the first evidence for celastrol’s ability to inhibit the catalytic activity of sPLA2IIA, 5-LOX and COX-2 enzymes. Celastrol significantly inhibited the catalytic activity of sPLA2IIA (IC50=6μM) in vitro, which is independent of substrate and calcium concentration. In addition, celastrol inhibited the catalytic activities of 5-LOX (IC50=5μM) and COX-2 (IC50=20μM) in vitro; sPLA2IIA-induced edema and carrageenan-induced edema in mice; and lipopolysaccharide-stimulated production of PGE2 in human neutrophils. Thus, celastrol modulates inflammatory responses by targeting multiple enzymes of AA pathway.

Novel N-substituted indole Schiff bases as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes: Synthesis, biological activities in vitro and docking study

Two new series of N-substituted indole derivatives 4a–l and 5a–h were synthesized. Their chemical structures were confirmed using spectroscopic tools including IR, 1H NMR, 13C NMR mass spectroscopy and elemental analyses. The results showed no significant cytotoxic activity on either cancer or normal human cells. Anti-inflammatory activity for all target compounds was evaluated in vitro. Compounds 5a–h were found to have better anti-inflammatory activity than 4a–l. The inhibitory activity of COX-2 and 5-LOX were tested for 5a–h. Three compounds, 5c, 5d and 5f showed excellent COX-2 inhibitory activity with IC50 ranging from 0.98 to 1.23 μM compared to the reference celecoxib (1.54 μM). These compounds had a reasonable selectivity index between 7.03 and 8.05. Additionally, p-methylbenzoyl derivative 5g (IC50 = 5.78 μM) had superior 5-LOX inhibitory activity, higher than quercetin. 5e was close to quercetin in its LOX inhibitory activity. Compounds 5a–h were docked inside the active site of COX-2 and 5-LOX enzymes.

Abstract 2604: Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention

Abstract Colorectal cancer (CRC) protective effects of NSAIDs and COX-2 inhibitors are well established; however, they are associated with gastrointestinal (GI) bleeding and cardiovascular risk. Mechanistic studies suggest that sparing COX-1/2, prostaglandin (PG)I2 synthase and selectively targeting microsomal PG synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) would inhibit PGE2 and Leukotrienes (LTs), respectively. To design and develop selective inhibitors of mPGES-1/5-LOX, we used insilico small molecular docking simulation approaches, and identified LFA-9 as a novel selective dual mPGES-1/5-LOX inhibitor among &amp;gt;20 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess inhibitory effects of LFA-9 on mPGES-1 and 5-LOX ex-vivo. At 7.5 μM, LFA-9 inhibited the mPGES-1 and 5-LOX activities by ∼57% and ∼68%, respectively. Maximum tolerable dose (100 -1,600ppm) of LFA-9 in AIN-76A diet on male C57Bl/6 mice were tested and observed that &amp;lt;1,600 ppm dietary LFA-9 to be safe and free from liver, GI and hematological toxicities. In AOM/DSS-induced colonic inflammation in rat, LFA-9 at ≥200ppm abolished the inflammation and suppressed mPGES-1 and 5-LOX activities in a dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed in AOM-rat carcinogenesis with colonic Aberrant Crypt Foci, (ACF) as surrogate marker in male F344 rats and intestinal tumor inhibition in APCMin/+ mice. In rats, colonic ACF were induced by AOM/DSS treatment and two week after the AOM/DSS treatment, LFA-9 (200, 400, and 600ppm) was fed by diet and ACF were evaluated after six-weeks. LFA-9 showed dose-response (P&amp;lt;0.0001) inhibitory effect on ACFs formation. At 600ppm, LFA-9 significantly inhibited colonic total ACF and multi-crypts by &amp;gt;60% (P&amp;lt;0.0001). In APCMin/+ mice study, six-week-old male and female mice (10/group) were fed diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks. Male and female APCMin/+ mice fed control diet developed 58±3.8 (Mean±SEM) and 59±5.9 polyps, respectively. LFA-9 administration at 350 and 700ppm in APCMin/+ mice significantly (p&amp;lt;0.0001) reduced total intestinal tumor multiplicity and size dose-dependently (31.6± 5.9 and 24.3±3.7, male mice; and 26.2±5.6 and 15.5±1.8, female mice, respectively). At the high dose both male and female mice showed a &amp;gt; 80% suppression of polyps with &amp;gt;2mm size. Mice fed 350ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed 700ppm LFA-9 showed 91% inhibition of colon tumors. LFA-9 showed significant suppression of markers of proliferation and inflammation. Overall, above results suggest LFA-9 as a novel dual mPGES-1/5-LOX inhibitor, a safer agent than other NSAIDs and has a potential for prevention of CRC in high-risk patients. (Grant Support: NCI N01-CN 25001-26). Citation Format: Naveena B. Janakiram, Altaf Mohammed, Gopal Pathuri, Venkateshwar Madka, Rebekah Ritchie, Taylor Bryant, Yuting Zhang, Qian Li, Stan Lightfoot, Hariprasad Gali, Steele E. Vernon, Chen S. Suen, Chinthalapally V. Rao. Chemoprevention of colorectal cancer by LFA-9, a novel dual mPGES-1/5-LOX inhibitor: safer approaches to chemoprevention. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2604.

The inhibitory activity of cocoa phenolic extract against pro-inflammatory mediators secretion induced by lipopolysaccharide in RAW 264.7 cells.

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 E2 (PGE2), 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 PGE2, 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.

Inhibition of Nitric Oxide Production in LPS-Stimulated RAW 264.7 Macrophages and 15-LOX Activity by Anthraquinones from Pentas schimperi.

The anti-inflammatory activity of a coumarin and nine anthraquinone derivatives, 3-hydroxy-1-methoxy-2-methylanthraquinone (1), 2-hydroxymethyl anthraquinone (2), schimperiquinone B (3), cleomiscosin A (4), damnacanthal (5), 1,2-dihydroxy anthraquinone (6), damnacanthol (7), 3-hydroxy-2-hydroxymethyl anthraquinone (8), 1-hydroxy-2-methoxyanthraquinone (9), and 2-hydroxymethyl-3-O-prenylanthraquinone (10), isolated from the roots of Pentas schimperi were determined. The anti-15-lipoxygenase activity and nitric oxide production inhibition on lipopolysaccharide-activated macrophages RAW 264.7 cells were determined as indicators of anti-inflammatory activity. The Griess assay was used to measure nitric oxide production and the ferrous oxidation-xylenol orange assay was used to determine the 15-lipoxygenase inhibitory activity. All the compounds significantly decreased nitrite + nitrate accumulation in lipopolysaccharide-stimulated RAW 264.7 cells in a concentration-dependent manner with 85.67 % to 119.75 % inhibition of nitrite + nitrate production at 20 µg/mL. Most of the compounds had a moderate inhibitory effect on 15-lipoxygenase activity. Compounds 8 and 10 were the most potent inhibitor both in nitrite + nitrate production with respective IC50 values of 1.56 µM and 6.80 µM. Compounds 2, 7, and 8 had good anti-15-lipoxygenase activity with respective IC50 values of 13.80 µM, 14.80 µM, and 15.80 µM compared to quercetin, which was used as a standard lipoxygenase inhibitor (IC50 of 16.80 µM). Our study revealed 3-hydroxy-2-hydroxymethyl anthraquinone and damnacanthol as potent inhibitors of both 15-lipoxygenase activity and nitric oxide production. Further studies are needed in order to envisage its possible future use as a therapeutic alternative against inflammatory diseases.

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance.

Impact of simultaneous stimulation of 5-lipoxygenase and myeloperoxidase in human neutrophils

Human neutrophil 5-lipoxygenase (5-LOX) oxidizes arachidonic acid (AA) to 5S-hydro(pero)xy-6E,8Z,11Z, 14Z-eicosatetraenoic acid (5-H(p)ETE) and leukotriene (LT)A4, which is further converted to the chemoattractant LTB4. These cells contain also the heme enzyme myeloperoxidase (MPO) producing several potent oxidants such as hypochlorous acid (HOCl). Previously, it was shown that MPO-metabolites influence 5-LOX product formation. Here, we addressed the question, whether a simultaneous activation of MPO and 5-LOX in neutrophils results in comparable changes of 5-LOX activity.Human neutrophils were stimulated with H2O2 or phorbol 12-myristate 13-acetate (PMA) for MPO activation and subsequently treated with calcium ionophore A23187 inducing 5-LOX product formation on endogenous AA. Special attention was drawn to neutrophil vitality, formation of MPO-derived metabolites and redox status. The pre-stimulation with H2O2 resulted in a concentration-dependent increase in the ratio of 5-HETE to the sum of LTB4+6-trans-LTB4 in consequence of MPO activation. Thereby no impairment of cell vitality and only a slightly reduction of total glutathione level was observed. An influence of MPO on 5-LOX product formation could be suggested using an MPO inhibitor. In contrast, the pre-stimulation with PMA resulted in different changes of 5-LOX product formation leading to a reduced amount of 5-HETE unaffected by MPO inhibition. Furthermore, impaired cell vitality and diminished redox status was detected after PMA stimulation. Nevertheless, a MPO-induced diminution of LTB4 was obvious. Further work is necessary to define the type of 5-LOX modification and investigate the effect of physiological MPO activators.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

5,7-dihydroxy-2-(3-hydroxy-4, 5-dimethoxy-phenyl)-chromen-4-one-a flavone from Bruguiera gymnorrhiza displaying anti-inflammatory properties.

Objective:Bruguiera gymnorrhiza (BRG) (L.) Lamk (Rhizophoraceae), a mangrove species, is widely distributed in the Pacific region, eastern Africa, Indian subcontinent, and subtropical Australia. The leaves of this plant are traditionally used for treating burns and inflammatory lesions. This study isolates the bioactive compound from the methanol extract of BRG leaves and evaluates the possible mechanisms of anti-inflammatory activity involved.Materials and Methods:Bioassay-guided fractionation of BRG was performed to identify the bioactive fraction (displaying inhibition of cyclooxygenase 2 [COX2] - 5-lipoxygenase (5-LOX) activities and tumor necrosis factor-alpha (TNF-α) production at the tested concentrations of 100 and 10 μg/ml). The fractionation was performed by solvent extraction and preparative high-performance liquid chromatography. The bioactive compound was characterized by ultraviolet–visible, liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The antioxidant potential was evaluated by electron spin resonance spectrum of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 250 μM. The effect of the compound was also studied on TNF-α converting enzyme and nuclear factor kappa B (NF-κB) activities at the concentrations 100, 10 and 1 μg/ml.Results:Bioassay-guided purification of BRG revealed the presence of a flavone (5,7-dihydroxy-2- [3-hydroxy-4,5-dimethoxy-phenyl]-chromen-4-one) of molecular weight 330Da. It demonstrated more than 80% inhibition against COX2, 5-LOX activities and TNF-α production at 100 μg/ml. It also displayed 40% inhibition against DPPH radical at the tested concentration along with 23.1% inhibition of NF-κB activity at 100 μg/ml.Conclusions:The isolated methoxy-flavone may play a predominant role in the anti-inflammatory properties displayed by BRG leaves. Such activity may involve multiple mechanisms, namely (a) modulation of oxidative stress (b) inhibition of arachidonic acid metabolism and (c) downregulation of pro-inflammatory cytokines probably through NF-κB inhibition.

Dual inhibition of COX-2/5-LOX blocks colon cancer proliferation, migration and invasion in vitro.

Inflammation is emerging as a new hallmark of cancer. Arachidonic acid (AA) metabolism, the family of cyclooxygenases (COXs) and lipoxygenase (LOX) play important roles in AA-related inflammatory cascades. In 94 colorectal cancer samples collected from the Han population, the immunohistochemical results indicated that 68% of the patients with colorectal cancer had a co-expression of both COX-2 and 5-LOX, while both displayed low expression in the matched normal tissues. In cell lines, three colorectal cancer cell lines exhibited high expression of COX-2 and 5-LOX. During stable silencing of the expression of COX-2 or 5-LOX in LoVo cancer cells, we found that downregulation of either COX-2 or 5-LOX significantly diminished the growth, migration and invasion of the colon cancer cells and specifically, downregulation of COX-2 could elicit upregulation of 5-LOX protein and vice versa. The above results suggested that the simultaneous blocking of COX-2 and 5-LOX activity may bring more potential benefits in managing the progression of colon cancer. Therefore, we sought to explore the effectiveness of a dual COX-2/5-LOX inhibitor darbufelone on the proliferation, migration, invasion and apoptosis of colon cancer cells, as well as the underlying mechanism of action. The results indicated that darbufelone significantly decreased the proliferative and invasive abilities of the colon cancer cells, in a dose-dependent manner. During the study of the related mechanisms, we found an upregulation of p27 and downregulation of cyclin D1 as well as CDK4 after darbufelone treatment, which indicated that darbufelone could arrest the cell cycle of LoVo cells at the G0/G1 phase. Furthermore, the activation of caspase-3 and -9, upregulation of Bax and downregulation of Bcl-2 demonstrated the occurrence of apoptosis by darbufelone. Finally, darbufelone also prevented the migration and invasion of LoVo cells, which may be ascribed to the upregulation of E-cadherin and ZO-1. In summary, our data suggest that the inhibition of both COX-2/5-LOX may be an effective therapeutic approach for colon cancer management, particularly for those patients with high expression of COX-2/5-LOX.

Exploring the role of chloro and methyl substitutions in 2-phenylthiomethyl-benzoindole derivatives for 5-LOX enzyme inhibition

Following the results we previously reported on a series of ethyl 2-phenylthiomethyl 5-hydroxyindole-3-carboxylate derivatives as 5-lipoxygenase (5-LOX) inhibitors, in order to obtain a more selective compound with respect to the previous generation of derivatives, we decided to modify the structure of the core ligand.The first level of structural modification involved the annelation of benzene to the indole, yielding corresponding benzo[g]indole derivatives, systematic optimization of methyl or chlorine groups in meta-, ortho- and ortho/para-position of 2-phenylthiomethyl moiety were applied. The reported results show that extension of the aromatic core led to a great enhancement of activity, especially in cell-free assay, and the accurate structure-based design provided compounds 6f, 6g and 6l that block 5-LOX activity in cell-free assays with IC50 ranging from 0.17 to 0.22 μM, and suppress 5-LOX product synthesis in polymorphonuclear leukocytes with IC50 ranging from 0.19 to 0.37 μM. Moreover we have identified 6f and 6l as dual 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors and compound 6l significantly reduces inflammatory reactions in the carrageenan-induced mouse paw oedema. The reported in vivo analysis, together with the accessible synthetic procedure, stimulate for the generation of further potent antinflammatory benzoindoles-based agents.

Anti-inflammatory activity of benzophenone and xanthone derivatives isolated from Garcinia (Clusiaceae) species

Species of the genus Garcinia have been the source of many benzophenone and xanthone derivatives. Recent data regarding potent biological properties of natural compounds in Garcinia species led us to investigate the in vitro anti-inflammatory effect of three known xanthones, lichexanthone (1), 1,3,6,7-tetrahydroxyxanthone (2), 1,3,5,6-tetrahydroxyxanthone (3), two new xanthones 1-hydroxy-3,6,7-tri-O,O,O-triprenylxanthone (4), 1,6-dihydroxy-3,7-di-O,O-diprenylxanthone (5) and two benzophenones isoxanthochymol (6), guttiferone E (7), isolated from Garcinia nobilis and Garcinia punctata. The Griess assay was used for the measurement of nitric oxide (NO) production in RAW264.7 macrophages and the ferrous oxidation-xylenol orange assay was used to determine the 15-lipoxygenase (15-LOX) inhibitory activity. All the compounds had inhibitory effect on 15-LOX activity to different extents. Compound (7) had the highest anti-LOX activity with an IC50 value of 43.05μg/mL. At the highest studied concentration (25μg/mL), compound (4) had the most potent inhibitory activity against NO release with a% of inhibition of 95.42% and less cytotoxic effect on RAW264.7 cells (% of cell viability of 81.40).The results presented here suggest that 1,3,5,6-tetrahydroxyxanthone (3) and guttiferone E (7) are promising inhibitors of NO production and 15-LOX activity. Further studies should be considered in order to elucidate the mechanism by which these compounds exert their inhibitory activities.

Euphorbia supina inhibits inflammatory mediators in mouse bone marrow-derived mast cells and macrophages

Euphorbia supina has been traditionally used for the treatment of furuncle and bloody diarrhea relevant to the inflammatory process. It has been proven to have a variety of pharmacological efficacies including antiarthritic, detoxification, hemostatic, and diuretic activities. RAW 264.7 macrophages and bone marrow-derived mast cells (BMMCs) were used to determine the anti-inflammatory and anti-allergic effects of E. supina (ES). NO production was assayed by measuring the nitrite content of the supernatants of cultured RAW 264.7 cells. β-hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis. ELISA was used for the analysis of interleukin-6 expression, and Western blotting was used to analyze 5-LOX, iNOS, and MAPK activation. The relevant gene expression upon ES treatment was measured by RT-PCR. ES inhibited inducible nitric oxide synthase (iNOS) in RAW 264.7 cells, and IL-6 and LTC4 production in PMA- and A23187-induced BMMCs along with the downregulation of 5-LOX gene expression. Furthermore, in the present study, a decrease in p-ERK, p-JNK, and p-P38 expression, as well as the suppression of degranulation, were observed by treatment with ES. Further in vivo study revealed that ES treatment also remarkably inhibited xylene-induced mouse ear edema and MPO levels in mice ears. This study demonstrates that ES has a potential regulatory effect on the expression of inflammatory mediators through the inhibition of both the phosphorylation of MAPK signaling and the activation of degranulation.

The 5-lipoxygenase (5-LOX) Inhibitor Zileuton Reduces Inflammation and Infarct Size with Improvement in Neurological Outcome Following Cerebral Ischemia.

Stroke is one of the most frequent causes of death and disability worldwide causing a major clinical and socioeconomic impact. Although the pathophysiology of brain ischemia and reperfusion is complex, the inflammatory process plays an important role in pathogenesis, contributing to the expansion of brain injury. The 5-lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of the leukotrienes and has been implicated and in the central nervous system (CNS) disorders such as Alzheimer's disease and acute ischemic stroke. Zileuton, a selective 5-LOX inhibitor, has antiinflammatory properties and exerts an inhibitory effect on inflammatory diseases. The objective of this study was to evaluate the effects of blocking 5-LOX activity in a murine model of transient and global brain ischemia. Zileuton improved neurological deficits and significantly decrease volume and density of lesion, compared to vehicle-ischemic animals measured by magnetic resonance imaging (MRI). In addition, the blockage of 5-LOX reduced infarct area and histopathological changes. Furthermore, by enzyme immunoassay (ELISA) increased brain levels of tumor necrosis factor-alpha (TNFalpha), interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5) were detected in the vehicle-ischemic group, whereas in Zileuton-ischemic group presented reduction of these mediators. The concentration of the antiinflammatory cytokine interleukin-10 (IL-10) was increased after 5-LOX inhibition. Our results suggest that Zileuton decreases brain damage and reduces inflammatory cytokines expression in the CNS which contributes, at least in part, to improve the neurological outcome of brain ischemia.

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

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