Cysteinyl Leukotriene Receptor Research Articles

Prospective Phase Ii Trial of Montelukast to Treat Bronchiolitis Obliterans Syndrome after Hematopoietic Cell Transplantation and Investigation into Bos Pathogenesis

Background: Bronchiolitis obliterans syndrome (BOS) is a severe manifestation of chronic graft-versus-host disease (cGVHD) following hematopoietic cell transplantation (HCT). Montelukast interrupts cysteinyl leukotriene activity and may diminish the activation and homing of cells to bronchioles and subsequent fibrosis. Methods: We performed a prospective phase II trial to test whether montelukast altered lung decline for patients with BOS after HCT in a single arm, open-label, multi-institutional study with primary endpoints of: i) FEV1 stability or improvement (<15% decline) and ii) slope of FEV1<1 point decline after six months treatment. Findings: 25 patients enrolled with moderate to severe lung disease after three months of stable cGVHD therapy. Montelukast was well-tolerated and no patient required escalation of BOS-directed therapy. At the primary endpoint, all evaluable patients (n=23) met criteria for treatment success using FEV1% predicted, and all but one had stable or improved FEV1 slope. Secondary endpoints included symptoms, function, and immune correlates. In those with > 5% FEV1 improvement, clinically meaningful improvements were seen in the Lee scores of breathing, energy, and mood, and in the Human Activity Profile, and 6MW for <5% FEV1 decline. Overall survival was 87% at two-years. Immune correlates showed elevated leukotriene receptor levels on blood eosinophils and monocytes vs. healthy controls, elevated urine leukotrienes in 45%, and cysteinyl leukotriene receptors on bronchoalveolar lavage subsets, all pre-treatment. Trial Registration: This study was approved with registration number: NCT00656058. Interpretation: These data suggest that montelukast may safely halt progression of BOS after HCT and that leukotrienes may play a role in the biology of BOS. Funding: NIH, Tucker Foundation, Ben’s Run. Declaration of Interest: KMW, EWC, DGJ, KB, LEC, BMG, FTH, SMS, DEF, DGJ, JR, JCGB, DA, SAM, GSC, REG have no disclosures. SJL reports Research funding: Amgen, AstraZeneca, Incyte, Kadmon, Novartis, Pfizer; Syndax, Takeda; Provision of study medication: Janssen; Steering committee for a clinical trial: Incyte; Consulting fees from Mallinckodt, Amgen. SZP reports research funding through the collaborative and clinical trials agreements executed between the NCI CCR and Kadmon, Pharmacyclics, Celgene, Eli Lilly, Actelion, CTI, and Mereo. PJM reports: other from AbGenomics (now AltruBio), personal fees from Pfizer, Inc. , personal fees from Pediatric Transplantation and Cellular Therapy Consortium, personal fees from Genentech, Inc, personal fees from Pharmacyclics, personal fees from Neovii, personal fees from Enlivex Therapeutics, personal fees from Mesoblast, personal fees from Rigel, personal fees from Talaris, personal fees from Janssen, non-financial support from Janssen, personal fees from Mount Sinai School of Medicine, outside the submitted work. Ethical Approval: Patients were prospectively enrolled at Fred Hutchinson Cancer Research Center and National Institutes of Health after obtaining institutional review board approval with informed consent

Montelukast: The New Therapeutic Option for the Treatment of Epilepsy.

Currently, there is no definitive cure for epilepsy. The available medications relieve symptoms and reduce seizure attacks. The major challenge with the available antiepileptic medication is safety and affordability. The repurposing of montelukast for epilepsy can be an alternative medication with a better safety profile. Montelukast is a leukotriene receptor antagonist that binds to the cysteinyl leukotrienes (CysLT) receptors used in the treatment of bronchial asthma and seasonal allergies. Emerging evidence suggests that montelukast’s anti-inflammatory effect can help to maintain BBB integrity. The drug has also neuroprotective and anti-oxidative activities to reduce seizure incidence and epilepsy. The present review summarizes the neuropharmacological actions of montelukast in epilepsy with an emphasis on the recent findings associated with CysLT and cell-specific effects.

Structure-Based Virtual Screening of Ultra-Large Library Yields Potent Antagonists for a Lipid GPCR.

Cysteinyl leukotriene G protein-coupled receptors, CysLT1R and CysLT2R, regulate bronchoconstrictive and pro-inflammatory effects and play a key role in allergic disorders, cardiovascular diseases, and cancer. CysLT1R antagonists have been widely used to treat asthma disorders, while CysLT2R is a potential target against uveal melanoma. However, very few selective antagonist chemotypes for CysLT receptors are available, and the design of such ligands has proved to be challenging. To overcome this obstacle, we took advantage of recently solved crystal structures of CysLT receptors and an ultra-large Enamine REAL library, representing a chemical space of 680 M readily available compounds. Virtual ligand screening employed 4D docking models comprising crystal structures of CysLT1R and CysLT2R and their corresponding ligand-optimized models. Functional assessment of the candidate hits yielded discovery of five novel antagonist chemotypes with sub-micromolar potencies and the best Ki = 220 nM at CysLT1R. One of the hits showed inverse agonism at the L129Q constitutively active mutant of CysLT2R, with potential utility against uveal melanoma.

Small-wedge synchrotron and serial XFEL datasets for Cysteinyl leukotriene GPCRs

Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing.A Correction to this paper has been published: https://doi.org/10.1038/s41597-020-00759-w

Zafirlukast prevented ox-LDL-induced formation of foam cells

Atherosclerosis (AS), a common arterial disease, is one of the main pathological roots of cardiovascular disease. The formation and accumulation of foam cells is an important event in early AS. An imbalance between cholesterol uptake and efflux is the primary cause of foam cell formation. Although research has focused on preventing the formation of foam cells, a safe and effective therapy has to be found. Zafirlukast is a widely useful type 1 cysteinyl leukotriene receptor (CysLT1R) antagonist with a good safety profile. Zafirlukast is the most used for the treatment of asthma and allergic rhinitis. However, the effect of zafirlukast on preventing the formation of foam cells has not been determined. The aim of this study was to investigate whether zafirlukast prevented macrophages from transforming into foam cells. Our data show that zafirlukast reduced the expression of CD36 and lipoprotein receptor-1 (LOX-1), which are responsible for lipid uptake. In addition, zafirlukast enhanced the activity of ATP-Binding Cassette A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1), leading to the acceleration of cholesterol efflux. Furthermore, zafirlukast influenced the activity of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, which mediates the expression of ABCA1 and ABCG1. In summary, our data indicate that zafirlukast might be a potential treatment strategy for AS by mediating lipid metabolism and preventing the formation of foam cells.

Cysteinyl leukotriene receptor 1 modulates autophagic activity in retinal pigment epithelial cells

The retinal pigment epithelium (RPE), which is among the tissues in the body that are exposed to the highest levels of phagocytosis and oxidative stress, is dependent on autophagy function. Impaired autophagy and continuous cellular stress are associated with various disorders, such as dry age-related macular degeneration (AMD), a disease for which effective therapies are lacking. Cysteinyl leukotriene receptor (CysLTR) 1 is a potential modulator of autophagy; thus, the aim of this study was to investigate the role of CysLTR1 in autophagy regulation in the RPE cell line ARPE-19. The polarized ARPE-19 monolayer exhibited expression of CysLTR1, which was colocalized with β-tubulin III. In ARPE-19 cells, autophagic activity was rhythmically regulated and was increased upon CysLTR1 inhibition by Zafirlukast (ZK) treatment. H2O2 affected the proautophagic regulatory effect of ZK treatment depending on whether it was applied simultaneously with or prior to ZK treatment. Furthermore, mRNA levels of genes related to the leukotriene system, autophagy and the unfolded protein response were positively correlated. As CysLTR1 is involved in autophagy regulation under basal and oxidative stress conditions, a dysfunctional leukotriene system could negatively affect RPE functions. Therefore, CysLTR1 is a potential target for new treatment approaches for neurodegenerative disorders, such as AMD.

Transcriptomic-based toxicological investigations of ethanol to human umbilical vein endothelial cells.

Ethanol has a complex effect on the cardiovascular system in humans, but the systemic effects of ethanol to endothelial cells were rarely investigated. In this study, we exposed human umbilical vein endothelial cells (HUVECs) to 5- or 50-mM ethanol and performed transcriptomics to investigate the systemic effects of ethanol. While these concentrations of ethanol did not significantly affect HUVEC viability, 5-mM ethanol significantly upregulated and downregulated 59 and 73 genes, respectively, whereas 50-mM ethanol significantly upregulated and downregulated 50 and 80 genes, respectively. Totally, 37 genes were shared by the two concentrations of ethanol. The most significantly altered gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway after 5-mM ethanol treatment were nucleic acid binding (GO:0003676) and Herpes simplex virus 1 infection (ko05168), respectively, whereas the most significantly altered GO term and KEGG pathway by 50-mM ethanol treatment were aryl sulfotransferase activity (GO:0004062) and chemical carcinogenesis (ko05204). We further verified that ethanol treatment downregulated the mRNA levels of CD38 molecule (CD38), ORAI calcium release-activated calcium modulator 2 (ORAI2), cysteinyl leukotriene receptor 2 (CYSLTR2), key genes involved in calcium signaling pathway (ko04020), as well as integrin subunit alpha 2 (ITGA2), and cAMP responsive element binding protein 3 like 2 (CREB3L2), key genes involved in PI3K-Akt signaling pathway (ko04151). The results from this study suggested that ethanol could induce systemic effects and alter signaling pathways in HUVECs.

High Cysteinyl Leukotriene Receptor 1 Expression Correlates with Poor Survival of Uveal Melanoma Patients and Cognate Antagonist Drugs Modulate the Growth, Cancer Secretome, and Metabolism of Uveal Melanoma Cells.

Simple SummaryThis research investigates the disease relevance and therapeutic potential of cysteinyl leukotriene receptors in uveal melanoma (UM), a rare eye cancer that often spreads to the liver. Unfortunately, there are no therapies available to stop the spread of UM and patients are often faced with an extremely poor prognosis. We assess whether the cysteinyl leukotriene receptors (CysLT1 and CysLT2) are relevant to the progression of UM. Using UM patient samples, we identified that increased levels of CysLT1 in tumours is associated with reduced patient survival. Using UM cell lines and zebrafish models, we found that drugs targeting CysLT1, but not CysLT2, can alter hallmarks of cancer including cell growth, proliferation, and metabolism. This study is the first to examine the relationship of the CysLT receptors with clinical features of UM. Our data strengthen the importance of CysLT signalling in UM and suggest that antagonism of CysLT1 may be of therapeutic interest in the disease.Metastatic uveal melanoma (UM) is a rare, but often lethal, form of ocular cancer arising from melanocytes within the uveal tract. UM has a high propensity to spread hematogenously to the liver, with up to 50% of patients developing liver metastases. Unfortunately, once liver metastasis occurs, patient prognosis is extremely poor with as few as 8% of patients surviving beyond two years. There are no standard-of-care therapies available for the treatment of metastatic UM, hence it is a clinical area of urgent unmet need. Here, the clinical relevance and therapeutic potential of cysteinyl leukotriene receptors (CysLT1 and CysLT2) in UM was evaluated. High expression of CYSLTR1 or CYSLTR2 transcripts is significantly associated with poor disease-free survival and poor overall survival in UM patients. Digital pathology analysis identified that high expression of CysLT1 in primary UM is associated with reduced disease-specific survival (p = 0.012; HR 2.76; 95% CI 1.21–6.3) and overall survival (p = 0.011; HR 1.46; 95% CI 0.67–3.17). High CysLT1 expression shows a statistically significant (p = 0.041) correlation with ciliary body involvement, a poor prognostic indicator in UM. Small molecule drugs targeting CysLT1 were vastly superior at exerting anti-cancer phenotypes in UM cell lines and zebrafish xenografts than drugs targeting CysLT2. Quininib, a selective CysLT1 antagonist, significantly inhibits survival (p < 0.0001), long-term proliferation (p < 0.0001), and oxidative phosphorylation (p < 0.001), but not glycolysis, in primary and metastatic UM cell lines. Quininib exerts opposing effects on the secretion of inflammatory markers in primary versus metastatic UM cell lines. Quininib significantly downregulated IL-2 and IL-6 in Mel285 cells (p < 0.05) but significantly upregulated IL-10, IL-1β, IL-2 (p < 0.0001), IL-13, IL-8 (p < 0.001), IL-12p70 and IL-6 (p < 0.05) in OMM2.5 cells. Finally, quininib significantly inhibits tumour growth in orthotopic zebrafish xenograft models of UM. These preclinical data suggest that antagonism of CysLT1, but not CysLT2, may be of therapeutic interest in the treatment of UM.

A simple and sensitive HPLC-MS/MS assay for the quantitation of montelukast in cell-based systems in vitro pulmonary drug permeability study

Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor 1 subtype (CysLT1) and widely used in the form of oral tablets and granules for asthma prophylaxis and treatment. Recently, due to the pulmonary inhaled administration can limit montelukast distribution in the systemic circulation, avoid the first-pass metabolism and have better therapeutic effects in respiratory disease treatment, explore alternative routes of administration, like delivery of montelukast via an inhaled, is a new research trend for montelukast. The aim of the current study was to develop and validate a simple, accurate, highly sensitive and selective liquid chromatography-tandem mass spectrometry method (LC–MS/MS) for determination of montelukast in an in vitro cell-based pulmonary pharmacokinetics system model, which can be used to be a better understanding the fate of inhaled montelukast in the lungs. In this study, montelukast was extracted by protein precipitation with acetonitrile containing labeled montelukast. The chromatography was performed on an Agilent Eclipse plus C8 column (4.6 mm × 100 mm, 3.5 μm, Darmstadt, Germany) operating at 35 ◦C. The mobile phase consisted of acetonitrile: 20 mM ammonium formate buffer (80: 20, v/v), was delivered at a flow rate of 0.5 mL/min. montelukast and the internal standard were both eluted at 4.2 min. A linear (1/x2) relationship was used to perform the calibration over an analytical range from 0.5 to 600 ng/mL. The intra- and inter-batch precision expressed as CV for four QC samples including LLOQ range from 1.14 % to 6.25 %. The intra- and inter-batch accuracy for four concentrations of montelukast were in the range of 95.19%–104.1%. All the values for accuracy and precision were within the acceptance range. The method met all the bioanalytical method validation requirements by ICH and was suitable for the assay of montelukast which in the in vitro cell-based pulmonary pharmacokinetics system model.

Ecchymosis: An Unexpected Side Effect of Montelukast

Montelukast is a leukotriene receptor antagonist that is used to treat allergy and asthma. It acts as a cysteinyl leukotriene receptor antagonist that blocks the action of leukotrienes and decreases inflammation. This agent is generally well tolerated in clinical practice. Although montelukast is generally considered as a safe drug, it can cause a few adverse drug reactions. In this case study, a rare side effect of montelukast that has been reported only twice before is presented. The importance of this case report is that the youngest patient who had ecchymosis due to the use of montelukast treatment is reported

Trichomonas vaginalis-secreted cysteinyl leukotrienes promote migration, degranulation and MCP-1 production in mast cells.

Trichomonas vaginalis, a flagellated extracellular protozoan parasite that infects the human genitourinary tract, is usually transmitted by sexual contact. Our previous study showed that the leukotriene B4 (LTB4 ), a Tvaginalis-secreted lipid mediator, induces interleukin (IL)-8 production and promotes mast cell degranulation and migration via BLT1 in human. In this study, we investigated whether Tvaginalis produces another leukotrienes and whether it causes increased MCP-1 production, mast cell migration and degranulation by activating mast cells. We found that cysteinyl leukotrienes (CysLTs) were contained in Tvaginalis-derived secretory product (TvSP) by ELISA. The TvSP-stimulated human mast cell line (HMC-1) exhibited significantly increased monocyte chemoattractant protein-1 (MCP-1) secretion compared to the unstimulated cells. Inhibition of NOX2 activation of cells by treatment of NOX inhibitor or NOX2 siRNA reduced TvSP-stimulated MCP-1 production in HMC-1 cells. It was also confirmed that the receptor for CysLTs is expressed in mast cells. The CysLT receptor (CysLTR) antagonist inhibited TvSP-stimulated MCP-1 production of mast cells, as well as ROS production, migration and degranulation of mast cells, and reduced phospho-NF-kB expression. These results suggest that Tvaginalis-secreted CysLTs promote migration, degranulation and MCP-1 production in human mast cells through CysLT receptor-mediated NOX2 activation.

Sarcoidosis exosomes stimulate monocytes to produce pro-inflammatory cytokines and CCL2

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities, but cell sources and mechanisms have not been identified. In the current study, we found that BALF exosomes from sarcoidosis patients, but not from healthy individuals, induced a dose-dependent elevation of intracellular IL-1β in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1β, IL-6 and TNF from both PBMCs and enriched monocytes, suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients, and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast, a cysteinyl leukotriene receptor antagonist. Further, reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis.

Exosomes from MSCs overexpressing microRNA-223-3p attenuate cerebral ischemia through inhibiting microglial M1 polarization mediated inflammation

AimsTo explore the therapeutic effect and possible mechanism of exosomes from MSCs overexpressing miR-223 on cerebral ischemia and microglia polarization mediated inflammation. Main methodsRats after middle cerebral artery occlusion and reperfusion (MCAO/R) surgery and microglia BV-2 exposed to oxygen and glucose deprivation (OGD) and cysteinyl leukotrienes (CysLTs) stimulation were subject to exosomes from miR-223-3p transfected MSCs treatment, respectively. Behavioral tests were applied to assess the rats' neurological function. FACS was used to analyze M1/M2 microglia BV-2. production of cytokines in the ischemic hemisphere and BV-2 was detected by ELISA or qRT-PCR. Western blotting and qRT-PCR were also used to examine the expression of cysteinyl leukotriene receptor 2 (CysLT2R) in vivo and in vitro. Key findingsExosomes from MSCs over expressing miR-223-3p decreased MCAO/R induced cerebral infarct volume, improved neurological deficits, promoted learning and memorizing abilities. They suppressed pro-inflammatory factors expression and promoted anti-inflammatory factors secretion in the ischemic cortex and hippocampus. In vitro, exosomal miR-223-3p exhibited a more evident impact on modulating mRNA expression and protein production of cytokines. It promoted M2 microglia transformation of M1 microglia induced by NMLTC4 with a concentration-dependent manner. Western blot and qRT-PCR also revealed exosomal miR-223-3p decreased mRNA and protein expression of CysLT2R in vitro and in vivo. SignificanceExosomal miR-223-3p from MSCs attenuated cerebral ischemia/reperfusion injury through inhibiting microglial M1 polarization mediated pro-inflammatory response, which may be related with inhibitory effect of exosomal miR-223-3p on CysLT2R.

Eicosanoids

This article describes the pathways of eicosanoid synthesis, eicosanoid receptors, the action of eicosanoids in different physiological systems, the roles of eicosanoids in selected diseases, and the major inhibitors of eicosanoid synthesis and action. Eicosanoids are oxidised derivatives of 20-carbon polyunsaturated fatty acids (PUFAs) formed by the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (cytP450) pathways. Arachidonic acid (ARA) is the usual substrate for eicosanoid synthesis. The COX pathways form prostaglandins (PGs) and thromboxanes (TXs), the LOX pathways form leukotrienes (LTs) and lipoxins (LXs), and the cytP450 pathways form various epoxy, hydroxy and dihydroxy derivatives. Eicosanoids are highly bioactive acting on many cell types through cell membrane G-protein coupled receptors, although some eicosanoids are also ligands for nuclear receptors. Because they are rapidly catabolised, eicosanoids mainly act locally to the site of their production. Many eicosanoids have multiple, sometimes pleiotropic, effects on inflammation and immunity. The most widely studied is PGE2. Many eicosanoids have roles in the regulation of the vascular, renal, gastrointestinal and female reproductive systems. Despite their vital role in physiology, eicosanoids are often associated with disease, including inflammatory disease and cancer. Inhibitors have been developed that interfere with the synthesis or action of various eicosanoids and some of these are used in disease treatment, especially for inflammation.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4380.

Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer\u2019s Disease mice

Leukotrienes (LTs) contribute to the neuropathology of chronic neurodegenerative disorders including Alzheimer’s Disease (AD), where they mediate neuroinflammation and neuronal cell-death. In consequence, blocking the action of Leukotrienes (LTs) ameliorates pathologies and improves cognitive function in animal models of neurodegeneration. Surprisingly, the source of Leukotrienes (LTs) in the brain is largely unknown. Here, we identified the Leukotriene (LT) synthesis rate-limiting enzyme 5-Lipoxygenase (5-Lox) primarily in neurons and to a lesser extent in a subpopulation of microglia in human Alzheimer´s Disease (AD) hippocampus brain sections and in brains of APP Swedish PS1 dE9 (APP-PS1) mice, a transgenic model for Alzheimer´s Disease (AD) pathology. The 5-Lipoxygenase (5-Lox) activating protein (FLAP), which anchors 5-Lipoxygenase (5-Lox) to the membrane and mediates the contact to the substrate arachidonic acid, was confined exclusively to microglia with the entire microglia population expressing 5-Lipoxygenase activating protein (FLAP). To define the contribution of microglia in the Leukotriene (LT) biosynthesis pathway, we ablated microglia using the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 in wildtype (WT) and APP-PS1 mice. Microglia ablation not only diminished the expression of FLAP and of the Leukotriene (LT) receptor Cysteinylleukotriene receptor 1 (CysLTR1), as expected based on their microglia cell type-specific expression, but also drastically reduced 5-Lipoxygenase (5-Lox) mRNA expression in the brain and its protein expression in neurons, in particular in wildtype (WT) mice. In conclusion i) microglia are key in Leukotriene (LT) biosynthesis, and ii) they regulate neuronal 5-Lipoxygenase (5-Lox) expression implying a yet unknown signaling mechanism between neurons and microglia.

Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)–GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2−/− colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

Methylation of cysteinyl leukotriene receptor 1 genes associates with lung function in asthmatics exposed to traffic-related air pollution

ABSTRACT Air pollution is associated with early declines in lung function and increased levels of asthma-related cysteinyl leukotrienes (CysLT) but a biological pathway linking this rapid response has not been delineated. In this randomized controlled diesel exhaust (DE) challenge study of 16 adult asthmatics, increased exposure-attributable urinary leukotriene E4 (uLTE4, a biomarker of cysteinyl leukotriene production) was correlated (p = 0.04) with declines in forced expiratory volume in 1-second (FEV1) within 6 hours of exposure. Exposure-attributable uLTE4 increases were correlated (p = 0.02) with increased CysLT receptor 1 (CysLTR1) methylation in peripheral blood mononuclear cells which, in turn, was marginally correlated (p = 0.06) with decreased CysLTR1 expression. Decreased CysLTR1 expression was, in turn, correlated (p = 0.0007) with FEV1 declines. During the same time period, increased methylation of GPR17 (a negative regulator of CysLTR1) was observed after DE exposure (p = 0.02); this methylation increase was correlated (p = 0.001) with decreased CysLTR1 methylation which, in turn, was marginally correlated (p = 0.06) with increased CysLTR1 expression; increased CysLTR1 expression was correlated (p = 0.0007) with FEV1 increases. Collectively, these data delineate a potential mechanistic pathway linking increased DE exposure-attributable CysLT levels to lung function declines through changes in CysLTR1-related methylation and gene expression.

Leukotriene receptors (version 2020.3) in the IUPHAR/BPS Guide to Pharmacology Database

The leukotriene receptors (nomenclature as agreed by the NC-IUPHAR subcommittee on Leukotriene Receptors [34, 37]) are activated by the endogenous ligands leukotrienes (LT), synthesized from lipoxygenase metabolism of arachidonic acid. The human BLT1 receptor is the high affinity LTB4 receptor whereas the BLT2 receptor in addition to being a low-affinity LTB4 receptor also binds several other lipoxygenase-products, such as 12S-HETE, 12S-HPETE, 15S-HETE, and the thromboxane synthase product 12-hydroxyheptadecatrienoic acid. The BLT receptors mediate chemotaxis and immunomodulation in several leukocyte populations and are in addition expressed on non-myeloid cells, such as vascular smooth muscle and endothelial cells. In addition to BLT receptors, LTB4 has been reported to bind to the peroxisome proliferator activated receptor (PPAR) α [196] and the vanilloid TRPV1 ligand-gated nonselective cation channel [217]. The receptors for the cysteinyl-leukotrienes (i.e. LTC4, LTD4 and LTE4) are termed CysLT1 and CysLT2 and exhibit distinct expression patterns in human tissues, mediating for example smooth muscle cell contraction, regulation of vascular permeability, and leukocyte activation. There is also evidence in the literature for additional CysLT receptor subtypes, derived from functional in vitro studies, radioligand binding and in mice lacking both CysLT1 and CysLT2 receptors [37]. Cysteinyl-leukotrienes have also been suggested to signal through the P2Y12 receptor [96, 243, 272], GPR17 [57] and GPR99 [168].

Cysteinyl leukotriene receptor 1 promotes 5-fluorouracil resistance and resistance-derived stemness in colon cancer cells.

Colon cancer is a therapy-resistant cancer with a low 5-year survival frequency. The drug 5-fluorouracil (5-FU) has been used as a first-line therapy in metastatic colon cancer in combination with leucovorin or oxaliplatin with a >40% resistance rate. High CysLT1R expression in tumors is associated with poor survival of colon cancer patients. We sought to examine the role of CysLT1R in 5-FU resistance and established 5-FU-resistant (5-FU-R) colon cancer cells. These 5-FU-R-cells expressed increased levels of CysLT1R and showed increased survival and migration compared to nonresistant cells. Increases in thymidylate synthase and active β-catenin were also observed in the 5-FU-R-cells. LTD4/CysLT1R signaling was further increased and abolished after CYSLTR1 CRISPR-Cas9-knockdown and reduced in CysLT1R-doxycycline-knockdown experiments and CysLT1R-antagonist montelukast/5-FU-treated cells. Montelukast and 5-FU resulted in synergistic effects by reducing HT-29cell and 5-FU-R-HT-29cell migration and zebrafish xenograft metastasis. An increase in the stem cell markers in 5-FU-R-cells and 5-FU-R-cell-derived colonospheres and in CysLT1R-Dox-knockdown cells increased colonosphere formation and stem cell markers was noticed after 5-FU treatment. IL-4-mediated stemness in both HT-29-colonospheres and 5-FU-R-cell derived colonospheres was abolished by montelukast or montelukast + 5-FU-treatment. Targeting CysLT1R signaling by montelukast might reverse drug resistance and decrease resistance-derived stemness in colon cancer patients.