Dysregulated Leukotriene Metabolism in Patients with COVID-19.

This study aimed to examine the leukotriene metabolism during COVID-19. In total, 180 participants were included in this study, of which 60 were healthy controls, 60 required intensive care units (ICU), and 60 did not require intensive care (non-ICU). The serum levels of 5-lipoxygenase (5-LO), 5-LO activating protein (ALOX5AP), and cysteinyl leukotriene (CYSLT) were measured, and the mRNA expression levels of 5-LO, ALOX5AP, and cysteinyl leukotriene receptor 1 (CYSLTR1) were investigated. Compared with the control group, both the non-ICU and ICU groups had lower levels of 5-LO and mRNA expression. ICU patients had lower levels of 5-LO and mRNA expression than non-ICU patients. CYSLTR1 mRNA expression was highest in the ICU group, followed by the non-ICU group, and healthy controls had the lowest mRNA expression levels. CYSLT levels were higher in the control group than in the non-ICU and ICU groups. CYSLTR1 expression was higher in patients than in controls; therefore, selective leukotriene receptor blockers can be used as treatment options. CYSLTR1 expression was higher in the ICU group than in the non-ICU group. Furthermore, CYSLTR1 mRNA expression may be a promising biomarker of COVID-19 severity.

GPR146 regulates pulmonary vascular remodeling by promoting pulmonary artery smooth muscle cell proliferation through 5-lipoxygenase.

The pathological feature of hypoxic pulmonary hypertension (PH) is pulmonary vascular remodeling (PVR), primarily attributed to the hyperproliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Existing PH-targeted drugs have difficulties in reversing PVR. Therefore, it is vital to discover a new regulatory mechanism for PVR and develop new targeted drugs. G protein-coupled receptor 146 (GPR146) is believed to participate in this process. This study aimed to investigate the role of GPR146 in PASMCs during PH. We investigated the role of GPR146 in PVR and its underlying mechanism using hypoxic PASMCs and mouse model (Sugen 5416 (20 mg/kg)/hypoxia). In our recent study, we have observed a significant increase in the expression of GPR146 protein in animal models of PH as well as in patients diagnosed with pulmonary arterial hypertension (PAH). Through immunohistochemistry, we found that GPR146 was mainly localized in the smooth muscle and endothelial layers of the pulmonary vasculature. GPR146 deficiency induction exhibited protective effects against hypoxia-induced elevation of right ventricular systolic blood pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling in mice. In particular, the deletion of GPR146 attenuated the hypoxia-triggered proliferation of PASMCs. Furthermore, 5-lipoxygenase (5-LO) was related to PH development. Hypoxia and overexpression of GPR146 increased 5-LO expression, which was reversed through GPR146 knockdown or siRNA intervention. Our study discovered that GPR146 exhibited high expression in the pulmonary vessels of pulmonary hypertension. Subsequent research revealed that GPR146 played a crucial role in the development of hypoxic PH by promoting lipid peroxidation and 5-LO expression. In conclusion, GPR146 may regulate pulmonary vascular remodeling by promoting PASMCs proliferation through 5-LO, which presents a feasible target for PH prevention and treatment.

Leukotriene metabolism and proiflammatory cytokines in Crimean Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging acute viral infection disease, yet its pathophysiology remains largely uncharacterized. Lipid mediators are molecules that play numerous roles in the physiologic and pathophysiologic conditions in certain viral diseases. No previous study evaluated the status of cysteinyl leukotrienes (CYSLT) and 5-lipoxygenase (5-LO) and their relationship with proinflammatory cytokines in CCHF. A total of 90 subjects including 60 CCHF patients and 30 healthy controls were enrolled the study. Serum CYSLT, 5-LO, interleukin-6 (IL-6), and ferritin levels were determined in the study population. Lower median 5-LO level was determined in patients compared to healthy controls (p = 0.0004). Higher ferritin (p < 0.001) and IL-6 (p < 0.001) levels in patients than healthy controls. No statistically significant difference was observed between patients and controls in terms of CYSLT levels. No statistically significant differences were observed between mild, moderate, and severe groups in terms of both 5-LO and CYSLT levels. IL-6 and ferritin levels were higher in severe group compared mild and moderate groups. In conclusion, changes in 5-LO enzyme and increased inflammation are related with the disease molecular mechanism. Higher inflammatory status contributes to the impaired hemostatic balance in CCHF. Thus, treatment strategies to reduce inflammation may help to prevent bleeding and DIC in patients. IL-6 and ferritin can be used to as an additional biomarker in the estmation of the prognosis and diagnosis of the patients.

In silico investigations of some Cyperus rotundus compounds as potential anti-inflammatory inhibitors of 5-LO and LTA4H enzymes.

The present study aimed to experimentally identify the essential oil of Algerian Cyperus rotundus L. and to model the interaction of some known anti-inflammatory molecules with two key enzymes involved in inflammation, 5-Lypoxygenase (5-LO) and leukotriene A4 hydrolase (LTA4H). Gas chromatography/gas chromatography-mass spectrometry (GC/GC-MS) revealed that 92.7% of the essential oil contains 35 compounds, including oxygenated sesquiterpenes (44.2%), oxygenated monoterpenes (30.2%), monoterpene hydrocarbons (11.8%) and sesquiterpene hydrocarbons (6.5%). The major identified oxygenated terpenes are humulene oxide II, caryophyllene oxide, khusinol, agarospirol, spathulinol and trans-pinocarveol Myrtenol and α-terpineol are known to exhibit anti-inflammatory activities. Several complexes obtained after docking the natural terpenes with 5-LO and LTA4H have shown strong hydrogen bonding interactions. The best docking energies were found with α-terpineol, Myrtenol and khusinol. The interaction between the natural products and amino-acid residues HIS367, ILE673 and GLN363 appears to be critical for 5-LO inhibition, while the interaction with residues GLU271, HIS295, TYR383, TYR378, GLU318, GLU296 and ASP375 is critical for LTA4H inhibition. Molecular dynamics (MD) trajectories of the selected docked complexes showed stable backbone root mean square deviation (RMSD), supporting the stability of the natural product-enzyme interaction.Communicated by Ramaswamy H. Sarma.

Conditioned media from blue light-emitting diode-exposed fibroblasts have an anti-inflammatory effect in vitro.

We have previously reported the protective effects of blue light-emitting diode (BLED)-stimulated cell metabolites on cell injury. To further examine the effect of conditioned media (CM) derived from BLED (5 J/cm2)-exposed human normal fibroblasts (CMBL5) for clinical application, we have used the choline chloride and phenol red-free media and then concentrated CMBL5 using a centrifugal filter unit. The collected CMBL5-lower part (CMBL5-LO) has evaluated the inflammatory protein expression profile in LPS-stimulated RAW264.7 cells. Comprehensive metabolomic profiling of CMBL5-LO was carried out using hybrid tandem mass spectrometry. Treatment with CMBL5-LO showed the cytoprotective effect on apoptotic cell death, but rather increased apoptotic cells after treatment with CMBL5-upper part (CMBL5-UP). In addition, CMBL5-LO inhibited several chemo-attractants, including interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, chemokine (C-C motif) ligand 5 (CCL5), granulocyte colony-stimulating factor (GCSF), and monocyte chemoattractant protein-1 (MCP-1) expression. Pro-inflammatory nitric oxide was decreased after CMBL5-LO treatment, but not by CMBL5-UP treatment. Interestingly, treatment with CMBL5-LO stimulated expression of heme oxygenase-1, indicating its anti-inflammatory property. Most endoplasmic reticulum (ER) stress proteins except for transcription factor C/EBP homologous protein (CHOP) were highly expressed after irradiation with BLED in cells. Further studies are needed to examine the precise mechanism by CMBL5-LO in cells.

Evidence of human cytomegalovirus infection and expression of 5-lipoxygenase in borderline ovarian tumors.

Borderline ovarian tumors (BOTs) belong to a group of tumors that are distinctly different from ovarian carcinomas. There is an increased risk of BOTs in patients with pelvic inflammatory disease. Human cytomegalovirus (HCMV) has been detected in ovarian cancer tissue specimens. This virus favors the inflammatory milieu by inducing expression of the potent inflammatory factor 5-lipoxygenase (5LO), which stimulates cellular viability, cellular proliferation and activates antiapoptotic signaling pathways. Here, we aimed to examine presence of HCMV and 5LO in BOTs. Expression levels of HCMV proteins (IE and pp65) and 5LO were examined in paraffin embedded BOT tissue sections by immunohistochemistry staining and HCMV immunoglobulin M and immunoglobulin G (IgG) levels were determined by serology in blood samples obtained from 15 patients with BOTs identified in a prospective study at Karolinska University Hospital. Extensive expression of HCMV-IE, pp65, and 5LO were detected in 87%, 40%, and 90% of examined BOT tissue sections, respectively. HCMV-IgG prevalence and antibody levels were significantly higher in patients with BOT compared to age matched healthy women (83.3% vs. 65,6%, respectively, p = .01). Whether HCMV can induce inflammation and affect the pathogenesis of BOTs should therefore be further investigated.

Zileuton ameliorates depressive-like behaviors, hippocampal neuroinflammation, apoptosis and synapse dysfunction in mice exposed to chronic mild stress.

Our previous study has found that zileuton, a selective 5-lipoxygenase (5LO) inhibitor, abrogated lipopolysaccharide-induced depressive-like behaviors and hippocampal neuroinflammation. Herein, we further extended our curiosity to investigate effects of zileuton on stress-induced depressive-like behaviors. Our data indicated that zileuton significantly ameliorated depressive-like behaviors in mice subjected to chronic mild stress (CMS), as shown in the tail suspension test, forced swimming test and novelty-suppressed feeding test. The further studies indicated that zileuton suppressed hippocampal neuroinflammation, evidenced by lower levels of TNF-α, IL-1ß and nuclear NF-κB p65 as well as decreased number of Iba1-positive cells. It also significantly ameliorated hippocampal apoptosis, indicated by deceased number of TUNEL-positive cells, deceased ratio of cleaved caspase-3/procaspase-3 and increased ratio of Bcl-2/Bax. More importantly, zileuton increased the level of synaptic proteins PSD-95 and SYN and the number of NeuN+/BrdU+ cells in the hippocampus. Over all, zileuton alleviated CMS-induced depressive-like behaviors, neuroinflammatory and apoptotic responses, abnormalities of synapse and neurogenesis in the hippocampus, suggesting that it might has beneficial effects on depression.

A combination of LCPUFAs regulates the expression of miRNA-146a-5p in a murine asthma model and human alveolar cells.

BACKGROUND: LCPUFAs are suggestive of having beneficial effects on inflammatory diseases such as asthma. However, little is known about the modulative capacity of omega-(n)-3 and n-6 LCPUFAs within the epigenetic regulation of inflammatory processes. OBJECTIVE: The aim of this study was to investigate whether a specific combined LCPUFA supplementation restores disease-dysregulated miRNA-profiles in asthmatic mice. In addition, we determined the effect of the LCPUFA supplementation on the interaction of the most regulated miRNA expression and oxygenase activity in vitro. METHODS: Sequencing of miRNA was performed by NGS from lung tissue of asthmatic and control mice with normal diet, as well as of LCPUFA supplemented asthmatic mice. Network analysis and evaluation of the biological targets of the miRNAs were performed by DIANA- miRPath v.3 webserver software, TargetScanMouse 7.2, and tool String v.10, respectively. Expression of hsa-miRNA-146a-5p and activity of COX-2 and 5-LO in LCPUFA-treated A549 cells were assessed by qPCR and flow cytometry, respectively. RESULTS: In total, 62 miRNAs were dysregulated significantly in murine allergic asthma. The LCPUFA combination restored 21 of these dysregulated miRNAs, of which eight (mmu-miR-146a-5p, -30a-3p, -139-5p, -669p-5p, -145a-5p, -669a-5p, -342-3p and -15b-5p) were even normalized compared to the control levels. Interestingly, six of the eight rescued miRNAs are functionally implicated in TGF-ß signaling, ECM-receptor interaction and fatty acid biosynthesis. Furthermore, in vitro experiments demonstrated that upregulation of hsa-miRNA-146a-5p is accompanied by a reduction of COX-2 and 5-LO activity. Moreover, transfection experiments revealed that LCPUFAs inhibit 5-LO activity in the presence and absence of anti-miR-146a-5p. CONCLUSION: Our results demonstrate the modulative capacity of LCPUFAs on dysregulated miRNA expression in asthma. In addition, we pointed out the high regulative potential of LCPUFAs on 5-LO regulation and provided evidence that miR-146a partly controls the regulation of 5-LO.

Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase inhibitor that induces apoptosis and may impair autophagic flux in RCC4 renal cancer cells.

The inhibition of 5-lipoxygenase (5-LO), the key enzyme for the biosynthesis of leukotrienes (LTs), has generated increasing enthusiasm as anti-inflammatory and antitumor strategies in recent years. Based on our previous studies, we synthesized a series of dihydroxycinnamic acid-based analogs that might be 5-LO inhibitors. LTs biosynthesis inhibition in HEK293 cells and polymorphonuclear leukocytes (PMNL) was measured and antitumor activities were investigated in Renal Cell Carcinoma (RCC). Results showed that the 2,5-dihydroxycinnamic acid phenethyl ester (10b) was the best 5-LO inhibitor and was 7-fold more potent than Zileuton (1), the only clinically approved 5-LO inhibitor. 2,5-Dihydroxy substitution was more favorable to 5-LO inhibition since compound 10b is twice as active as CAPE (2) which is a 3,4-dihydroxylcinnamic acid ester. Meanwhile, 10b reduced the cell viability of renal cancer cells  and was more selective toward RCC4 and 786.0 cells which are deficient for the Von Hippel-Lindau (VHL) tumor suppressor gene. As to the underlying cell-death mechanisms, 10b induced apoptosis in VHL-deficient RCC4 cells. Also, increases in LC3B and p62 expression suggest a blockage of the autophagic flux in RCC in response to 10b.

Evaluation on monoamine neurotransmitters changes in depression rats given with sertraline, meloxicam or/and caffeic acid.

Inflammation drives the development of depression and may affect neurotransmitters and thus neurocircuits increase the risk of depression. To investigate the influence of inhibition of inflammatory pathways on the biogenic amine neurotransmitters metabolism in depressive rats, sertraline, and meloxicam, the inhibitors of arachidonic acid - cyclooxygenase-2/lipoxygenase (AA-COX-2/5-LO) pathways, were given to depressive rats. After the development of depression model by chronic unpredictable mild stress (CUMS) for 6 weeks, Successful modeling rats were selected and randomly divided into CUMS group and medication administration group. After given medicine, The biogenic amine neurotransmitters in rat cortex and hippocampus were measured by high-performance liquid chromatography equipped with an electrochemical detector (HPLC-ECD). Compared with the normal group, the concentration of norepinephrine (NE) significantly decreased and the concentrations of Tyrosine (Tyr), Tryptophan (Trp), 3,4-dihydroxyphenyl acetic acid (DOPAC), 3-methoxy-4-hydroxyphenylglycol (MHPG), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) significantly increased in the CUMS group. Sertraline significantly inhibited the elevation of 5-HIAA. Meloxicam inhibited the decrease of NE level in CUMS-induced rat and the increase of Trp, MHPG, and 5-HIAA level in a dose-dependent manner. Caffeic acid inhibited the decrease of NE and the increase of Trp and MHPG in a dose-dependent manner. The inhibition of AA-COX-2/5-LO pathways can improve the behaviors of depression rats and suppress CUMS-induced changes in biogenic amines. Compared with the single-dose lipoxygenase (5-LO) or Cyclooxygenase-2 (COX-2) inhibitor, the combination treatment with meloxicam 1 mg/kg and caffeic acid 10 mg/kg have no significant improvement in CUMS-induced depression behavior and the level of cortical monoamine neurotransmitters and their metabolites.

Human cytomegalovirus infection is correlated with enhanced cyclooxygenase-2 and 5-lipoxygenase protein expression in breast cancer.

PURPOSE: While enhanced expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) and their derived metabolites is associated with breast cancer (BC) risk, the precise link between BC carcinogenesis and enhanced inflammatory activity remains to be clarified. Human Cytomegalovirus (HCMV) may induce expression of COX-2 and 5-LO and is frequently found in breast cancer biopsies. Thus, we investigated whether there is an association between HCMV proteins and expression of COX-2 and 5-LO in human BC tissue and BC cell lines. MATERIALS AND METHODS: Paraffin embedded biopsies obtained from 49 patients with breast cancer and 26 tissue samples from adjacent, benign breast tissues were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA), COX-2, and 5-LO proteins by immunohistochemistry. In vitro, uninfected and HCMV-infected BC cell lines were examined for COX-2 and 5-LO transcripts and proteins by PCR and flow cytometry. RESULTS: Extensive expression of COX-2, 5-LO and HCMV-IE proteins were preferentially detected in BC samples. We found a statistically significant concordant correlation between extensive HCMV-IE and COX-2 (P < 0.0001) as well as with HCMV-IE and 5-LO (P = 0.0003) in infiltrating BC. In vitro, HCMV infection induced COX-2 and 5-LO transcripts and COX-2 proteins in MCF-7 cells (P =0.008, P =0.018, respectively). In MDA-MB-231 cells that already had high base line levels of COX-2 expression, HCMV induced both COX-2 and 5-LO proteins but not transcripts. CONCLUSION: Our findings demonstrate a significant correlation between extensive HCMV-IE protein expression and overexpression of COX-2 and 5-LO in human breast cancer.

Lens cholesterol biosynthesis inhibition: A common mechanism of cataract formation in laboratory animals by pharmaceutical products.

CJ-12,918, a 5-lipoxygenase (5-LO) inhibitor, caused cataracts during a 1-month safety assessment studies in rats whereas the structurally similar ZD-2138 was without effect. For CJ-12,918 analogs, blocking different sites of metabolic liability reduced (CJ-13,454) and eliminated (CJ-13,610) cataract formation in both rats and dogs. Using this chemical series as a test set, models and mechanisms of toxicity were first explored by testing the utility of ex vivo rat lens explant cultures as a safety screen. This model overpredicted the cataractogenic potential of ZD-2138 due to appreciably high lens drug levels and was abandoned in favor of a mechanism-based screen. Perturbations in lens sterol content, from a decline in lathosterol content, preceded cataract formation suggesting CJ-12,918 inhibited lens cholesterol biosynthesis (LCB). A 2-day bioassay in rats using ex vivo LCB assessments showed that the level of LCB inhibition was correlated with incidence of cataract formation in animal studies by these 5-LO inhibitors. Thereafter, this 2-day bioassay was applied to other pharmaceutical programs (neuronal nitric oxide synthase, sorbitol dehydrogenase inhibitor, squalene synthetase inhibitor and stearoyl-CoA desaturase-1 inhibitors/D4 antagonists) that demonstrated cataract formation in either rats or dogs. LCB inhibition >40% was associated with a high incidence of cataract formation in both rats and dogs that was species specific. Bioassay sensitivity/specificity were further explored with positive (RGH-6201/ciglitazone/U18666A) and negative (tamoxifen/naphthalene/galactose) mechanistic controls. This body of work over two decades shows that LCB inhibition was a common mechanism of cataract formation by pharmaceutical agents and defined a level of inhibition >40% that was typically associated with causing cataracts in safety assessment studies typically ≥1 month.

HMGB1 enhances AGE-mediated VSMC proliferation via an increase in 5-LO-linked RAGE expression.

Receptors for advanced glycation end-product (RAGE) play a pivotal role in the progression of proliferative vascular diseases. However, the precise mechanisms regulating RAGE expression in vascular smooth muscle cells (VSMCs) of the injured vasculatures is unclear. Given the potential importance of 5-lipoxygenase (5-LO) derived mediators in cellular responses mediated by RAGE, this study aimed to evaluate in VSMCs treated with high mobility group box 1 (HMGB1): 1) the RAGE expression; 2) the AGE-induced VSMC proliferation; 3) the role of 5-LO signaling in HMGB1-induced RAGE expression. In cultured human VSMCs stimulated with HMGB1 (100 ng/ml), RAGE mRNA and protein expression were markedly increased along with an increase in AGE-mediated VSMC proliferation. Both of these effects were markedly attenuated in cells pretreated with zileuton (1-10 µM), a 5-LO inhibitor, as well as in cells transfected with 5-LO siRNA, suggesting a potential involvement of 5-LO signaling in HMGB1-mediated RAGE expression in VSMCs. Moreover, 5-LO expression, accompanied by production of leukotrienes was markedly increased in HMGB1-stimulated VSMCs, which was attenuated in cells deficient of TLR2 or RAGE. Taken together, our results suggest that HMGB1-induced increase in 5-LO expression enhances RAGE expression in VSMCs, which stimulates AGE-mediated VSMC proliferation. Thus, the 5-LO-RAGE signaling axis in VSMCs might serve as a potential therapeutic target for vascular remodeling in the injured vasculature.

C-590T Promoter Polymorphism of the Interleukin (IL)-4 Gene Is Associated with an Increased usceptibility to Nasal Polyposis.

BACKGROUND: Recent studies have shown interleukin 4 (IL-4) and 5 lipoxygenase (5-LO) to play an important role in development of nasal polyposis. Investigation into the genetic factors associated with allergic and non-allergic nasal polyposis has been examined for more than fifteen years. Despite these efforts, the genetic factors underlying the development of nasal polyposis have yet to be clearly understood. The current study examined the relationship between C-590T promoter polymorphisms of the IL-4 gene and the presence of nasal polyps. Additionally, we examined the levels of 5-LO expression in nasal polyp tissue and its association with the IL-4 promoter gene polymorphisms. METHODS: A total of 320 subjects were enrolled in the study, of which 256 were healthy controls and 64 were patients with nasal polyps. The Real-Time PCR HRM-based method was used to determine the genotypes of IL-4 C-590T. The expression of 5-LO within the 64 samples of nasal polyp tissue was determined by immunohistochemical staining to examine the association of 5-LO with the IL-4 C-590T genotype. RESULTS: Genetic analysis showed a significant difference in the frequencies of the IL-4 polymorphisms at C-590T in patients with nasal polyps as compared with controls (p<0.001). No significant difference was seen in the expression of 5-LO among genotypes in patients with nasal polyps (p=0.139). CONCLUSION: The results suggest that the inheritance of TT and CT genotypes at the IL-4 C-590T promoter gene is associated with nasal polyps however, there is no association between the expression of 5-LO in nasal polyp tissues and IL-4 C-590T genotypes in patients with nasal polyps.

Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

Sinapic acid phenethyl ester as a potent selective 5-lipoxygenase inhibitor: Synthesis and structure-activity relationship.

Given the hepatotoxicity and an unfavorable pharmacokinetic profile of zileuton (Zyflo® ), currently the only approved and clinically used 5-Lipoxygenase (5-LO) inhibitor, the search for potent and safe 5-LO inhibitors is highly demanded. The action of several phenolic acid phenethyl esters as potential 5-Lipoxygenase (5-LO) inhibitors has been investigated. For this purpose, a series of 14 phenethyl esters was synthesized and their impact on 5-LO inhibition was evaluated. The effects of position and number of hydroxyl and methoxy groups on the phenolic acid were investigated. The shortening of the linker between the carbonyl and the catechol moiety as well as the presence of the α,ß-unsaturated carbonyl group was also explored. The sinapic acid phenethyl ester (10), which can be named SAPE (10) by analogy to caffeic acid phenethyl ester (CAPE), inhibited 5-LO in a concentration-dependent manner and outperformed both zileuton (1) and CAPE (2). With an IC50 of 0.3 µm, SAPE (10) was threefold more potent than CAPE (2) and 10-fold more potent than zileuton (1), the only 5-LO inhibitor approved for clinical use. Unlike CAPE (2), SAPE (10) had no effect on 12-lipoxygenase (12-LO) and less effect on cyclooxygenase 1 (COX-1) which makes it a more selective 5-LO inhibitor.

MiR-216a-3p inhibits colorectal cancer cell proliferation through direct targeting COX-2 and ALOX5.

Colorectal Cancer (CRC) is a most common digestive system malignant tumor. Despite recent advance in CRC treatment, searching for efficient biomarker and individual treatment therapy remains an urgent need. Cyclooxygenase-2 (COX-2) plays a critical role in the development and progression of CRC. In addition, shunting of arachidonic acid metabolism to the 5-lipoxygenase (ALOX5, 5-LO) pathway has also been reported to be implicated in the CRC pathogenesis. Cancer cell viability is promoted by ALOX5 through several mechanisms that are similar to those of COX-2. In recent years, it has been widely recognized that through inhibition of target genes, miRNAs can exert both oncogenic and tumor suppressive functions, depending on circumstances. In the present study, we screened for candidate microRNAs (miRNAs) which were predicted to regulate COX-2 and ALOX5 by online tools. Among the candidate miRNAs, miR-216a-3p expression was down-regulated in CRC tissues and cell lines; a higher miR-216a-3p expression was correlated with longer overall survival in patients with CRC. Moreover, ectopic miR-216a-3p expression significantly suppressed CRC cell proliferation. Using luciferase reporter gene, real-time PCR, and western blot assays, we confirmed the miR-216a-3p regulation of COX-2 and ALOX5 through direct targeting; further verified that miR-216a-3p could inhibit COX-2 and ALOX5 expression in CRC cells, thus to affect CRC cell proliferation. Taken together, miR-216a-3p presents a novel target of CRC treatment; rescuing miR-216a-3p expression in CRC might be a promising strategy for CRC treatment.

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

Association study between serum level of 5-LO and Hcy and carotid atherosclerotic plaque stability / 重庆医学

Objective To explore the correlation between 5-LO,Hcy and the stability of carotid artery arteriosclerosis (CAS).Methods A total of 176 patients diagnosis as CAS were assigned as study group then subdivided into stable plaques SP (group) and instable plaques IP(group).108 healthy volunteers were assigned as control group.The serum levels of 5-LO and Hcy were measured and the relationship between the two groups were analyzed.The risk factors of CAS were investigated by Logistic regression analysis and 5-LO and Hcy were used to predict the stability of carotid atherosclerotic plaque by drawing ROC curve.Results The levels of 5-LO and Hcy in the IP group were higher than those in the SP group and the control group(P＜0.05).The level of Hcy in SP group was higher than that in the control group (P＜0.05),while there is no statistical significance between SP and control group in the level of serum 5-LO(P＞0.05).Logistic regression analysis showed that 5-LO,Hcy and diabetes were the risk factors of CAS (P＜0.05).The ROC curve indicate that the optimal cut-off concentration of 5-LO was 232.89 pg/mL for discriminating the IP from SP,the sensibility and specificity were 84.4％ and 81.8％ respectively.And the optimal cut-off concentration of Hcy was 12.53 μmol/L and the sensibility and specificity were 70.1％ and 66.7％ respectively.Conclusion Serum 5-LO and Hcy are risk factors for predicting the stability of CAS plaques;regulating both levels may be a potential target for clinically stable CAS.

Improved Antitumor Activity of a Therapeutic Melanoma Vaccine through the Use of the Dual COX-2/5-LO Inhibitor Licofelone.

Immune-suppressive cell populations impair antitumor immunity and can contribute to the failure of immune therapeutic approaches. We hypothesized that the non-steroidal anti-inflammatory drug licofelone, a dual cyclooxygenase-2/5-LO inhibitor, would improve therapeutic melanoma vaccination by reducing immune-suppressive cell populations. Therefore, licofelone was administered after tumor implantation, either alone or in combination with a peptide vaccine containing a long tyrosinase-related protein 2-peptide and the adjuvant α-galactosylceramide, all formulated into cationic liposomes. Mice immunized with the long-peptide vaccine and licofelone showed delayed tumor growth compared to mice given the vaccine alone. This protection was associated with a lower frequency of immature myeloid cells (IMCs) in the bone marrow (BM) and spleen of tumor-inoculated mice. When investigating the effect of licofelone on IMCs in vitro, we found that the prostaglandin E2-induced generation of IMCs was decreased in the presence of licofelone. Furthermore, pre-incubation of BM cells differentiated under IMC-inducing conditions with licofelone reduced the secretion of cytokines interleukin (IL)-10 and -6 upon lipopolysaccharides (LPS) stimulation as compared to untreated cells. Interestingly, licofelone increased IL-6 and IL-10 secretion when administered after the LPS stimulus, demonstrating an environment-dependent effect of licofelone. Our findings support the use of licofelone to reduce tumor-promoting cell populations.