Lipoxygenase Metabolites Research Articles

Comprehensive lipid profiling of bleomycin-induced lung injury.

Drug-induced lung injury is an adverse effect of drug treatment that can result in respiratory failure. Because lipid profiling could provide cutting-edge understanding of the pathophysiology of toxicological responses, we performed lipidomic analyses of drug-induced lung injury. We used a mouse model of bleomycin-induced lung injury and followed the physiological responses at the acute inflammatory (day 2), inflammatory-to-fibrosis (day 7) and fibrosis (day 21) phases. The overall lipid profiles of plasma, lung and bronchoalveolar lavage fluid (BALF) revealed that drastic changes in lipids occurred in the lung and BALF, but not in the plasma, after 7 and 21days of bleomycin treatment. In the lung, the levels of ether-type phosphatidylethanolamines decreased, while those of phosphatidylcholines, bismonophosphatidic acids and cholesterol esters increased on days 7 and 21. In BALF, the global lipid levels increased on days 7 and 21, but only those of some lipids, such as phosphatidylglycerols/bismonophosphatidic acids and phosphatidylinositols, increased from day 2. The lung levels of prostaglandins, such as prostaglandin D2 , were elevated on day 2, and those of 5- and 15-lipoxygenase metabolites of docosahexaenoic acid were elevated on day 7. In BALF, the levels of 12-lipoxygenase metabolites of polyunsaturated fatty acids were elevated on day 7. Our comprehensive lipidomics approach suggested anti-inflammatory responses in the inflammatory phase, phospholipidosis and anti-inflammatory responses in the inflammatory-to-fibrosis phase, and increased oxidative stress and/or cell phenotypic transitions in the fibrosis phase. Understanding these molecular changes and potential mechanisms will help develop novel drugs to prevent or treat drug-induced lung injury.

СОСТАВ ЖИРНЫХ КИСЛОТ И УРОВЕНЬ ИХ МЕТАБОЛИТОВ ПРИ ЧАСТИЧНО КОНТРОЛИРУЕМОЙ БРОНХИАЛЬНОЙ АСТМЕ НА ФОНЕ КОМПЛЕКСНОГО ЛЕЧЕНИЯ С ПРИМЕНЕНИЕМ МИЛЛИМЕТРОВОЙ ТЕРАПИИ

To assess the anti-inflammatory effect of millimeter therapy in the treatment of partially controlled asthma, the fatty acid composition of erythrocyte membranes and their oxygenase derivatives were studied. Eighty-five subjects aged 18 to 45 years old were examined, 60 of them were diagnosed with partially controlled asthma and 25 were a control group, which consisted of conditionally healthy subjects without bronchopulmonary pathology. Two study groups were formed. Patients of the 1st group (n=30) received only basic medical treatment: inhalation glucocorticosteroid and β2-agonists of short action on demand. Patients of the 2nd group (n=30) underwent complex treatment: basic medical treatment in combination with millimeter therapy according to the developed technique. Clinical symptoms (frequency of nocturnal and daily asthma attacks per week, severity of cough, need for short-acting bronchodilators during the day), results of the asthma symptom control questionnaire (ACQ-5 test), lung function, composition of fatty acid (FA) in erythrocyte membranes, level of eicosanoids were assessed. The dynamics of clinical-functional and laboratory indicators in patients was evaluated one month after the start of treatment. The study established a modification of FA erythrocyte membranes composition, characterized by an increase in the proportion of some representatives of saturated and monounsaturated FA, increased production of arachidonic acid and its cyclooxygenase and lipoxygenase metabolites (thromboxane B2, leukotriene B4) against the background of deficiency of eicosapentaenoic and docosapentaenoic acids. A significant decrease in the level of oxygenated derivatives of fatty acids in the blood plasma of patients was revealed against the background of positive dynamics of clinical course of the disease in the 2nd group receiving complex treatment, which indicates the normalization of metabolic transformations of oxygenated derivatives of fatty acids, and as a consequence the extinction of allergic inflammation under the influence of electromagnetic waves of the millimeter range of extremely high frequency.

Serum LTB4, HODE13 and PLA2G7 as biomarkers of cardiovascular disease in SLE

Abstract Background Cardiovascular complications are the most serious ones associated with lupus, and are thought to be caused by inflammation which results from autoimmunity. This study uses a metabolomic screen to identify metabolites that are elevated in SLE patients with and without CVD. Four of metabolites elevated in the initial screen have been selected for validation by ELISA. Methods 27 serum samples (10 healthy controls, 8 with SLE and plaque progression, 9 with SLE and no plaque progression) were screened for elevated metabolites. Four of the most significantly elevated metabolites were selected for validation in another cohort of 85 serum samples (25 healthy controls, 53 with lupus nephritis, 7 with both lupus nephritis and cardiovascular disease). Results Metabolomic screening of the initial cohort demonstrated that several lipoxygenase metabolites (and HODE 13) were elevated in SLE patients who developed CVD compared to other SLE patients (p < .05) and healthy controls (p < .05). Based on this, LOX15, LTB4, and PLA2G7 (as well as HODE13) were selected for ELISA validation. Serum PLA2G7 correlated positively with number and increase of atherosclerotic plaques, as well as smoking history (p<.05 and <.01, respectively). LTB4 correlated positively with coronary artery disease (p<.05) and HODE13 correlated positively with hypertension and SLEDAI (p<.05). Conclusions Serum LTB4, HODE13 and PLA2G7 exhibit potential as biomarkers distinguishing between SLE patients who are or are not at risk of developing cardiovascular complications. Longitudinal studies are in progress to assess the clinical utility of these novel markers.

Bioactive lipids serve as biomarkers of host immune response during severe influenza infection in pediatric patients

Abstract Influenza virus infects 5–20% of the population annually, leading to considerable morbidity and mortality especially in children. Robust biomarkers, reflecting the host immune response, are needed to identify patients at risk of prolonged illness or death. Eicosanoids, a family of bioactive arachidonic acid metabolites produced by the host in response to influenza infection, have pro- and anti-inflammatory properties. This study aimed to assess the relationship between the host lipid response at ICU admission with clinical outcomes in a multicenter cohort of 105 children with influenza-related illness (PICFLU study). We developed a mass spectrometry-based method to quantify >100 lipid metabolites from nasopharyngeal swabs and endotracheal aspirates. The bioactive lipid profile in influenza infected children was impacted by multiple factors including patient age and bacterial coinfection. Patients coinfected with methicillin-resistant Staphylococcus aureus and influenza had significantly elevated levels of pro-inflammatory lipids and concomitantly lower levels of anti-inflammatory 15-lipoxygenase metabolites. Interestingly, children that presented with high levels of lipids including prostaglandin E2 and 12-hydroxyeicosatetraenoic acid were more likely to have fatal outcomes despite reports indicating these lipids have opposing immunomodulatory activity. These results suggest children at risk of mortality have an amplified bioactive lipid immune response that may negatively impact disease resolution. Our study provides evidence that bioactive lipids are potential prognostic biomarkers of patient immune response phenotype, and may help to identify children at risk of influenza-related death.

12‐LOX Oxylipins Derived from Omega‐3 Supplementation with Docosahexanoic Acid Potently Regulate Human Blood Platelets

Cardiovascular disease (CVD) is the leading cause of mortality in the United States in both men and women. Thrombotic‐associated ischemic injuries due to excessive platelet activation are largely responsible for the high level of death associated with CVD. Therapies that seek to pharmacologically inhibit platelet activation, aggregation, or clot stabilization, have the potential to reduce the high mortality rates associated with CVD but are also associated with an increased risk of bleeding. The oxygenated products of polyunsaturated fatty acids—oxylipins—have been shown to be critical regulators of platelet function. Previously, it has been shown that Docosahexanoic Acid (DHA) has antiplatelet activity; therefore, we hypothesize that DHA regulates platelet function at least in part through its 12‐lipoxygenase (12‐LOX) metabolites, 11‐ and 14‐ hydroxydocosahexaenoic acid (11‐ and 14‐HDHA). Our study sought to investigate the role that 12‐LOX derived products of DHA, 11‐ and 14‐ HDHA, have on human blood platelet aggregation and activation. Platelet aggregation, granule secretion, and integrin αIIbβ3 activation were measured following agonist stimulation in the presence of DHA, 11‐ or 14‐ HDHA. Both 11‐ and 14‐ HDHA were observed to dose‐dependently attenuate aggregation in response to sub‐maximal concentrations of either thrombin or collagen. Additionally, both 11‐ and 14‐ HDHA were observed to attenuate intracellular signaling as demonstrated by a decrease in granule secretion in platelets treated with DHA compared to vehicle control treated platelets. To further the study, the effects of DHA supplementation in vivo were investigated in wild‐type mice as well as mice lacking 12‐LOX. DHA was found to play an important role in regulation of platelet function in vivo. Future studies will help to elucidate the mechanism by which DHA and its metabolites inhibit platelet function and play a role in supplementation‐dependent regulation of blood clotting. Once completed this will be the first study to clearly define a role for DHA and its 12‐LOX metabolites in the regulation of platelet aggregation and give important insight into the benefits and risks of fish oil supplementation on the cardiovascular system.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Altered eicosanoid production and phospholipid remodeling during cell culture

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.

Effect of Omega-3 Fatty Acid Supplementation on Oxylipins in a Routine Clinical Setting.

Omega-6 polyunsaturated fatty acid (n-6 PUFA) is the predominant polyunsaturated fatty acid (PUFA), especially in Western diet. A high omega-6/omega-3 ratio in Western diets is implicated in the development of cardiovascular diseases and inflammatory processes. Studies in animal models and in humans have demonstrated beneficial effects of omega-3 PUFA (n-3 PUFA) in a variety of diseases, including cardiac arrhythmias and inflammatory diseases, as well as breast and colon cancer. The molecular mechanisms underlying the effects of n-3 PUFA are still not well understood. Possible mechanisms include competition between n-3 and n-6 PUFAs at the cyclooxygenase (COX) and lipoxygenase (LOX) and cytochrome P450 levels, and subsequent formation of oxylipins with specific anti-inflammatory or anti-arrhythmic effects. In this study, we report the impact of routine long-term treatment with prescription-grade n-3 PUFA (either 840 mg or 1680 mg per day) on blood cell membrane fatty acid composition, as well as plasma oxylipin patterns, in a patient population with severe hyperlipidemia and cardiovascular disease who are on standard lipid-lowering and cardioprotective medications. Lipidomics analyses were performed by LC/ESI-MS/MS. Supplementation led to a dose-dependent increase in n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the blood cell fraction. We also observed a dose-dependent increase in EPA- and DHA-derived epoxy metabolites, whereas the effect of n-3 PUFA supplementation on LOX-dependent EPA- and DHA-derived hydroxy metabolites was less pronounced, with a tendency towards lower metabolites in subjects with higher n-3 PUFA levels. These data thus generally confirm effects of n-3 PUFA supplementation observed previously in healthy individuals. Additionally, they indicate a suppressive effect of high n-3 PUFA supplementation on the formation of LOX metabolites in the context of concomitant aspirin medication.

Method Development by Design of Experiments for Quantification of Lipoxygenase Metabolites in Human Cancer Cells with UPLC-MS/MS

Method Development by Design of Experiments for Quantification of Lipoxygenase Metabolites in Human Cancer Cells with UPLC-MS/MS

Effects of licofelone, a novel 5-LOX inhibitor, in comparison to celecoxib on gastric mucosa of dogs

Despite the extensive application of nonsteroidal anti-inflammatory drugs (NSAIDs), the use of these drugs is limited due to their adverse effects especially on gastric mucosa. Dual inhibitors that inhibit both cyclooxygenase (COX) and lipoxygenase (LOX) metabolites are considered to have less gastric toxicity in comparison to non-selective and COX-2 selective inhibitors. In this study, fifteen mixed breed dogs were randomly divided into three groups: group 1 (n=5) received placebo, group 2 (n=5) licofelone, an inhibitor of COX- 1, COX-2, and 5-LOX (2.5 mg/kg; twice daily) and group 3 (n=5) celecoxib, a COX-2 selective inhibitor (3 mg/kg; twice daily) per os for 14 days. All dogs underwent blinded gastroscopies on days 0, 7, 14 and one week after cessation of treatment and gastric lesions were scored. Examinations to detect fecal occult blood were performed daily. Results showed that licofelone is significantly better tolerated than celecoxib in terms of gastric side effects (P=0.008). Therefore, it seems that licofelone can be an appropriate alternative in dogs when NSAID therapy is necessary. Occult blood was not detected in any dog during the study.

Linoleic acid metabolite leads to steroid resistant asthma features partially through NF-\u03baB

Studies have highlighted the role of nutritional and metabolic modulators in asthma pathobiology. Steroid resistance is an important clinical problem in asthma but lacks good experimental models. Linoleic acid, a polyunsaturated fatty acid, has been linked to asthma and glucocorticoid sensitivity. Its 12/15–lipoxygenase metabolite, 13-S-hydroxyoctadecadienoic acid (HODE) induces mitochondrial dysfunction, with severe airway obstruction and neutrophilic airway inflammation. Here we show that HODE administration leads to steroid unresponsiveness in an otherwise steroid responsive model of allergic airway inflammation (AAI). HODE treatment to allergic mice further increased airway hyperresponsiveness and goblet metaplasia. Treatment with dexamethasone was associated with increased neutrophilic inflammation in HODE treated allergic mice; unlike control allergic mice that showed resolution of inflammation. HODE induced loss of steroid sensitivity was associated with increased p-NFkB in mice and reduced GR-α transcript levels in cultured human bronchial epithelia. In summary, HODE modifies typical AAI to recapitulate many of the phenotypic features seen in severe steroid unresponsive asthma. We speculate that since HODE is a natural metabolite, it may be relevant to the increased asthma severity and steroid insensitivity in patients who are obese or consume high fat diets. Further characterization of HODE induced steroid insensitivity may clarify the mechanisms.

ОСОБЛИВОСТІ ВМІСТУ ДЕЯКИХ МЕТАБОЛІЧНИХ ПОКАЗНИКІВ ТА ТУМОРНЕКРОТИЧНОГО ФАКТОРУ У ХВОРИХ НА КОМОРБІДНУ ПАТОЛОГІЮ

РЕЗЮМЕ. Відомо, що в регуляції трофічних та репаративних процесів тканин значну роль відіграють метаболіти арахідонової кислоти та ліпоперекисні похідні, що підкреслює їх важливу роль у рецидивуванні та/ або хронізації запального процесу. Окрім того, не виключається їх значно більший вплив на перебіг захворювань в умовах коморбідності.Мета дослідження – вивчення вмісту ліпооксигеназних метаболітів арахідонової кислоти (лейкотрієни (LT) С4 і В4; радіоімунний метод), проміжної та кінцевої ліпоперекисних сполук (дієнові кон'югати (DC) та малоновий діальдегід (MDA); спектрофотометричний метод), а також сироваткового туморнекротичного фактора α (TNFα; імуноферментний метод) у ході лікування 40 хворих (вік 37 – 49 років, 25 чоловіків та 15 жінок) на пептичну виразку (ПВ), у 23 з яких вона була коморбідною із хронічним бронхітом; референтною нормою були показники 20 практично здорових осіб (групи не відрізнялись за віком і статтю).Виявилось, що у коморбідних хворих після лікування рівень LTВ4, порівняно з хворими на ПВ ((108,5±20,6) пг/ мл), був підвищеним у 1,3 раза (у здорових – ((53,2±11,4) пг/мл; р<0,001) і більш ніж в 1,4 раза перевищував рівень LTС4 (у хворих на ПВ – (98,7±18,9) пг/мл; референтна норма – (39,7±10,4) пг/мл; р<0,001). З’ясовано, що підвищення вмісту ліпооксигеназних метаболітів АК у хворих на коморбідну патологію супроводжувалось також суттєвим підвищенням рівня проміжних і кінцевих ліпоперекисних сполук, при цьому рівень DC у хворих з коморбідною патологією перевищував такий у хворих на ПВ майже на третину ((6,81±0,33) мкмоль/л, р<0,001), а вміст MDA – більш ніж на чверть ((9,53±0,41) мкмоль/л, р<0,001). Вказані зміни метаболічних показників у коморбідних хворих після лікування супроводжував більш ніж удвічі підвищений вміст сироваткового TNFα ((51,0±2,8) пг/мл; у здорових – (24,5±3,2) пг/мл; р<0,001), що було також у 1,34 раза вище, ніж у хворих на ПВ після лікування. Виявлені особливості вмісту ліпооксигеназних метаболітів арахідонової кислоти, проміжного та кінцевого ліпоперекисних сполук та прозапального TNFα після лікування у хворих на коморбідну патологію свідчать про збереження у них високої прозапальної активності крові, що передбачає розробку заходів з оптимізації лікування та профілактики означеної патології у вказаної категорії хворих.

NLRP3 inflammasome as a novel target for docosahexaenoic acid metabolites to abrogate glomerular injury

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis during hyperhomocysteinemia (hHcys). However, it remains unclear whether the NLRP3 inflammasome can be a therapeutic target for treatment of hHcys-induced kidney injury. Given that DHA metabolites-resolvins have potent anti-inflammatory effects, the present study tested whether the prototype, resolvin D1 (RvD1), and 17S-hydroxy DHA (17S-HDHA), an intermediate product, abrogate hHcys-induced podocyte injury by targeting the NLRP3 inflammasome. In vitro, confocal microscopy demonstrated that 17S-HDHA (100 nM) and RvD1 (60 nM) prevented Hcys-induced formation of NLRP3 inflammasomes, as shown by reduced colocalization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) or caspase-1. Both DHA metabolites inhibited Hcys-induced caspase-1 activation and interleukin-1β production. However, DHA had no significant effect on these Hcys-induced changes in podocytes. In vivo, DHA lipoxygenase metabolites substantially inhibited podocyte NLRP3 inflammasome formation and activation and consequent glomerular sclerosis in mice with hHcys. Mechanistically, RvD1 and 17S-HDHA were shown to suppress Hcys-induced formation of lipid raft redox signaling platforms and subsequent O2·− production in podocytes. It is concluded that inhibition of NLRP3 inflammasome activation is one of the important mechanisms mediating the beneficial action of RvD1 and 17S-HDHA on Hcys-induced podocyte injury and glomerular sclerosis

Purification and Characterization of LTC 4 Synthase from Sheep Uterus

Purification and Characterization of LTC 4 Synthase from Sheep UterusPurification and Characterization of LTC 4 Synthase from Sheep UterusEicosanoids, the oxygenated metabolites of eicosapolyenoic fatty acids such as arachidonic acid via the cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase (EPOX) pathways, are generated in response to specific stimuli, elicit the response and are then quickly metabolized. Hence, these are rightly termed as “local hormones” or “autocoids”. They are involved in the regulation of a variety of physiological as well as pathological processes, including reproduction. While there are extensive studies on the role of COX metabolites, such as prostaglandins, in reproduction, not much is known on the role of LOX metabolites in reproduction. Earlier, we have identified abundant LOX activity in sheep uterus and the highly purified enzyme was found to be a homo-dimeric protein with a molecular weight of 66 kDa. When incubated with arachidonic acid, the enzyme showed two lipoxygenase activities producing both 12- and 15-Hydroxyeicosatetraenoic acid (15-HPETEs) at the optimum pH of 5.5. The relative concentration of 12- and 15-HPETEs, however, changed with the pH of the reaction, 12-Hydroxyeicosatetraenoic acid (HETE) being higher in the alkaline range and 15-HETE being the abundant in the acidic range. Furthermore, the enzyme showed the dual lipoxygenase based 14,15-LTA 4 synthase activity as evidenced by the formation of 8,15-diHETEs, the hydrolysis products of 14,15-LTA 4 . In the present study, leukotriene C 4 synthase (LTC 4 S) enzyme was purified on Q-Sepharose column after solubilization of microsomes utilizing a combination of CHAPS and taurocholate. The purified enzyme showed activity with 5, 6-LTA 4 and 14, 15-LTA 4 , with slight preference towards the latter, and converting them to corresponding LTC 4 s. Both methyl esters and free acids of LTA 4 served as substrates, though the activity was more with methyl esters. However, the enzyme showed no activity with I-chloro-2, 4-dinitrobenzene (CDNB), the conventional substrate of glutathione S-transferases. Western blot analysis of sheep uterine microsomal proteins with LTC 4 synthase specific-peptide as well as whole protein antibodies showed strong cross reactivity with two closely migrating 70 kDa proteins. While showing similarity with the known LTC 4 synthases, sheep uterine LTC 4 synthase thus appears to be quite different in terms of molecular weight, as most LTC 4 synthases reported are 18 kDa proteins. In view of its association with the microsomal membranes and involvement in eicosanoid and glutathione metabolism, sheep uterine LTC 4 synthase may form a member of MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism) superfamily.

Regulation of δ Opioid Receptor-Mediated Signaling and Antinociception in Peripheral Sensory Neurons by Arachidonic Acid-Dependent 12/15-Lipoxygenase Metabolites.

The function of δ opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and κ opioid receptor (KOR) agonist-mediated inhibition of prostaglandin E2 (PGE2)-stimulated cAMP accumulation, but not inhibition, by the 5-HT1 receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE2-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

Cross-Talk between Cancer Cells and the Tumour Microenvironment: The Role of the 5-Lipoxygenase Pathway.

5-lipoxygenase is an enzyme responsible for the synthesis of a range of bioactive lipids signalling molecules known collectively as eicosanoids. 5-lipoxygenase metabolites such as 5-hydroxyeicosatetraenoic acid (5-HETE) and a number of leukotrienes are mostly derived from arachidonic acid and have been shown to be lipid mediators of inflammation in different pathological states including cancer. Upregulated 5-lipoxygenase expression and metabolite production is found in a number of cancer types and has been shown to be associated with increased tumorigenesis. 5-lipoxygenase activity is present in a number of diverse cell types of the immune system and connective tissue. In this review, we discuss potential routes through which cancer cells may utilise the 5-lipoxygenase pathway to interact with the tumour microenvironment during the development and progression of a tumour. Furthermore, immune-derived 5-lipoxygenase signalling can drive both pro- and anti-tumour effects depending on the immune cell subtype and an overview of evidence for these opposing effects is presented.

Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3.

Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPV3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPV3+/+ mice compared with ICRTRPV3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation.

Peripheral neuronal activation by inflammatory mediators is a multifaceted physiological response that involves a multitude of regulated cellular functions. One key pathway that has been shown to be involved in inflammatory pain is Gq/GPCR, whose activation by inflammatory mediators is followed by the regulated response of the cation channel transient receptor potential vanilloid 1 (TRPV1). However, the mechanism that underlies TRPV1 activation downstream of the Gq/GPCR pathway has yet to be fully defined. In this study, we employ pharmacological and molecular biology tools to dissect this activation mechanism via perforated-patch recordings and calcium imaging of both neurons and a heterologous system. We showed that TRPV1 activity downstream of Gq/GPCR activation only produced a subdued current, which was noticeably different from the robust current that is typical of TRPV1 activation by exogenous stimuli. Moreover, we specifically demonstrated that 2 pathways downstream of Gq/GPCR signaling, namely endovanilloid production by lipoxygenases and channel phosphorylation by PKC, converge on TRPV1 to evoke a tightly regulated response. Of importance, we show that only when both pathways are acting on TRPV1 is the inflammatory-mediated response achieved. We propose that the requirement of multiple signaling events allows subdued TRPV1 activation to evoke regulated neuronal response during inflammation.-Kumar R., Hazan, A., Geron, M., Steinberg, R., Livni, L., Matzner, H., Priel, A. Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation.

Decreases in 15-lipoxygenase metabolites in Olmsted syndrome model rats

BackgroundOlmsted syndrome (OS) is a congenital dermatosis characterized by palmoplantar keratoderma and periorificial keratotic plaque. TRPV3 (transient receptor potential vanilloid subtype 3) encodes a thermosensitive Ca2+ channel and is the causative gene of OS. However, the molecular mechanism that causes the pathological development of OS is unclear. ObjectiveWe aimed to investigate the molecular mechanisms underlying OS pathology from the perspective of lipid metabolism. MethodsComprehensive lipidomics and microarray analyses were conducted on tissue samples from a non-lesional skin area of OS model rats (Ht rats) and from wild type (WT) rats as the control. ResultsInfiltration of leukocytes such as eosinophils and neutrophils and an increase in the fibrotic region were detected in the unaffected skin area of Ht rats compared with the WT rats. Among about 600 lipid species examined, the levels of 15-lipoxygenase (LOX) metabolites, the precursors of anti-inflammatory and pro-resolving lipid mediators, and dihydroceramides decreased by ≥16-fold in Ht rats compared with WT rats. Consistent with the decreases in the 15-LOX metabolites, expression levels of the genes that encode the 15-LOXs, Alox15 and Alox15b, were largely reduced. Conversely, increased expression levels were detected of Il36b, Ccl20, Cxcl1, and Cxcl2, which encode cytokines/chemokines, and S100a8 and S100a9, which encode the Ca2+ binding proteins that are implicated in epidermal proliferation. ConclusionThe pro-inflammatory state in the unaffected skin of Ht rats caused by decreases in 15-LOX metabolites and increases in cytokines/chemokines may contribute to the pathogenesis of OS.

Alterations in Epidermal Eicosanoid Metabolism Contribute to Inflammation and Impaired Late Differentiation in FLG-Mutated Atopic Dermatitis

Loss-of-function mutations in the FLG gene cause ichthyosis vulgaris (IV) and represent the major predisposing genetic risk factor for atopic dermatitis (AD). Although both conditions are characterized by epidermal barrier impairment, AD also exhibits signs of inflammation. This work was aimed at delineating the role of FLG loss-of-function mutations on eicosanoid metabolism in IV and AD. Using human epidermal equivalents (HEEs) generated with keratinocytes isolated from nonlesional skin of patients with FLG wild-type AD (WT/WT), FLG-mutated AD (FLG/WT), IV (FLG/FLG), or FLG WT control skin, we assessed the potential autocrine role of epidermal-derived eicosanoids in FLG-associated versus FLG-WT AD pathogenesis. Ultrastructural analyses demonstrated abnormal stratum corneum lipid architecture in AD and IV HEEs, independent of FLG genotype. Both AD (FLG/WT) and IV (FLG/FLG) HEEs showed impaired late epidermal differentiation. Only AD (FLG/WT) HEEs exhibited significantly increased levels of inflammatory cytokines. Analyses of lipid mediators revealed increased arachidonic acid and 12-lipoxygenase metabolites. Whereas treatment of control HEEs with arachidonic acid increased expression of inflammatory cytokines, 12-hydroxy-eicosatetraenoic acid attenuated expression of late differentiation markers. Thus, FLG mutations lead to alterations in epidermal eicosanoid metabolism that could serve as an autocrine trigger of inflammation and impaired late epidermal differentiation in AD.

The 1,4 benzoquinone-featured 5-lipoxygenase inhibitor RF-Id induces apoptotic death through downregulation of IAPs in human glioblastoma cells.

BackgroundEmbelin is a potent dual inhibitor of 5-lipoxigenase (5-LOX) and microsomal prostaglandin E2 synthase (mPGES)-1 that suppresses proliferation of human glioma cells and induces apoptosis by inhibiting XIAP and NF-κB signaling pathway. Synthetic structural modification yielded the derivative 3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (RF-Id), an embelin constrained analogue, with improved efficiency against 5-LOX in human neutrophils and anti-inflammatory activity in vivo. Taking into account that lipoxygenase (LOX) metabolites, from arachidonic acid and linoleic acid, have been implicated in tumor progression, here, we determined whether RF-Id was able to hinder glioblastoma (GBM) cancer cell growth and the related mechanisms.MethodsU87MG and LN229 cells were plated in 96-wells and treated with increasing concentrations of RF-Id. Cell viability was evaluated by MTT assay. The effects of the compounds on cell cycle, apoptosis, oxidative stress and autophagy were assessed by flow cytometry (FACS). The mode of action was confirmed by Taqman apoptosis array and evaluating caspase cascade and NFκB pathway by western blotting technique.ResultsHere, we found that RF-Id induced a stronger inhibition of GBM cell growth than treatment with embelin. Flow cytometry analysis showed that RF-Id induced about 30 % apoptosis and a slight increase of autophagy after 72 h on U87-MG cells. Moreover, the compound induced an increase in the percentage of cells in G2 and S phase that was paralleled by an increase of p21 and p27 expression but no significant changes of the mitochondrial membrane potential; array analysis showed a significant upregulation of CASP8 and a downregulation of IAP family and NFκB genes in cells treated with RF-Id. RF-Id induced a significant cleavage of caspases 8, 9, 3 and 7, blocked c-IAP2/XIAP interaction by inducing XIAP degradation and inhibited NFκB pathway.Conclusions RF-Id induced a caspase-dependent apoptosis in GBM cells by inhibiting IAP family proteins and NFκB pathway and represents a promising lead compound for designing a new class of anti-cancer drugs with multiple targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0440-x) contains supplementary material, which is available to authorized users.