High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer.

In normal colon tissue, oestrogen receptor alpha (ERα) is expressed at low levels, while oestrogen receptor beta (ERß) is considered the dominant subtype. However, in colon carcinomas, the ERα/ß ratio is often increased, an observation that prompted us to further investigate ERα's role in colorectal cancer (CRC). Here, we assessed ERα nuclear expression in 351 CRC patients. Among them, 119 exhibited positive ERα nuclear expression, which was significantly higher in cancer tissues than in matched normal tissues. Importantly, patients with positive nuclear ERα expression had a poor prognosis. Furthermore, positive ERα expression correlated with increased levels of the G-protein coupled cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear ß-catenin, both known tumour promoters. In mouse models, ERα expression was decreased in Cysltr1-/- CAC (colitis-associated colon cancer) mice but increased in ApcMin/+ mice with wild-type Cysltr1. In cell experiments, an ERα-specific agonist (PPT) increased cell survival via WNT/ß-catenin signalling. ERα activation also promoted metastasis in a zebrafish xenograft model by affecting the tight junction proteins ZO-1 and Occludin. Pharmacological blockade or siRNA silencing of ERα limited cell survival and metastasis while restoring tight junction protein expression. In conclusion, these findings highlight the potential of ERα as a prognostic marker for CRC and its role in metastasis.

Mast Cells in Aspirin-Exacerbated Respiratory Disease.

PURPOSE OF REVIEW: Aspirin-exacerbated respiratory disease (AERD) is a syndrome of high type 2 inflammation and is known to critically involve mast cell activation. The mast cell is an important cell in the baseline inflammatory processes in the upper and lower airway by maintaining and amplifying type 2 inflammation. But it also is prominent in the hypersensitivity reaction to COX-1 inhibition which defines this condition. RECENT FINDINGS: Recent work highlights the mast cell as a focal point in AERD pathogenesis. Using AERD as a specific model of both high type 2 asthma and chronic sinusitis, the role of mast cell activity can be better understood in other aspects of airway inflammation. Further dissecting out the mechanism of COX-1-mediated mast cell activation in AERD will be an important next phase in our understanding of NSAID-induced hypersensitivity as well as AERD pathophysiology.

Potential of Anti-Leukotriene Drugs as New Therapeutic Agents for Inhibiting Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA) is a cancer with a poor prognosis due to difficulties in diagnosis and limited treatment options, highlighting the urgent need for new targeted therapies. In a clinical setting, we found that leukotriene levels in bile were higher than in serum. Immunohistochemical analysis of surgically resected samples also revealed that CysLT receptor 1 (CysLTR1) was more highly expressed in CCA than in normal bile duct tissue, prompting us to investigate leukotriene as a potential therapeutic target in CCA. In vitro studies using CCA cell lines expressing CysLTR1 showed that leukotriene D4, a major ligand of CysLTR1, promoted cell proliferation, with increased phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Additionally, treatment with two clinically available anti-allergic drugs-zileuton, an inhibitor of CysLT formation, and montelukast, a CysLTR1 inhibitor-had inhibitory effects on cell proliferation and migratory capacity, accompanied by the reduced phosphorylation of AKT and ERK1/2. Furthermore, the simultaneous administration of both drugs synergistically enhanced the inhibitory effect on cell proliferation. Our study suggests that use of these drugs may represent a novel approach to treat CCA through drug repositioning.

Exploring the neuroprotective effects of montelukast on brain inflammation and metabolism in a rat model of quinolinic acid-induced striatal neurotoxicity.

BACKGROUND: One intrastriatal administration of quinolinic acid (QA) in rats induces a lesion with features resembling those observed in Huntington's disease. Our aim is to evaluate the effects of the cysteinyl leukotriene receptor antagonist montelukast (MLK), which exhibited neuroprotection in different preclinical models of neurodegeneration, on QA-induced neuroinflammation and regional metabolic functions. METHODS: The right and left striatum of Sprague Dawley and athymic nude rats were injected with QA and vehicle (VEH), respectively. Starting from the day before QA injection, animals were treated with 1 or 10 mg/kg of MLK or VEH for 14 days. At 14 and 30 days post-lesion, animals were monitored with magnetic resonance imaging (MRI) and positron emission tomography (PET) using [18F]-VC701, a translocator protein (TSPO)-specific radiotracer. Striatal neuroinflammatory response was measured post-mortem in rats treated with 1 mg/kg of MLK by immunofluorescence. Rats treated with 10 mg/kg of MLK also underwent a [18F]-FDG PET study at baseline and 4 months after lesion. [18F]-FDG PET data were then used to assess metabolic connectivity between brain regions by applying a covariance analysis method. RESULTS: MLK treatment was not able to reduce the QA-induced increase in striatal TSPO PET signal and MRI lesion volume, where we only detected a trend towards reduction in animals treated with 10 mg/kg of MLK. Post-mortem immunofluorescence analysis revealed that MLK attenuated the increase in striatal markers of astrogliosis and activated microglia in the lesioned hemisphere. We also found a significant increase in a marker of anti-inflammatory activity (MannR) and a trend towards reduction in a marker of pro-inflammatory activity (iNOS) in the lesioned striatum of MLK-compared to VEH-treated rats. [18F]-FDG uptake was significantly reduced in the striatum and ipsilesional cortical regions of VEH-treated rats at 4 months after lesion. MLK administration preserved glucose metabolism in these cortical regions, but not in the striatum. Finally, MLK was able to counteract changes in metabolic connectivity and measures of network topology induced by QA, in both lesioned and non-lesioned hemispheres. CONCLUSIONS: Overall, MLK treatment produced a significant neuroprotective effect by reducing neuroinflammation assessed by immunofluorescence and preserving regional brain metabolism and metabolic connectivity from QA-induced neurotoxicity in cortical and subcortical regions.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

The tumor promoter cysteinyl leukotriene receptor 1 regulates PD-L1 expression in colon cancer cells via the Wnt/ß-catenin signaling axis.

Immunotherapy targeting programmed death-ligand 1 (PD-L1) or PD-1 in solid tumors has been shown to be clinically beneficial. However, in colorectal cancer (CRC), only a subset of patients benefit from PD-1/PD-L1 treatment. Previously, we showed that high cysteinyl leukotriene receptor 1 (CysLT1R) levels are associated with poor prognosis in CRC patients. Recently, we have revealed the role of the tumor promoter CysLT1R in drug resistance and stemness in colon cancer (CC) cells. Here, we show the role of the CysLT1R/Wnt/ß-catenin signaling axis in the regulation of PD-L1 using both in vitro and in vivo preclinical model systems. Interestingly, we found that both endogenous and IFNγ-induced PD-L1 expression in CC cells is mediated through upregulation of CysLT1R, which enhances Wnt/ß-catenin signaling. Therapeutic targeting of CysLT1R with its antagonist montelukast (Mo), as well as CRISPR/Cas9-mediated or doxycycline-inducible functional absence of CysLT1R, negatively regulated PD-L1 expression in CC cells. Interestingly, an anti-PD-L1 neutralizing antibody exhibited stronger effects together with the CysLT1R antagonist in cells (Apcmut or CTNNB1mut) with either endogenous or IFNγ-induced PD-L1 expression. Additionally, mice treated with Mo showed depletion of PD-L1 mRNA and protein. Moreover, in CC cells with combined treatment of a Wnt inhibitor and an anti-PD-L1 antibody was effective only in ß-catenin-dependent (APCmut) context. Finally, analysis of public dataset showed positive correlations between the PD-L1 and CysLT1R mRNA levels. These results elucidate a previously underappreciated CysLT1R/Wnt/ß-catenin signaling pathway in the context of PD-L1 inhibition in CC, which might be considered for improving the efficacy of anti-PD-L1 therapy in CC patients. Video Abstract.

Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Deficiency of astrocyte CysLT1R ameliorates depression-like behaviors in mice by modulating glutamate synaptic transmission.

Our previous study suggests that hippocampal cysteinyl leukotriene receptor 1 (CysLT1R) could be involved in depression. Herein we hypothesize that CysLT1R may regulate depression by affecting synaptic glutamate cycling based on existence of CysLT1R in the astrocytes that participate in occurrence of depression. We found that CysLT1R expression was significantly increased in the astrocyte of chronic unpredictable mild stress (CUMS)-induced depression-like mice, CysLT1R astrocyte-specific conditional knockout (AcKO) significantly improved depression-like behaviors, as indicated by decreased immobility time in the forced swimming test and tail suspension test and increased sucrose preference in the sucrose preference test, and knockdown of CysLT1R in the astrocyte of dentate gyrus (DG), the region with the most significant increase of CysLT1R in the astrocyte of depression-like mice, produced similar effects. Correspondingly, overexpression of CysLT1R in the astrocyte of DG induced depression-like behaviors in mice. The further study showed that CysLT1R AcKO ameliorated synaptic plasticity impairment, as reflected by increased synapse, LTP and PSD95, and promoted glutamate transporter 1 (GLT-1) expression by inhibiting NF-κB p65 nuclear translocation mediated by ß-arestin2 and clatrhin, subsequently decreased glutamate in synaptic cleft and GluN2B on postsynaptic membrane in depression-like mice. The present study also showed that GLT-1 agonist or NF-κB inhibitor ameliorated depressive-like behaviors induced by overexpression of the astrocyte CysLT1R of DG. Our study demonstrated that astrocyte CysLT1R regulated depression by modulating glutamate synaptic transmission, suggesting that CysLT1R could be a potential target for developing novel drugs of anti-depression.

Leukotriene D4 accelerates antigen-mediated mast cell responses via the cysteinyl leukotriene 1 receptor.

Cysteinyl leukotrienes (CysLTs), released from mast cells (MCs), are important mediators in allergy. Type 1 receptors for CysLTs (CysLT1R) are involved in accelerating IgE-mediated MC activation. In this study, we aimed to elucidate the mechanisms underlying CysLT1R-mediated MC activation. The CysLT1R agonist/antagonist was applied to two types of major MC models-RBL-2H3 cells and bone marrow-derived MCs (BMMCs). The use of CysLT1R and CysLT2R inhibitors revealed that CysLT1R plays a major role in the acceleration of MC activation. The administration of the CysLT1R agonist leukotriene D4 upregulated IgE-mediated Akt and ERK phosphorylation and subsequently enhanced TNF-α expression, suggesting that CysLT1R regulates the downstream pathway of MC activation. However, these observations were not corroborated by CysLT1R knockdown using shRNA, suggesting a differential regulatory mechanism between the temporal and constitutive inhibitions of CysLT. In conclusion, CysLT1R enhances MC activation by accelerating IgE-induced signal transduction, which enables the co-regulation of rapid degranulation and delayed synthesis of inflammatory mediators in MCs.

Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis.

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2-/-) compared with WT and CysLT1R-null mice (Cysltr1-/-). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2-/- recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.

Cysteinyl leukotriene induces eosinophil extracellular trap formation via cysteinyl leukotriene 1 receptor in a murine model of asthma.

PURPOSE: Eosinophils are one of the main cells responsible to the inflammatory response in asthma by the release of inflammatory molecules such as cytokines, reactive oxygen species (ROS), cytotoxic granule, eosinophil extracellular trap (EET), and lipid mediators as cysteinyl leukotriene (cysLT). The interconnections between these molecules are not fully understood. Here, we attempted to investigate the cysLT participation in the mechanisms of EET formation in an asthma model of OVA challenge. MATERIALS AND METHODS: Before intranasal challenge with OVA, BALB/cJ mice were treated with a 5-lipoxygenase-activating protein (FLAP) inhibitor (MK-886), or with a cysLT1 receptor antagonist (MK-571) and the lung and bronchoalveolar lavage fluid (BALF) were analyzed. RESULTS: We showed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in inflammatory cells, goblet cells hyperplasia, and eosinophil peroxidase (EPO) activity in the airway. However, only OVA-challenged mice treated with MK-571 had an improvement in lung function. Also, treatments with MK-886 or MK-571 decreased Th2 cytokines levels in the airway. Moreover, we observed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in EET formation in BALF. We also verified that EET release was not due to cell death because the cell viability remained the same among the groups. CONCLUSION: We revealed that the decrease in cysLT production or cysLT1 receptor inhibition by MK-886 or/and MK-571 treatments, respectively reduced EET formation in BALF, showing that cysLT regulates the activation process of EET release in asthma.

Anti-Allergic Drug Suppressed Pancreatic Carcinogenesis via Down-Regulation of Cellular Proliferation.

Pancreatic cancer is a fatal disease, and thus its chemoprevention is an important issue. Based on the recent report that patients with allergic diseases have a low risk for pancreatic cancer, we examined the potential chemopreventive effect of anti-allergic agents using a hamster pancreatic carcinogenesis model. Among the three anti-allergic drugs administered, montelukast showed a tendency to suppress the incidence of pancreatic cancer. Further animal study revealed a significantly decreased incidence of pancreatic cancer in the high-dose montelukast group compared with controls. The development of the pancreatic intraepithelial neoplasia lesions was also significantly suppressed. The Ki-67 labeling index was significantly lower in pancreatic carcinomas in the high-dose montelukast group than in controls. In vitro experiments revealed that montelukast suppressed proliferation of pancreatic cancer cells in a dose-dependent manner with decreased expression of phospho-ERK1/2. Montelukast induced G1 phase arrest. Conversely, leukotriene D4 (LTD4), an agonist of CYSLTR1, increased cellular proliferation of pancreatic cancer cells with an accumulation of phospho-ERK1/2. In our cohort, pancreatic ductal adenocarcinoma patients with high CYSLTR1 expression showed a significantly unfavorable clinical outcome compared with those with low expression. Our results indicate that montelukast exerts a chemopreventive effect on pancreatic cancer via the LTD4-CYSLTR1 axis and has potential for treatment of pancreatic carcinogenesis.

Cysteinyl Leukotriene Synthesis via Phospholipase A2 Group IV Mediates Exercise-induced Bronchoconstriction and Airway Remodeling.

It is well known that the prevalence of asthma is higher in athletes, including Olympic athletes, than in the general population. In this study, we analyzed the mechanism of exercise-induced bronchoconstriction by using animal models of athlete asthma. Mice were made to exercise on a treadmill for a total duration of 1 week, 3 weeks, or 5 weeks. We analyzed airway responsiveness, BAL fluid, lung homogenates, and tissue histology for each period. In mice that were treated (i.e., the treatment model), treatments were administered from the fourth to the fifth week. We also collected induced sputum from human athletes with asthma and analyzed the supernatants. Airway responsiveness to methacholine was enhanced with repeated exercise stimulation, although the cell composition in BAL fluid did not change. Exercise induced hypertrophy of airway smooth muscle and subepithelial collagen deposition. Cysteinyl-leukotriene (Cys-LT) levels were significantly increased with exercise duration. Montelukast treatment significantly reduced airway hyperresponsiveness (AHR) and airway remodeling. Expression of PLA2G4 (phospholipase A2 group IV) and leukotriene C4 synthase in the airway epithelium was upregulated in the exercise model, and inhibition of PLA2 ameliorated AHR and airway remodeling, with associated lower levels of Cys-LTs. The levels of Cys-LTs in sputum from athletes did not differ between those with and without sputum eosinophilia. These data suggest that AHR and airway remodeling were caused by repeated and strenuous exercise. Cys-LTs from the airway epithelium, but not inflammatory cells, may play an important role in this mouse model.

MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication.

The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 µM and a maximum HCV RNA level reduction of approximatively 1 log10 MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

Combining 1,4-dihydroxy quininib with Bevacizumab/FOLFOX alters angiogenic and inflammatory secretions in ex vivo colorectal tumors.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer-related mortality worldwide with one in every five patients diagnosed with metastatic CRC (mCRC). In mCRC cases, the 5-year survival rate remains at approximately 14%, reflecting the lack of effectiveness of currently available treatments such as the anti-VEGF targeting antibody Bevacizumab combined with the chemotherapy folinic acid, fluorouracil and oxaliplatin (FOLFOX). Approximately 60% of patients do not respond to this combined treatment. Furthermore, Bevacizumab inhibits dendritic cell (DC) maturation in poor responders, a key process for tumor eradication. METHOD: Following drug treatment, secreted expression levels of angiogenic and inflammatory markers in tumor conditioned media generated from human ex vivo colorectal tumors were measured by ELISA. Dendritic cell phenotypic and maturation markers were assessed by flow cytometry. RESULTS: Our novel compound, 1,4-dihydroxy quininib, acts in an alternative pathway compared to the approved therapy Bevacizumab. 1,4-dihydroxy quininib alone, and in combination with Bevacizumab or FOLFOX significantly reduced TIE-2 expression which is involved in the promotion of tumor vascularization. Combination treatment with 1,4-dihydroxy quininib significantly increased the expression level of DC phenotypic and maturation markers. CONCLUSION: Our results indicate the anti-angiogenic small molecule 1,4-dihydroxy quininib could be an alternative novel treatment in combination therapy for CRC patients.

The synergistic effects of clopidogrel with montelukast may be beneficial for asthma treatment.

Platelets modulate asthma pathogenesis by forming the platelet-eosinophil aggregation (PEA), which facilitates the activation of eosinophils. Platelets exhibit the purinergic receptor (P2Y12R), which responds to cysteinyl leukotriene E4 (LTE4 ). We have suggested that the combination of an antiplatelet drug (clopidogrel, [Clo]) and montelukast (Mon) would synergistically suppress asthma. BALB/c mice were intraperitoneally sensitized with ovalbumin (OVA) on days 0 and 14 and subsequently challenged on days 28-30 and 42-44. Mice were administered with Clo (10 mg/kg), Mon (10 mg/kg) or both drugs (Clo/Mon) orally 30 minutes before the OVA (1%) challenge on days 42-44. Mice were assayed for airway hyper-responsiveness (AHR) to methacholine and airway inflammation. Clopidogrel and montelukast attenuated the increased AHR; the combined treatment was more effective than a single treatment for total and eosinophil counts (all P < 0.05). Levels of interleukin (IL)-4, IL-5, IL-13, platelet factor 4, eosinophil peroxidase and LTE4 increased in the bronchoalveolar lavage fluid of asthmatic mice, but these levels decreased in mice treated with Clo/Mon (all P < 0.05). Goblet cell hyperplasia decreased in response to Clo/Mon. Mouse platelets and eosinophils were isolated and co-cultured for an in vitro assay with 10 µmol/L adenosine diphosphate (ADP), LTE4 (200 nmol/L), Mon (1 µmol/L), Clo (1 µmol/L) and Clo/Mon (1 µmol/L). Flow cytometry revealed that the increased formation of the PEA (%) was fully mediated by ADP and partly mediated by LTE4 . Clo/Mon reduced ADP-induced PEA formation and P-selectin expression (P < 0.05). In conclusion, Clo/Mon synergistically relieved asthma by inhibiting ADP-mediated PEA formation.

Evaluation of oncogenic cysteinyl leukotriene receptor 2 as a therapeutic target for uveal melanoma.

Uveal melanoma is a rare, but deadly, form of eye cancer that arises from melanocytes within the uveal tract. Although advances have emerged in treatment of the primary tumour, patients are still faced with vision loss, eye enucleation and lethal metastatic spread of the disease. Approximately 50% of uveal melanoma patients develop metastases, which occur most frequently in the liver. Metastatic patients encounter an extremely poor prognosis; as few as 8% survive beyond 2 years. Understanding of the genetic underpinnings of this fatal disease evolved in recent years with the identification of new oncogenic mutations that drive uveal melanoma pathogenesis. Despite this progress, the lack of successful therapies or a proven standard-of-care for uveal melanoma highlights the need for new targeted therapies. This review focuses on the recently identified CYSLTR2 oncogenic mutation in uveal melanoma. Here, we evaluate the current status of uveal melanoma and investigate how to better understand the role of this CYSLTR2 mutation in the disease and implications for patients harbouring this mutation.

Evidence for the induction of Th2 inflammation by group 2 innate lymphoid cells in response to prostaglandin D2 and cysteinyl leukotrienes in allergic rhinitis.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play important roles in allergic inflammation. However, their roles in the pathophysiology of allergic rhinitis (AR) are poorly understood. OBJECTIVE: Prevalence of ILC2s in the inferior nasal turbinate (INT) tissues and the activating mechanisms of ILC2s were examined in patients with house dust mite (HDM)-induced AR. METHODS: Eighteen patients with HDM-induced AR and 13 control subjects were recruited. Fresh INT tissues and peripheral blood mononuclear cells (PBMCs) were analysed using flow cytometry. Nasal lavage fluids (NLF) were collected at 10 minutes after the nasal provocation test (NPT) with HDM disc, and released mediators were measured by ELISA. Sorted ILC2s were cultured and stimulated with mediators associated with AR. RESULTS: The prevalence of ILC2s was significantly increased in nasal mucosa of patients with HDM-induced AR, and it was positively correlated with the number of infiltrating eosinophils. ILC2s in the INT tissues expressed a prostaglandin D2 (PGD2 ) receptor, chemoattractant receptor-homologous molecule-expressed TH2 cells (CRTH2) and a cysteinyl leukotriene (cysLTs) receptor, CysLT1. After NPT, the number of eosinophils and concentrations of PGD2 and cysLTs were significantly increased in the NLF from AR patients. PGD2 and cysLTs significantly induced IL-5 production from cultured PBMC-derived ILC2s dose-dependently. PGD2 -induced and cysLTs-induced productions of IL-5 and IL-13 from ILC2s were completely inhibited by ramatroban, a dual CRTH2 and thromboxane receptor antagonist, and montelukast, a CysLT1 antagonist, respectively. CONCLUSIONS: PGD2 -CRTH2 and cysLTs-CysLT1 axes may activate tissue-resident ILC2s to produce Th2 cytokines, IL-5 and IL-13, leading to the development of allergic inflammation in AR.

The Role of Leukotrienes as Potential Therapeutic Targets in Allergic Disorders.

Leukotrienes (LTs) are lipid mediators that play pivotal roles in acute and chronic inflammation and allergic diseases. They exert their biological effects by binding to specific G-protein-coupled receptors. Each LT receptor subtype exhibits unique functions and expression patterns. LTs play roles in various allergic diseases, including asthma (neutrophilic asthma and aspirin-sensitive asthma), allergic rhinitis, atopic dermatitis, allergic conjunctivitis, and anaphylaxis. This review summarizes the biology of LTs and their receptors, recent developments in the area of anti-LT strategies (in settings such as ongoing clinical studies), and prospects for future therapeutic applications.

Antagonism of cysteinyl leukot-riene receptor 1 (cysLT1R) by montelukast regulates differentiation of MC3T3-E1 cells under overloaded mechanical environment.

Long-term exposure to overloaded mechanical environment induces bone fatigue damage symptoms and osteoblast damages. Montelukast is a selective cysteinyl leukot-riene receptor 1 (cysLT1R) antagonist, which has been used for the treatment of bronchial asthma in clinics. In the current study, we have identified a novel pharmacological role of montelukast by finding that it has protective properties against overload damage in osteoblastic MC3T3-E1 cells. Firstly, our results show that CysLT1R is expressed in MC3T3-E1 cells. Mechanical tensile strain of 5000-7000 µÎµ resulted in a significant upregulation of CysLT1R in osteoblastic MC3T3-E1 cells in an intensity dependent manner. Secondly, MTT assay indicates that loading with 5000 µÎµ mechanical strain inhibited cell proliferation, which was suppressed by montelukast treatment. Furthermore, montelukast promotes cell differentiation by increasing the expression of ALP and RUNX2. Alizarin Red S staining assay showed that montelukast abolished the inhibitory effects of overload mechanics on osteoblast mineralization. Mechanistically, the effect of montelukast on osteoblastic differentiation acted by activating the extracellular regulated protein kinases (ERK) pathway. The obtained results suggested that montelukast promotes proliferation and differentiation in osteoblasts exposed to overload mechanics.