5-LOX Activity Research Articles

Jasonia glutinosa (L.) DC., a traditional herbal medicine, reduces inflammation, oxidative stress and protects the intestinal barrier in a murine model of colitis.

Jasonia glutinosa (L.) DC., known as rock tea (RT), is traditionally used in Spain as a digestive due to its beneficial properties in bowel disorders. The pharmacological nature of these properties has not been established yet. The aim of this work was to evaluate the therapeutic utility of RT in experimental colitis and to identify chemical constituents with anti-inflammatory and/or anti-oxidative properties. RT extract was prepared with ethanol in a Soxhlet apparatus and analysed by HPLC-DAD. Superoxide radical scavenging properties, xanthine oxidase and lipoxygenase (5-LOX) inhibitory activity, and capability to lower nitric oxide (NO) and tumor necrosis factor α (TNF-α) levels were measured in cell-free and cell-based assays. In the 2.5%-dextran-sodium sulphate (DSS) injury-repair model of ulcerative colitis (UC), mice were daily treated with sulfasalazine (SSZ, as reference drug, 100mg/kg bw), RT (5, 25 and 50mg/kg bw, p.o.), or vehicle over 20 days. Colitis was scored daily. Colon samples were examined macroscopically and histopathologically. Protein levels of myeloperoxidase (MPO), interleukins 6, and 10 (IL-6, IL-10), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied as markers of oxidative stress and inflammatory activity. The integrity of the apical epithelial layer was assessed by immunofluorescence staining of zonula ocludens-1 (ZO-1). Finally, intestinal contractility was also evaluated by isometric myography. Fifteen phenolic compounds and three pigments were identified and quantified, of which caffeoylquinic acids, and the flavonoid, quercetin-3-O-galactoside, were the most abundant. RT extract significantly scavenged superoxide radicals, inhibited 5-LOX activity, and lowered NO and TNF-α levels. DSS-treated mice receiving RT scored clinically lower than controls during the first 3 days of DSS treatment and during the recovery period. SSZ was less effective than RT. Anatomical and histological examination of colon samples revealed that RT significantly prevented colon shortening, increased colon thickness, and lowered the macroscopic damage score. RT also significantly prevented the increase of MPO activity, IL-6 levels, iNOS and COX-2 expression, the loss of ZO-1 apical expression, and normalized contractility disturbances. In conclusion, daily administration of RT showed therapeutic properties in the DSS-model of UC. The benefits of RT can likely be attributed to its anti-inflammatory and antioxidant phenolic and flavonoid constituents.

Phenolic rich Cocos nucifera inflorescence extract ameliorates inflammatory responses in LPS-stimulated RAW264.7 macrophages and toxin-induced murine models

Anti-inflammatory and antinociceptive effects of the acetone extract of Cocos nucifera (CnAE), an important ingredient in several traditional drugs, have been studied using different in vitro and in vivo models. CnAE did not show any observable toxicity in RAW264.7 macrophages by MTT assay. The calorimetric analysis (total COX, 5-LOX, MPO, iNOS and NO), ELISA (IL-1β, IL-6, TNF-α and PGE2) and qRT-PCR (IL-1β, IL-6, TNF-α and NF-κB) were performed in LPS-induced RAW264.7 macrophages. Phosphorylation of NF-κBp65 and IκB was determined by western blotting. CnAE (100µg/mL) remarkably inhibited total COX (68.67%) and 5-LOX (63.67%) activities, and subsequent release of iNOS, NO and PGE2 (p ≤ 0.05) in RAW264.7 cells treated with LPS. ELISA showed CnAE markedly decreased the level of pro-inflammatory cytokines IL-1β (p ≤ 0.001), IL-6 (p ≤ 0.001) and TNF-α (p ≤ 0.001) in LPS treated RAW264.7 cells. CnAE (100µg/mL) also significantly down-regulated the mRNA expressions of pro-inflammatory cytokines (IL-1β, p ≤ 0.05; IL-6, p ≤ 0.01 and TNF-α, p ≤ 0.001) and NF-κB (p ≤ 0.001) against LPS-induction. Moreover, LPS-induced phosphorylation of IκB-α and NF-κB p65 was significantly inhibited by CnAE (100µg/mL). In vivo anti-inflammatory studies showed that CnAE (400mg/kg) significantly inhibited carrageenan-induced acute paw oedema (59.81%, p ≤ 0.001) and formalin-induced chronic paw oedema (52.90%, p ≤ 0.001) in mice. CnAE at a dose of 400mg/kg also showed a significant anti-nociceptive effect on acetic acid-induced writhing (48.21%, p ≤ 0.001) and Eddy's hot plate methods. These findings suggest that CnAE has significant anti-inflammatory and anti-nociceptive properties, mainly attributed to the inhibition of NF-κB/IκB signalling cascade.

Early increase of specialized pro-resolving lipid mediators in patients with ST-elevation myocardial infarction

BackgroundTermination of acute inflammation is an active process orchestrated by lipid mediators (LM) derived from polyunsaturated fatty acids, referred to as specialized pro-resolving mediators (SPM). These mediators also provide novel therapeutic opportunities for treating inflammatory disease. However, the regulation of these molecules following acute myocardial infarction (MI) remains of interest. MethodsIn this prospective observational study we aimed to profile plasma levels of SPMs in ST-elevation MI (STEMI) patients during the first week following MI. Plasma LM concentrations were measured in patients with STEMI (n = 15) at three time points and compared with stable coronary artery disease (CAD; n = 10) and healthy controls (n = 10). FindingsOur main findings were: (i) Immediately after onset of MI and before peak troponin T levels, STEMI patients had markedly increased levels of SPMs as compared with healthy controls and stable CAD patients, with levels of these mediators declining during follow-up. (ii) The increase in SPMs primarily reflected an increase in docosapentaenoic acid- and docosahexaenoic acid-derived protectins. (iii) Several individual protectins were correlated with the rapid increase in neutrophil counts, but not with CRP. (iv) A shift in 5-LOX activity from the leukotriene B4 pathway to the pro-resolving RvTs was observed. InterpretationThe temporal regulation of SPMs indicates that resolution mechanisms are activated early during STEMI as part of an endogenous mechanism to initiate repair. Thus strategies to boost the activity and/or efficacy of these endogenous mechanisms may represent novel therapeutic opportunities for treatment of patients with MI. FundThis work was supported by grants from the South-Eastern Norwegian regional health authority, the European Research Council under the European Union's Horizon 2020 research and innovation program, a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society, and the Barts Charity.

Protective effect of piceatannol and bioactive stilbene derivatives against hypoxia-induced toxicity in H9c2 cardiomyocytes and structural elucidation as 5-LOX inhibitors

Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies including cardiovascular diseases. The present research was performed to investigate the potential protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar concentrations, and this effect was more evident in piceatannol-treated cells, that significantly increased MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally, taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for 5-LOX activity. Among the synthesized analogues ( 3–7), compound 7 was the most effective in improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the inflammatory and oxidative pathway.

Iron-Dependent Trafficking of 5-Lipoxygenase and Impact on Human Macrophage Activation.

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

Impact of food polyphenols on oxylipin biosynthesis in human neutrophils

The intake of food polyphenols is associated with beneficial impacts on health. Besides anti-oxidative effects, anti-inflammatory properties have been suggested as molecular modes of action, which may result from modulations of the arachidonic acid (AA) cascade. Here, we investigated the effects of a library of food polyphenols on 5-lipoxygenase (5-LOX) activity in a cell-free assay, and in human neutrophils. Resveratrol, its dimer (ε-viniferin), and its imine analogue (IRA) potently blocked the 5-LOX-mediated LT formation in neutrophils with IC50 values in low μM-range. Among the tested flavonoids only the isoflavone genistein showed potent 5-LOX inhibition in neutrophils (IC50 = 0.4 ± 0.1 μM), however was ineffective on isolated 5-LOX. We exclude an interference with the 5-LOX-activating protein (FLAP) in HEK_5-LOX/±FLAP cells and suggest global effects on intact immune cells. Using LC-MS based targeted oxylipin metabolomics, we analyzed the effects of 5-LOX-inhibiting polyphenols on all branches of the AA cascade in Ca2+-ionophore-challenged neutrophils. While ε-viniferin causes a clear substrate shunt towards the remaining AA cascade enzymes (15-LOX, cyclooxygenase – COX-1/2, cytochrome P450), resveratrol inhibited the COX-1/2 pathway and showed a weak attenuation of 12/15-LOX activity. IRA had no impact on 15-LOX activity, but elevated the formation of COX-derived prostaglandins, having no inhibitory effects on COX-1/2. Overall, we show that food polyphenols have the ability to block 5-LOX activity and the oxylipin pattern is modulated with a remarkable compound/structural specificity. Taken the importance of polyphenols for a healthy diet and their concentration in food supplements into account, this finding justifies further investigation.

Macrophage migration inhibitory factor (MIF) controls cytokine release during respiratory syncytial virus infection in macrophages.

Respiratory syncytial virus (RSV) is the major cause of infection in children up to 2 years old and reinfection is very common among patients. Tissue damage in the lung caused by RSV leads to an immune response and infected cells activate multiple signaling pathways and massive production of inflammatory mediators like macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. Therefore, we sought to investigate the role of MIF during RSV infection in macrophages. We evaluated MIF expression in BALB/c mice-derived macrophages stimulated with different concentrations of RSV by Western blot and real-time PCR. Additionally, different inhibitors of signaling pathways and ROS were used to evaluate their importance for MIF expression. Furthermore, we used a specific MIF inhibitor, ISO-1, to evaluate the role of MIF in viral clearance and in RSV-induced TNF-α, MCP-1 and IL-10 release from macrophages. We showed that RSV induces MIF expression dependently of ROS, 5-LOX, COX and PI3K activation. Moreover, viral replication is necessary for RSV-triggered MIF expression. Differently, p38 MAPK in only partially needed for RSV-induced MIF expression. In addition, MIF is important for the release of TNF-α, MCP-1 and IL-10 triggered by RSV in macrophages. In conclusion, we demonstrate that MIF is expressed during RSV infection and controls the release of pro-inflammatory cytokines from macrophages in an in vitro model.

Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase.

The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44 (+), CD133 (+) cell subpopulations were isolated from human prostate tumors which exhibit stem-like properties showing therapeutic-resistance, capacity of self-renewal, and exact recapitulation of the original tumor in vivo. Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

QSAR classification-based virtual screening followed by molecular docking studies for identification of potential inhibitors of 5-lipoxygenase

Developments of novel inhibitors to prevent the function of 5-lipoxygenase (5-LOX) proteins that are responsible for a variety of inflammatory and allergic disease are a major challenge in the scientific community. In this study, robust QSAR classification models for predicting 5-LOX activity were developed using machine learning algorithms. The Support Vector Machines (SVM), Logistic Regression, k-Nearest Neighbour (NN) and Decision Trees were adopted to improve the prediction ability of the classification models. The most informative molecular descriptors that contribute to the prediction of 5-LOX activity are screened from e-Dragon, Ochem, PowerMV and Combined databases using Filter-based feature selection methods such as Correlation Feature Selection (CFS) and Information Gain (IG). Performances of the models were measured by 5-fold cross-validation and external test sets prediction. Evaluation of performance of feature selection revealed that the CFS method outperforms the IG method for all descriptor databases except for PowerMV database. The best ensemble classification model was obtained with the IG filtered ‘PowerMV’ descriptor database using kNN (k = 5) algorithm which displayed an overall accuracy of 76.6% for the training set and 77.9% for the test set. Finally, we employed this model as a virtual screening tool for identifying potential 5-LOX inhibitors from the e-Drug3D drug database and found 43 potential hit candidates. This top screened hits containing one known 5-LOX inhibitors zileuton as well as novel scaffolds. These compounds further screened by applying molecular docking simulation and identified four potential hits such as Belinostat, Masoprocol, Mefloquine and Sitagliptin having a comparable binding affinity to zileuton.

A 5‑lipoxygenase-specific sequence motif impedes enzyme activity and confers dependence on a partner protein

Leukotrienes (LT) are lipid mediators of the inflammatory response that play key roles in diseases such as asthma and atherosclerosis. The precursor leukotriene A4 (LTA4) is synthesized from arachidonic acid (AA) by 5‑lipoxygenase (5-LOX), a membrane-associated enzyme, with the help of 5‑lipoxygenase-activating protein (FLAP), a nuclear transmembrane protein. In lipoxygenases the main chain carboxylate of the C-terminus is a ligand for the non-heme iron and thus part of the catalytic center. We investigated the role of a lysine-rich sequence (KKK653–655) 20 amino acids upstream of the C-terminus unique to 5-LOX that might displace the main-chain carboxylate in the iron coordination sphere. A 5-LOX mutant in which KKK653–655 is replaced by ENL was transfected into HEK293 cells in the absence and presence of FLAP. This mutant gave ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type 5-LOX. Co-expression of the enzymes with FLAP led to an equalization of 5-LOX products detected, with wild-type 5-LOX product levels increased and those from the mutant enzyme decreased. These data suggest that the KKK motif limits 5-LOX activity and that this attenuated activity must be compensated by the presence of FLAP as a partner protein for effective LT biosynthesis.

Zileuton suppresses cholangiocarcinoma cell proliferation and migration through inhibition of the Akt signaling pathway.

BackgroundInflammatory lipid mediators play an important role in several cancer types. Leukotrienes (LTs), pro-inflammatory lipid mediators, are involved in chronic inflammation and cancer progression. They are derived from arachidonic acid by 5-lipoxygenase (5-LOX) activity. On the other hand, 15-lipoxygenase (15-LOX-1) converts LTs into lipoxins (LXs), pro-resolving lipid mediators. LXs are involved in the attenuation of inflammation and cancer development.PurposeWe aimed to investigate the lipid mediator pathways, especially the LTs and LXs pathways, by studying 5-LOX and 15-LOX-1 expression in human cholangiocarcinoma (CCA) tissue. We also investigated the efficiency of zileuton (5-LOX inhibitor) treatment and BML-111 (LXA4 analog) addition on CCA cell lines properties.Patients and methodsThe expression of 5-LOX and 15-LOX-1 in fifty human cholangiocarcinoma (CCA) tissue was analyzed using immunohistochemical staining. In addition, the effect of zileuton and BML-111 on CCA cell growth and migration was demonstrated using a cell viability assay and wound-healing assay, respectively. Furthermore, the molecular mechanism by which zileuton inhibits CCA cell migration was revealed using immunofluorescent staining and western blot analysis, respectively.ResultsWe demonstrate that the upregulation of 5-LOX is significantly correlated with CCA recurrent status. A positive 15-LOX-1 signal was significantly associated with a longer survival time in CCA patients. We found that co-expression of 5-LOX and 15-LOX-1 resulted in a relatively good prognosis in CCA patients. In addition, zileuton could inhibit CCA cell migration as well as BML-111. Interestingly, zileuton treatment not only downregulated 5-LOX, but also upregulated 15-LOX-1, together with reversing the epithelial-mesenchymal transition to mesenchymal-epithelial transition phenotype as observed in EMT marker western blot.ConclusionThese findings suggest that 5-LOX and 15-LOX-1 play a key role in CCA and may serve as targets for CCA therapy.

Terminalia bellirica (Gaertn.) Roxb fruit exerts anti-inflammatory effect via regulating arachidonic acid pathway and pro-inflammatory cytokines in lipopolysaccharide-induced RAW 264.7 macrophages.

Terminalia bellirica (Gaertn.) Roxb. (Family: Combretaceae), known as Bhibhitaki in Sanskrit and locally known as Behera in India, has been used for centuries in Ayurveda, a universal system of medicine in India. The dried fruit of T. bellirica is used for the treatment of several disorders. The present study aims to explore the anti-inflammatory effects of aqueous acetone extracts isolated from T. bellirica (AATB) in RAW 264.7 cell lines. The AATB was prepared from the fruits of T. bellirica. Different concentrations of AATB (6.25-100μg/ml) were used for MTT assay. The anti-inflammatory effect of AATB was evaluated by using different assays such as total cyclooxygenase (COX), 5-lipoxygenase (5-LOX) activity, nitrate and reactive oxygen species (ROS) production. The mRNA level expression of COX-2, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were studied in LPS stimulated RAW 264.7 cells. AATB treatment significantly diminished the elevated levels of inflammatory markers. Moreover, AATB downregulated the mRNA level expression of TNF-α, IL-6 and COX-2 genes. The result of our study suggest the use of AATBand is able to reduce inflammatory conditions associated with various diseases.

5-Lipoxygenase: Its involvement in gastrointestinal malignancies.

Lipoxygenases (LOXs) are dioxygenases that catalyze the peroxidation of linoleic acid (LA) or arachidonic acid (AA), in the presence of molecular oxygen. The existence of inflammatory component in the tumor microenvironment intimately links the LOXs to gastrointestinal (GI) cancer progression. Amongst the six-different human LOX-isoforms, 5-LOX is the most vital enzyme for leukotriene (LT) biosynthesis, which is the main inflammation intermediaries. As recent investigations have shown the association of 5-LOX with tumor metastasis, there has also been significant progress in discovering the function of 5-LOX pathway in GI cancer. Studies on GI cancer cells using the pharmacological drugs targeting 5-LOX pathway have shown antiproliferative and proapoptotic effects. Pharmacogenetic discoveries in other diseases have revealed strong heritable basis for the leukotriene pathway, which helps in exploring the mechanistic source of genetic alteration within the leukotriene pathway and offer insights into GI cancer pathogenesis and future prospects for treatment and prevention. This review recapitulates the current research status of 5-LOX activity in GI malignancies.

Intravenous anesthetic propofol and its analogue flopofol attenuated 5-lipoxygenase function and leukotriene B4 production

Abstract Anesthesia is a routine clinical practice in surgical and interventional procedures, provided by using volatile and intravenous anesthetics. A growing literature suggests that anesthetics affect immune cellular function, and it is of great importance to understand the mechanism given an increased number of immunocompromized patients undergo anesthesia in current practice. Propofol is a very common intravenous anesthetic. We found that propofol attenuated the production of leukotriene B4 (LTB4), a major neutrophil chemoattractant, in addition to other leukotrienes in our experimental sepsis model. Because leukotrienes are produced via 5-lipoxgenase (5-LOX) activity, we hypothesized that propofol would attenuate 5-LOX activity. Our in vitro experiments using N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophils and 5-LOX transfected HEK cells showed that propofol attenuated 5-LOX activity. Using photoactivable propofol called azi-Pm, we showed that propofol directly bound to 5-LOX at functionally active sites. Flopofol is a propofol analogue with the substitution of 1-hydroxyl group to fluorine, and previously shown to lack anesthetic effect. In contrast, this analogue also attenuated 5-LOX activity and reduced LTB4 production, suggesting the possibility to use anesthetic as a prototype of immunomodulator. In addition, the impact of propofol in neutrophil function needs to be delineated in clinical scenario.

Edaravone, a free radical scavenger, protects neuronal cells’ mitochondria from ischemia by inactivating another new critical factor of the 5-lipoxygenase pathway affecting the arachidonic acid metabolism

To investigate the neuroprotective effect of edaravone was dependent on 5-lipoxygenase (5-LOX) signalling pathway or not. Middle cerebral artery occlusion (MCAO) and oxygen glucose deprivation (OGD) were established in SD rats and PC12 cells to mimic ischemic injury. In vivo, edaravone can significantly reduce neurological deficit scores, infarct volume, ROS level and expression of 5-LOX. For in vitro experiment, reduced viability, cell death which occurred via necrosis and apoptosis were shown after OGD and even severer in OGD-reperfusion (OGD-R). Interestingly, edaravone (0.01, 0.1, 1 μmol/L) and caffeic acid (5-LOX inhibitor) can dramatically attenuate OGD/OGD-R injuries. Profoundly, mitochondrial transmembrane potential was ameliorated and cristae membranes (detected by electron microscope) were swollen in OGD/OGD-R cells; however, edaravone preserved the normal ultrastructure of mitochondria and reduced ROS. Astonishingly, immunohistochemistry analyses showed that 5-LOX was first located in the cytosol, dendrites and nuclei of control cells and then translocated to the nuclear membrane after OGD/OGD-R, which indicated the activation of 5-LOX pathway. Edaravone and caffeic acid can inhibit 5-LOX translocation to the nuclear membrane after OGD/OGD-R and reduce cysteinyl leukotrienes (CysLTs), which are metabolites of 5-LOX. Our results are the first to indicate that the protective action of edaravone may function, at least in part, by inhibiting 5-LOX activation, maintaining the ultrastructure and integrated function of mitochondria, thus protecting neuronal cells from ischemia. Furthermore, the instability of mitochondria may be another critical factor in 5-LOX activation.

MESUAFERRIN A-BIOACTIVE FLAVONOID ISOLATED FROM THE BARK OF MESUA FERREA L. AGAINST PHOSPHOLIPASE A2, CYCLOOXYGENASE AND LIPOXYGENASE: AN IN VITRO, IN VIVO AND IN SILICO APPROACH

Objective: The main objective of the present study was to evaluate the anti-inflammatory activity of isolated bioactive flavonoid Mesuaferrin-A from the bark of Mesuaferrea L. by in vitro, in vivo and in silico approach.Methods: To evaluate the effect of isolated bioactive flavonoid Mesuaferrin-A on arachidonic acid metabolizing enzymes (PLA2, COX-2 and 5-LOX) using in vitro methods, followed by carrageenan-induced paw edema model by in vivo and to determine the binding orientation and interactions of Mesuaferrin-A onarachidonic acid metabolizing enzymes (PLA2, COX-2 and 5-LOX) crystal proteins using molecular docking (in silico) studies.Results: Mesuaferrin-A exhibited a dose-dependent significant 5-LOX inhibitory and considerable COX-2 inhibitory activity by in vitro, The inhibitory activities of 5-LOX and COX-2 at 100µg/ml were found to be 78.67%, 81.03% with IC50 values of 45.22µg/ml and 35.74µg/ml respectively. Whereas Mesuaferrin-A showed less PLA2 inhibitory activity. Mesuaferrin-A showed 68.34% inhibitory activity at 400 mg/kg body weight at the late phase of carrageenan-induced paw edema, and In silico studies demonstrated that Mesuaferrin-A strongly binds with 5-LOX and COX-2, these strong binding affinity of Mesuaferrin-A on active site amino acids of 5-LOX and COX-2 may be responsible for inhibition of enzyme activity. Mesuaferrin-A showeda comparable 5-LOX and COX-2 inhibition activity with (positive control).Conclusion: It was concluded that Mesuaferrin-A act as 5-LOX and COX dual inhibitor, from the results it was suggests that Mesuaferrin-A, may be an effective preventive and therapeutic approach for patients with inflammatory-related diseases.

Abstract B42: Safer approaches to colorectal cancer 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 enhanced cardiovascular risk. Mechanistic studies suggest that upregulation of 15-PGDH, sparing COX-1/2 and prostaglandin (PG)I2 synthase and/or selectively targeting mPGES-1 and 5-LOX, would reduce the cardiovascular side effects and may improve the chemopreventive efficacy. One unexplored area in colorectal cancer chemoprevention towards safer drug development is limited or no studies on targeting mPGES-1/5LOX. To design and develop selective inhibitors of dual mPGES-1/5-LOX, we used in silico small-molecular docking simulations approaches, and identified LFA-9 as a novel dual mPGES-1/5-LOX inhibitor among >35 analogs. Azoxymethane (AOM)-induced rat colonic tumors were utilized to assess pharmacodynamic inhibitory effects of LFA-9 on mPGES-1 and 5-LOX by Radio-HPLC. At low μM LFA-9 inhibited the mPGES-1 and 5-LOX activities by ~57% and ~68%, respectively. We tested five different doses (100-1,600 ppm) of LFA-9 by modified AIN-76A diet on male C57 mice and observed that <1,600 ppm dietary LFA-9 was found to be safer and free from the liver and GI toxicities. In AOM/DSS-induced colonic inflammation in F344 rat, 200 ppm and above LFA-9 completely abolishes the colonic inflammation and suppresses mPGES-1 and 5-LOX in dose-response manner. Potential CRC preventive efficacy of LFA-9 was assessed AOM-rat carcinogenesis (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, dietary LFA-9 (200, 400, and 600 ppm) showed dose-response (P<0.0001) inhibitory effects on ACFs formation. In APCMin/+ mice study, diet containing 0, 350, and 700 ppm LFA-9 for 14 weeks administration significantly (p<0.0001) reduced total intestinal tumor multiplicity and size dose-dependently in both male and female mice. At high dose both male and female mice showed > 80% suppression of colonic polyps with >2 mm size. Importantly, mice fed with 350-ppm LFA9 showed colon tumor inhibition of 60% (male) and 90% (female). It is noteworthy that both male and female mice fed with 700-ppm LFA-9 showed 91% inhibition of colon tumors. Overall, the above results show that LFA-9 is a novel dual mPGES-1/5-LOX inhibitor, a safer agent and potential for prevention of CRC in high-risk patients. (Grant support: NCI N01-CN 25001-26 and 1R01CA213987-01.) Citation Format: Chinthalapally V. Rao, Altaf Mohammed, Naveena B. Janakiram. Safer approaches to colorectal cancer chemoprevention [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B42.

Altered Anandamide Metabolism in Microgravity: the “RESLEM” experiment

Altered Anandamide Metabolism in Microgravity: the “RESLEM” experiment

Rotula aquatica Lour attenuates secretion of proinflammatory mediators and cytokines in lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages.

Rotula aquatica belongs to the family Boraginaceae, and is reported to contain baunerol, steroids and alkaloids. In Ayurveda, R. aquatica has been used for the treatment of various diseases such as diabetes, treatment of piles, venereal disease, and cancer. The current study aims to investigate the anti-inflammatory effect of methanolic extract of R. aquatica (MERA) in RAW 264.7 cells. The cytotoxicity of MERA was analyzed by MTT assay. The total cyclooxygenase (COX) activity, 5-lipoxygenase (5-LOX) activity, myeloperoxidase activity, inducible nitric oxide synthase activity, nitrate level and reactive oxygen species production were studied in LPS-stimulated RAW 264.7 cells. The gene level expression of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were also evaluated in this study. The MERA did not show any cytotoxicity at different concentrations (6.25-100µg/ml). MERA (100μg/ml) inhibited total COX and 5-LOX activity at 50.53 and 62.03%, respectively, besides significantly (p<0.05) diminished nitrate and ROS generation, when compared with LPS control. Moreover, MERA down-regulated the mRNA expressions of inflammatory marker genes like TNF-α, IL-6, and COX-2 against LPS stimulation. Our results demonstrate that MERA is able to attenuate inflammatory response, possibly via ROS and NO suppression, inhibiting the production of arachidonic acid metabolites and modulation of proinflammatory mediators and cytokines release.