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Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A(3) adenosine receptor (A(3)AR) signaling. Supplementing adenosine signaling with selective A(3)AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A(3)AR in the spinal cord with an ADK inhibitor or A(3)AR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%–75%), cleaved caspase 1 (40%–60%), interleukin (IL)-1β (76%–80%), and tumor necrosis factor (50%–60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A(3)AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A(3)AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A(3)AR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the A(3)AR, resulting in NOD-like receptor pyrin domain-containing 3–interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by A(3)AR agonists, suggesting that A(3)AR may be a target for therapeutic intervention with selective A(3)AR agonist as opioid adjuncts.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N

Adenosine is a purine nucleoside, responsible for the regulation of multiple physiological and pathological cellular and tissue functions by activation of four G protein-coupled receptors (GPCR), namely A1, A2A, A2B, and A3 adenosine receptors (ARs). In recent years, extensive progress has been made to elucidate the role of adenosine in pain regulation. Most of the antinociceptive effects of adenosine are dependent upon A1AR activation located at peripheral, spinal, and supraspinal sites. The role of A2AAR and A2BAR is more controversial since their activation has both pro- and anti-nociceptive effects. A3AR agonists are emerging as promising candidates for neuropathic pain. Although their therapeutic potential has been demonstrated in diverse preclinical studies, no AR ligands have so far reached the market. To date, novel pharmacological approaches such as adenosine regulating agents and allosteric modulators have been proposed to improve efficacy and limit side effects enhancing the effect of endogenous adenosine. This review aims to provide an overview of the therapeutic potential of ligands interacting with ARs and the adenosinergic system for the treatment of acute and chronic pain.

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer&rsquo, s disease, Parkinson&rsquo, s disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin&ndash, chalcone hybrids were synthesized (compounds 1&ndash, 8) and screened in radioligand binding (hA1, hA2A and hA3) and adenylyl cyclase (hA2B) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin&ndash, chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA1 or hA3 subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA1, while the methoxy counterparts were selective for the hA3. The most potent hA1 ligand was compound 7 (Ki = 17.7 &micro, M), whereas compound 4 was the most potent ligand for hA3 (Ki = 2.49 &micro, M). In addition, docking studies with hA1 and hA3 homology models were established to analyze the structure&ndash, function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.

Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A3 adenosine receptor (AR) affinity/selectivity, e.g., ...

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Following our study of 4′-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5′-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (∼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and β-arrestin 2 (βarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.

Background MicroRNAs (miRNAs) are important post-translational regulators. Elevated levels of miR-206 in ulcerative colitis (UC) were associated with suppression of anti-inflammatory A3 adenosine receptor (A3AR) expression. However, the relationship of miR-206 to histologic remission in UC patients remains unknown. This study correlates expression levels of miR-206 with histologic remission in patients treated via long-term mesalamine treatment to identify a possible mode of action for this mainstay drug for UC. Methods Expression of miR-206 and its target A3AR were analyzed in HT29 cell line before and after mesalamine treatment (2 mM) at different time points (0, 4, 12, and 24 hours) by qRT-PCR and western blot analysis. Expression of miR-206 and pathological scores of colonoscopic biopsy specimens were studied in 10 UC patients treated with mesalamine treatment for 2 to 6 years. Results miR-206 transcripts decreased 2.23-fold (P = 0.0001) 4 hours after 2 mM mesalamine treatment in HT29 colon cells compared with untreated controls. However, the mRNA/protein levels of A3AR increased by 4-fold (P = 0.04) and 2-fold, respectively, in same cells. miR-206 relative expression decreased significantly in patients treated with 4.8 g of mesalamine (P = 0.002) but not with 2.4 g (P = 0.35). Tissue assessment of sequential mesalamine-treated colonoscopic biopsies indicate a strong correlation between downregulation of miR-206 and histologic improvement (R = 0.9111). Conclusion Mesalamine treatment has an effect on epithelial miRNAs. Downregulation of miR-206 by long-term mesalamine treatment may confer a protective effect in inducing and maintaining histologic remission. Thus, miR-206 expression levels can be utilized as a possible biomarker for therapeutic response to mesalamine treatment.

Vascular calcification is a typical feature of atherosclerosis and is associated with adverse cardiovascular events such as myocardial infarction and stroke. Several studies have suggested that adenosine, an ATP metabolite may function as an endogenous regulator of arterial calcification. However, its effects on vascular smooth muscle cell calcification have not been clarified. In this study, we investigated the inhibitory effects of adenosine on vascular calcification in vitro by utilizing the culture of human aortic smooth muscle cells (HASMCs). Osteoblastic differentiation of HASMCs was induced by the treatment with oncostatin M and osteogenic differentiation medium. Adenosine and its metabolically stable analogue, 2-chloroadenosine (CADO) significantly reduced matrix mineralization and alkaline phosphatase (ALP) activities in HASMCs. The mRNA expression of tissue non-specific alkaline phosphatase (TNAP) was down-regulated by adenosine and CADO, but the mRNA expression of other osteoblastic differentiation markers, such as Runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP)-II, was not significantly affected by these two reagents. Among the adenosine receptor (AR) subtype-selective agonists used, only IB-MECA (A

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage.

Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders. Adenosine elicits biological effects through four G protein-coupled receptors (A1, A2A, A2B, and A3). The A2A and A2B receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels, whereas A1 and A3 receptors inhibit AC and decrease cAMP levels. Several studies have investigated the effects of adenosine receptors (AdoRs) in glaucoma, because modulation of A1, A2A, or A3 receptor regulates intraocular pressure. In addition, AdoR-related phenomena may induce neuroprotective effects in retinal neurons. Notably, A1, A2A, and A3 receptor agonists reportedly inhibit retinal ganglion cell (RGC) death in in vitro and in vivo glaucoma models. However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we described the role of an AdoR subtype in neurite outgrowth and RGC axonal regeneration. The distribution of AdoRs in the retina was evaluated by immunohistochemical analysis. Using primary cultured rat RGCs in vitro and an optic nerve crush model in vivo, neurite elongation was evaluated after stimulation by the following AdoR agonists: CHA, an A1 receptor agonist; CGS21680, an A2A receptor agonist; BAY60-6583, an A2B receptor agonist; and 2-Cl-IB-MECA, an A3 receptor agonist. To determine the mechanism of neurite promotion, the candidate molecules of signal transduction associated with the neurite elongation of AdoRs were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. All four AdoRs (A1, A2A, A2B, and A3) were present in the inner retinal layers. Among the agonists for AdoR, only 2-Cl-IB-MECA significantly promoted neurite outgrowth in primary cultured RGCs. Signaling pathway analyses showed that 2-Cl-IB-MECA caused upregulated phosphorylation of Akt in cultured RGCs. Additionally, LY294002, an inhibitor of Akt, suppressed the neurite-promoting effects of the A3 receptor agonist in RGCs. Moreover, 2-Cl-IB-MECA increased the number of regenerating axons in the optic nerve crush model. Taken together, these data indicate that activation of the A3 receptor, not the A1 or A2 receptors, promotes in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, which is caused by the activation of an Akt-dependent signaling pathway. Therefore, AdoR activation may be a promising candidate for the development of novel regenerative modalities for glaucoma and other optic neuropathies.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease, and the current pharmacological treatment for DKD is limited to renin-angiotensin system (RAS) inhibitors. Adenosine is detectable in the kidney and is significantly elevated in response to cellular damage. While all 4 known subtypes of adenosine receptors, namely, A1AR, A2aAR, A2bAR, and A3AR, are expressed in the kidney, our previous study has demonstrated that a novel, orally active, species-independent, and selective A3AR antagonist, LJ-1888, ameliorates unilateral ureteral obstruction-induced tubulointerstitial fibrosis. The present study examined the protective effects of LJ-2698, which has higher affinity and selectivity for A3AR than LJ-1888, on DKD. In experiment I, dose-dependent effects of LJ-2698 were examined by orally administering 1.5, 5, or 10 mg/kg for 12 weeks to 8-week-old db/db mice. In experiment II, the effects of LJ-2698 (10 mg/kg) were compared to those of losartan (1.5 mg/kg), which is a standard treatment for patients with DKD. LJ-2698 effectively prevented kidney injuries such as albuminuria, glomerular hypertrophy, tubular injury, podocyte injury, fibrosis, inflammation, and oxidative stress in diabetic mice as much as losartan. In addition, inhibition of lipid accumulation along with increases in PGC1α, a master regulator of mitochondrial biogenesis, were demonstrated in diabetic mice treated with either LJ-2698 or losartan. These results suggest that LJ-2698, a selective A3AR antagonist, may become a novel therapeutic agent against DKD., Diabetic kidney disease: drug successfully targets key protein A therapeutic treatment targeting a protein involved in the progression of diabetic kidney disease (DKD) shows promise in mouse trials. Between 30 and 40 per cent of diabetic patients suffer from DKD, a common cause to fatal end-stage kidney disease. Protein receptors, commonly expressed on cell surfaces throughout the body, play both positive and negative roles in diseases. The A3 adenosine receptor (A3AR) is highly expressed in diabetic kidney tissue, and is linked to disease progression. Hunjoo Ha at Ewha Womans University in Seoul, Republic of Korea, and co-workers demonstrated the positive effect of a novel drug in targeting A3AR in mice with DKD. A 12-week treatment of the drug prevented kidney injury, lowered oxidative stress and inflammation, and improved kidney function. It may prove an invaluable drug, particularly in combination with an existing DKD drug.

Background: Bone and joint formation, maintenance, and regeneration are regulated by both chemical and physical signals. Among the physical signals there is an increasing realization of the role of pulsed electromagnetic fields (PEMF) in the treatment of nonunions of bone fractures. The discovery of the piezoelectric properties of bone by Fukada and Yasuda in 1953 in Japan established the foundation of this field. Pioneering research by Bassett and Brighton and their teams resulted in the approval by the Food and Drug Administration (FDA) of the use of PEMF in the treatment of fracture healing. Although PEMF has potential applications in joint regeneration in osteoarthritis (OA), this evolving field is still in its infancy and offers novel opportunities. Methods: We have systematically reviewed the literature on the influence of PEMF in joints, including articular cartilage, tendons, and ligaments, of publications from 2000 to 2016. Conclusions: PEMF stimulated chondrocyte proliferation, differentiation, and extracellular matrix synthesis by release of anabolic morphogens such as bone morphogenetic proteins and anti-inflammatory cytokines by adenosine receptors A2(A) and A3 in both in vitro and in vivo investigations. It is noteworthy that in clinical translational investigations a beneficial effect was observed on improving function in OA knees. However, additional systematic studies on the mechanisms of action of PEMF on joints and tissues therein, articular cartilage, tendons, and ligaments are required.

Chemical entities with structural diversity were introduced as candidates targeting adenosine receptor with different clinical activities, containing 3,7-dihydro-1H-purine-2,6-dione, especially adenosine 3 receptors (ADORA3). Our initial approach started with pharmacophore screening of ADORA3 modulators; to choose linagliptin (LIN), approved anti-diabetic drug as Dipeptidyl peptidase-4 inhibitors, to be studied for its modulating effect towards ADORA3. This was followed by generation, purification, analytical method development, and structural elucidation of oxidative degraded product (DEG). Both of LIN and DEG showed inhibitory profile against hepatocellular carcinoma cell lines with induction of apoptosis at G2/M phase with increase in caspase-3 levels, accompanied by a downregulation in gene and protein expression levels of ADORA3 with a subsequent increase in cAMP. Quantitative in vitro assessment of LIN binding affinity against ADORA3 was also performed to exhibit inhibitory profile at Ki of 37.7 nM. In silico molecular modelling showing binding affinity of LIN and DEG towards ADORA3 was conducted.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis.

A series of adenosine and 2′-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A(1), A(2A), A(2B) and A(3) adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A(3) AR over other ARs was observed for most tested ligands. In particular, 5′-ethylcarbamoyl-N(6)-(3-phenylpropyl)adenosine (18), N(6)-(3-phenylpropyl)-2-chloroadenosine (24) and N(6)-(3-phenylpropyl)adenosine (40) showed nanomolar A(3) affinity (K(i) 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A(3) affinity (K(i) 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A(3) receptor selectivity than the corresponding phenyl analogs: 7 vs. 8, 15 vs. 16, 17 vs. 18.

Mast cells (MCs) constitute an important part of the tumor microenvironment (TME). However, their underlying mechanisms of activation within the TME remain poorly understood. Here we show that recapitulating cell-to-cell contact interactions by exposing MCs to membranes derived from a number of cancer cell types, results in MC activation, evident by the increased phosphorylation of the ERK1/2 MAP kinases and Akt, in a phosphatidylinositol 3-kinase dependent fashion. Activation is unidirectional since MC derived membranes do not activate cancer cells. Stimulated ERK1/2 phosphorylation is strictly dependent on the ecto enzyme CD73 that mediates autocrine formation of adenosine, and is inhibited by knockdown of the A3 adenosine receptor (A3R) as well as by an A3R antagonist or by agonist-stimulated down-regulation of the A3R. We also show that cancer cell mediated triggering upregulates expression and stimulates secretion of interleukin 8 from the activated MCs. These findings provide evidence for a novel mode of unidirectional crosstalk between MCs and cancer cells implicating direct activation by cancer cells in MC reprogramming into a pro tumorigenic profile.

Regulation of blood flow depends on systemic and local release of vasoactive molecules such as insulin and adenosine. These molecules cause vasodilation by activation of plasma membrane receptors at the vascular endothelium. Adenosine activates at least four subtypes of adenosine receptors (A(1)AR, A(2A)AR, A(2B)AR, A(3)AR), of which A(2A)AR and A(2B)AR activation leads to increased cAMP level, generation of nitric oxide, and relaxation of the underlying smooth muscle cell layer. Vasodilation caused by adenosine also depends on plasma membrane hyperpolarization due to either activation of intermediate-conductance Ca2+-activated K+ channels in vascular smooth muscle or activation of ATP-activated K+ channels in the endothelium. Adenosine also causes vasoconstriction via a mechanism involving A(1)AR activation resulting in lower cAMP level and increased thromboxane release. Insulin has also a dual effect causing NO-dependent vasodilation, but also sympathetic activity-and increased endothelin 1 release-dependent vasoconstriction. Interestingly, insulin effects require or are increased by activation or inactivation of adenosine receptors. This is phenomenon described for D-glucose and L-arginine transport where A(2A)AR and A(2B)AR play a major role. Other studies show that A(1)AR activation could reduce insulin release from pancreatic beta-cells. Whether adenosine modulation of insulin biological effect is a phenomenon that depends on co-localization of adenosine receptors and insulin receptors, and adenosine plasma membrane transporters is something still unclear. This review summarizes findings addressing potential involvement of adenosine receptors to modulate insulin effect via insulin receptors with emphasis in the human vasculature. (C) 2016 Elsevier Ltd. All rights reserved.

Mast cells (MCs) play a critical role in allergic inflammation. Although purinergic signalling is implicated in the regulation of various immune responses, its role in MC function is not fully understood. In this study, we investigated the regulatory role of purinergic signalling in MC degranulation, using mouse bone marrow-derived mast cells (BMMCs). Notably, BMMCs expressed various functional P2 adenosine triphosphate (ATP) receptors, including ionotropic P2X4 and P2X7, involved in the regulation of BMMC degranulation. Thus, P2X7 receptor activation induced a marked degranulation from BMMCs directly. Although P2X4 receptor activation did not independently induce degranulation, it significantly potentiated the degranulation triggered by antigen-induced, high-affinity IgE receptor (FceRI) stimulation. In addition, ATP synergistically augmented degranulation induced by adenosine A3 receptor activation. Moreover, BMMCs highly expressed ecto-nucleotidase CD39, but not ecto-5'-nucleotidase (CD73), and were therefore unable to directly convert ATP to adenosine. However, in the presence of CD73-expressing cells, ATP-mediated BMMC stimulation caused a marked degranulation in a CD73- and adenosine-dependent manner. These results demonstrate that purinergic signalling plays an important role in MC degranulation through at least three distinct mechanisms: (1) higher ATP concentrations directly induce degranulation via P2X7 receptor activation, (2) lower ATP concentrations augment FceRI-mediated degranulation via P2X4 receptor activation, and (3) in an ecto-nucleotidase-enrich environment, ATP and the converted product adenosine induce a synergistic degranulation by P1 and P2 receptor co-activation.

Aim of the Study: The cell cycle, a vital process that involves in cells' growth and division, lies at the heart of cancer. It has been shown that IB-MECA, an A3 adenosine receptor agonist inhibits the proliferation of cancer cells by inducing cell cycle arrest in several tumors. In this study, we evaluated the role of IB-MECA inhibition in cell cycle progression in ovarian cancer cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in Caov-4 and OVCAR-3. Analysis of cell cycle distribution was carried out by flow cytometry. To determine the mechanisms of IB-MECA-mediated induction of cell cycle arrest, the expression of cell cycle regulatory proteins Cyclin D1 and cyclin-dependent kinase 4 (CDK4) was evaluated. Results: Our results showed that IB-MECA significantly reduced cell viability in a dose-dependent manner. Moreover, our results indicated that a low concentration of IB-MECA induced G1 cell cycle arrest. Reduction of Cyclin D1 and CDK4 protein levels was also observed after treating cancer cells with IB-MECA. Conclusion: This study demonstrated that IB-MECA induces G1 phase cell cycle arrest through Cyclin D1/CDK4-mediated pathway in ovarian cancer cells.

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.

Compounds able to simultaneously bind a biological target and be conjugated to a second specific moiety are attractive tools for the development of multi-purpose ligands useful as multi-target ligands, receptor probes or drug delivery systems, with both therapeutic and diagnostic applications. The human A(3) adenosine receptor is a G protein-coupled receptor involved in many physio-pathological conditions, e.g. cancer and inflammation, thus representing a promising research target. In this work, two series of conjugable hA(3)AR antagonists, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus, were developed. The introduction of an aromatic ring at the 5 position of the scaffold, before (phenylacetamido moiety) or after (1,2,3-triazole obtained by click chemistry) the conjugation is aimed to increase affinity and selectivity towards the hA(3)AR receptor. As expected, conjugable compounds showed good affinity towards the hA(3)AR. In order to prove their potential in the development of hA(3)AR ligands for different purposes, compounds were also functionalized with fluorescent probes. Unfortunately, conjugation decreased affinity and selectivity for the target as compared to the hA(2A)AR. Computational studies identified specific non-conserved residues of the extracellular loops which constitute a structural barrier able to discriminate between ligands, giving insights into the rational development of new highly selective ligands.

Adenosine receptors participate in many physiological functions. Molecules that may selectively interact with one of the receptors are favorable multifunctional chemical entities to treat or decelerate the evolution of different diseases. 3-Arylcoumarins have already been studied as neuroprotective agents by our group. Here, differently 8-substituted 3-arylcoumarins are complementarily studied as ligands of adenosine receptors, performing radioligand binding assays. Among the synthesized compounds, selective A

Adenosine A

We investigated the Gi-coupled A(3) adenosine receptor (A(3)AR) activation mechanism by running 7.2 μs of molecular dynamics (MD) simulations. Based on homology to G protein-coupled receptor (GPCR) structures, three constitutively active mutant (CAM) and the wild-type (WT) A(3)ARs in the apo form were modeled. Conformational signatures associated with three different receptor states (inactive R, active R*, and bound to Gi protein mimic) were predicted by analyzing and comparing the CAMs with WT receptor and by considering site-directed mutagenesis data. Detected signatures that were correlated with receptor state included: Persistent salt-bridges involving key charged residues for activation (including a novel, putative ionic lock), rotameric state of conserved W(6.48), and Na(+) ions and water molecules present. Active-coupled state signatures similar to the X-ray structures of β(2) adrenergic receptor-Gs protein and A(2A)AR-mini-Gs and the recently solved cryo-EM A(1)AR-Gi complexes were found. Our MD analysis suggests that constitutive activation might arise from the D107(3.49)-R108(3.50) ionic lock destabilization in R and the D107(3.49)-R111(3.53) ionic lock stabilization in R* that presumably lowers the energy barrier associated with an R to R* transition. This study provides new opportunities to understand the underlying interactions of different receptor states of other Gi protein-coupled GPCRs.

Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A

The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no experimental structure of A3R. Here, we have generated a molecular model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-d-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA). Molecular dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, molecular mechanics-generalized Born surface area calculations were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.

The Wnt/β-catenin pathway confers a chain of molecular events in livers affected by non-alcoholic steato-hepatitis (NASH). Namodenoson, a selective agonist of the A3 adenosine receptor (A3AR), which is highly expressed in pathological liver cells, induces a robust anti-inflammatory effect in the liver, mediated via the de-regulation of the Wnt/β-catenin pathway. Namodenoson also acts as a liver protective agent by inhibiting ischemia/reperfusion injury. Based on these unique characteristics, we investigated the anti-NASH effect of Namodenoson in murine models of steato-hepatitis and in the LX2 human hepatic stellate cell line (HSC). In the STAM model, Namodenoson significantly decreased the non-alcoholic fatty liver disease (NAFLD) activity score, NAS, demonstrating anti-inflammatory and anti-steatotic effects. In the carbon tetrachloride (CCl4) model, Namodenoson reversed alanine aminotransferase (ALT) to normal values and signifi-cantly improved liver inflammation and fibrosis, as well as the adiponectin and leptin levels. Namodenoson de-regulated the Wnt/β-catenin pathway in the liver extracts of the CCl4 model mice and in the LX2 HSCs, manifested by a decrease in the expression of phosphoinositide 3-kinase (PI3K), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), β-catenin, lymphoid enhancer-binding factor 1 (Lef-1) and cyclin D1, and an increase in the expression level of glycogen synthase kinase 3β (GSK-3β). The fibrosis marker, α-smooth muscle actin (α-SMA) was also de-regulated, supporting the anti-fibrotic effect of Namodenoson. On the whole, the findings of this study demonstrate that Namodenoson exerts an anti-NASH effect mediated via the de-regulation of the PI3K/NF-κB/Wnt/β-catenin signaling pathway. Thus, targeting A3AR may prove to be a novel direction in the pharmacotherapy of NAFLD/NASH.

The adenosine A

Adenosine acts as a powerful signaling molecule via four distinct G protein-coupled receptors, designated A

Adenosine receptors are a family of G protein-coupled receptors with increased attention as drug targets on different indications. We investigate the thermodynamics of ligand binding to the A3 adenosine receptor subtype, focusing on a recently reported series of diarylacetamidopyridine inhibitors via molecular dynamics simulations. With a combined approach of thermodynamic integration and one-step perturbation, we characterize the impact of the charge distribution in a central heteroaromatic ring on the binding affinity prediction. Standard charge distributions according to the GROMOS force field yield values in good agreement with the experimental data and previous free energy calculations. Subsequently, we examine the thermodynamics of inhibitor binding in terms of the energetic and entropic contributions. The highest entropy penalties are found for inhibitors with methoxy substituents in meta position of the aryl groups. This bulky group restricts rotation of aromatic rings attached to the pyrimidine core which leads to two distinct poses of the ligand. Our predictions support the previously proposed binding pose for the o-methoxy ligand, yielding in this case a very good correlation with the experimentally measured affinities with deviations below 4 kJ/mol.

Introduction: There is a vicious cycle of tumor hypoxia, high adenosine levels, immune suppression and cancer growth that involves the use of adenosine receptor ligands in tumors. After several years of research, the candidates emerging as promising new anticancer drugs are A3 adenosine receptor agonists and A2A receptor antagonists. Areas covered: The authors give an updated overview of the field related to A3 receptor agonists and A2A receptor antagonists in cancer and propose their perspectives on the status of these compounds in oncology. The rationale for the modulation of adenosine receptors in cancer is addressed, starting from the first in vitro evidence of their efficacy up to the animal and clinical studies. Expert opinion: A3 and A2A receptors are attractive targets in oncologic therapy due to their involvement in cancer progression and immune-resistance. Of relevance, the A3 subtype is also a tumor marker to be used in a personalized drug treatment program while the A2A receptor, playing a non-redundant role in immunomodulation, may be blocked in combination with checkpoint inhibitors to improve their efficacy. The future will reveal how successful this approach is in the fight against cancer.

Adenosine receptors (ARs) belongs to the family of G-protein coupled receptors (GPCR) that are responsible for the modulation of a wide variety of physiological functions. The ARs are also implicated in many diseases such as cancer, arthritis, cardiovascular and renal diseases. The adenosine A3 receptor (A3AR) has emerged as a potential drug target for the progress of new and effective therapeutic agents for the treatment of various pathological conditions. This receptor’s involvement in many diseases and its validity as a target has been established by many studies. Both agonists and antagonists of A3AR have been extensively investigated in the last decade with the goal of developing novel drugs for treating diseases related to immune disorders, inflammation, cancer, and others. In this review, we shall focus on the medicinal chemistry of A3AR ligands, exploring the diverse chemical classes that have been projected as future leading drug candidates. Also, the recent advances in the therapeuetic applications of A3AR ligands are highlighted.

Methotrexate is the most frequently administered first-line treatment for rheumatoid arthritis (RA). The disease-modifying effects of methotrexate are mainly associated with enhanced release of free adenosine. The downstream anti-inflammatory effects of adenosine are mediated via its binding to adenosine receptor 2A (ADORA2A) and 3 (ADORA3). Many clinically important single nucleotide polymorphisms (SNPs) were reported in ADORA2A and ADORA3 genes.To investigate whether tagging ADORA2A and ADORA3 polymorphisms influences methotrexate treatment in RA.In total, 212 RA patients treated with methotrexate were genotyped for tagging ADORA2A (rs2298383, rs8141793, rs2236624, rs5751876, rs35320474, and rs17004921) and ADORA3 SNPs (rs2298191, rs1544223, rs78594984, rs35511654, rs2229155, rs3393, and rs3394).RA patients who carried ADORA3 rs35511654 G allele showed a tendency toward better response to methotrexate treatment (P = 0.054). Carriers of ADORA2A polymorphic allele rs2298383 (P = 0.011), rs2236624 (P = 0.027), rs5751876 (P = 0.018), and rs35320474 (P = 0.026) were less likely to experience methotrexate induced adverse events. All associations remained significant after adjustment for clinical factors. The effects of these polymorphisms were also significant in haplotype analyses.Polymorphisms in the ADORA2A gene may influence methotrexate treatment response and may be considered as a potential biomarker for methotrexate treatment in rheumatoid arthritis.

Adenosine is a purine nucleoside which is an effective controller of inflammation. The inflammatory effect of adenosine is expressed via its four receptor subtypes viz. A1, A2A, A2B and A3. The various inflammatory conditions including rheumatoid arthritis (RA) are initiated by adenosine receptors of which A2A and A3 play a vital role. RA primarily is an auto-immune disorder which is manifested as chronic inflammation in the synovial lining of joints. In order to develop an effective treatment, the role of cytokines, IL–1, TNF-α and IL–6 is crucial. Besides, the knowledge of PI3K-PKB/Akt and NF-kB signaling pathway is also important to understand the antiinflammatory targets. Methotrexate along with various other molecules like, NSAIDs and DMARDs are presently used as treatment lines for controlling RA. The enhanced knowledge of the preclinical stages and pathogenesis along with recent potent therapeutics raises the hopes that RA can be prevented in the near future.

A series of novel 7-amino-5-oxo-2-substituted-aryl/hetero-aryl-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles (4a-4t) was synthesized, characterized and evaluated for their binding affinity and selectivity towards hA1 , hA2A , hA2B and hA3 adenosine receptors (ARs). Compound 4a with a phenyl ring at 2-position of the triazolo moiety of the scaffold showed high affinity and selectivity for hA1 AR (Ki hA1 = 0.076 μM, hA2A = 25.6 μM and hA3 > 100 μM). Introduction of various electron donating and withdrawing groups at different positions of the phenyl ring resulted in drastic reduction in affinity and selectivity towards all the ARs, except compound 4b with a 4-hydroxyphenyl group at 2-position. Interestingly, the replacement of the phenyl ring with a smaller heterocyclic thiophene ring (π excessive system) resulted in further improvement of affinity for hA1 AR of compound 4t (Ki hA1 = 0.051 μM, hA2A = 9.01 μM and hA3 > 13.9 μM) while retaining the significant selectivity against all other AR subtypes similar to compound 4a. The encouraging results for compounds 4a and 4t indicate that substitution at 2-position of the scaffold with π-excessive systems other than thiophene may lead to even more potent and selective hA1 AR antagonists.

Adenosine receptors ADORA

Recently, studies have focused on the antihyperalgesic activity of the A(3) adenosine receptor (A(3)AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A(3)AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A(3)AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A(3)AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca(2+)-activated K(+) (K(Ca)) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca(2+) influx via N-type voltage-dependent Ca(2+) channels, as Cl-IB-MECA–induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca(2+) currents, and its effect was inhibited by 56% in the presence of A(3)AR antagonist MRS1523, demonstrating that the majority of adenosine’s effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A(3)AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca(2+) measurements confirmed the inhibitory role of A(3)AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A(3)AR activation could be mediated through N-type Ca(2+) channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A(3)AR-based therapy as a viable approach to alleviate pain in different pathologies.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The clinical course may range from spontaneous regression towards ganglioneuroblastoma/ganglioneuroma or maturation to a very aggressive form characterized by an extensive hypoxic area. In solid tumors, extracellular microenvironment hypoxia induces the transcription of hypoxia-inducible factors (HIFs) leading to synthesis of pro-angiogenic factor, VEGF; also, it increases extracellular adenosine production from ATP breakdown. To date, the role of this nucleoside in the hypoxic/angiogenic pathway characterizing the core of cancer mass has not been investigated yet. Therefore, the aim of the present study was to analyze the adenosine effect on modulation of the HIF-1α/2α/VEGF pathway mediated through A

[Image: see text] The development of covalent ligands for G protein-coupled receptors (GPCRs) is not a trivial process. Here, we report a streamlined workflow thereto from synthesis to validation, exemplified by the discovery of a covalent antagonist for the human adenosine A(3) receptor (hA(3)AR). Based on the 1H,3H-pyrido[2,1-f]purine-2,4-dione scaffold, a series of ligands bearing a fluorosulfonyl warhead and a varying linker was synthesized. This series was subjected to an affinity screen, revealing compound 17b as the most potent antagonist. In addition, a nonreactive methylsulfonyl derivative 19 was developed as a reversible control compound. A series of assays, comprising time-dependent affinity determination, washout experiments, and [(35)S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hA(3)AR-homology model and site-directed mutagenesis study was performed to demonstrate that amino acid residue Y265(7.36) was the unique anchor point of the covalent interaction. This workflow might be applied to other GPCRs to guide the discovery of covalent ligands.

Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors’ ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3−6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.

Acupuncture has many advantages in the treatment of certain diseases as opposed to drug therapy. Besides, adenosine has been revealed to affect cellular progression including proliferation. Therefore, this study aimed at exploring the mechanism involving acupuncture stress and adenosine in fibroblast proliferation. The fibroblasts from fascia tissues of the acupoint area (Zusanli) were stimulated by different levels of stress, different concentrations of adenosine, and agonist or antagonist of A

Increasing evidence suggests that miRNAs are widely dysregulated in ulcerative colitis (UC), potentially affecting UC pathogenesis, diagnosis, and therapy. microRNA (miR) -206 has been reported to be upregulated in UC; however, its function and role in UC remain unknown. Here, we elucidate the function of miR-206 in the pathogenesis of UC. In patients with active-UC, miR-206 and adenosine A3 receptor (A3AR) levels were significantly upregulated and downregulated, respectively, and were inversely correlated. A3AR was expressed in the colon mucosa (particularly in colon epithelial-cell membranes). In HT-29 cells, miR-206 downregulated A3AR mRNA/protein expression by directly targeting the A3AR 3'-UTR; miR-206 overexpression and knockdown respectively increased and decreased TNF-α-induced nuclear NF-κB/p65, p-IκB-α, IKKα, p-IKKα and IL-8/IL-1β secretion. However, A3AR-siRNA reversed the miR-206 inhibitory effect. Furthermore, miR-206 increased dextran sodium sulphate-induced colitis severity (i.e., increased bodyweight loss, DAI score, colon shrinkage, and MPO activity), which was partially ameliorated by miR-206-antagomir treatment. miR-206-agomir treatment potently suppressed A3AR expression and increased NF-κB signalling and downstream cytokine (TNF-α/IL-8/IL-1β) expression in the mouse colon, in contrast to miR-206-antagomir administration. Taken together, our results demonstrated that miR-206 has a proinflammatory role in UC by downregulating A3AR expression and activating NF-κB signalling.

// Angelo Torres 1 , Yosselyn Vargas 1 , Daniel Uribe 1 , Catherine Jaramillo 1 , Alejandra Gleisner 2 , Flavio Salazar-Onfray 2 , Mercedes N. Lopez 2 , Romulo Melo 3 , Carlos Oyarzun 1 , Rody San Martin 1 , Claudia Quezada 1 1 Laboratorio de Patologia Molecular, Instituto de Bioquimica y Microbiologia, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 2 Instituto Milenio de Inmunologia e Inmunoterapia, Facultad de Medicina, Universidad de Chile, Santiago, Chile 3 Servicio de Neurocirugia, Instituto de Neurocirugia Dr. Asenjo, Santiago, Chile Correspondence to: Claudia Quezada, email: claudiaquezada@uach.cl Keywords: adenosine receptors, ATP-binding cassette (ABC) transporter superfamily, glioblastoma stem-like cells, multiple drug resistance Received: February 18, 2016 Accepted: August 29, 2016 Published: September 15, 2016 ABSTRACT MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A 3 receptor (A 3 AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A 3 AR to control MRP1 in GSCs. In vitro pharmacological blockade of A 3 AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A 3 AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A 3 AR blockade on GSCs.

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.

// Zhen-Hu Ren 1, 2, * , Cheng-Zhong Lin 1, 2, * , Wei Cao 1, 2 , Rong Yang 1, 2 , Wei Lu 1, 2 , Zhe-Qi Liu 1, 2 , Yi-Ming Chen 1, 2 , Xi Yang 1, 2 , Zhen Tian 3 , Li-Zhen Wang 3 , Jiang Li 3 , Xu Wang 1, 2 , Wan-Tao Chen 1, 2 , Tong Ji 1, 2 , Chen-Ping Zhang 1, 2 1 Department of Oral Maxillofacial-Head and Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China 2 Shanghai Research Institute of Stomatology and Shanghai Key Laboratory of Stomatology, Shanghai 200011, China 3 Department of Oral Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China * These authors contributed equally to this work Correspondence to: Chen-Ping Zhang, email: zhangcp9th@hotmail.com Tong Ji, email: jitong70@hotmail.com Keywords: CD73, adenosine receptor, EGFR, EMT, head and neck squamous cell carcinoma Received: April 18, 2016 Accepted: July 28, 2016 Published: August 20, 2016 ABSTRACT CD73 is a cell surface immunosuppressive enzyme involved in tumor progression and metastasis. While patients whose cancer cells express elevated CD73 are typically associated with an unfavorable outcome, the clinical impact of CD73 expression in patients with Head and neck squamous cell carcinoma (HNSCC) remains unclear. In the present study, we investigated the prognostic significance of CD73 in HNSCC using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with reduced overall survival in patients with HNSCC. We also investigated the functional role of CD73 in vitro and demonstrated that CD73 promotes HNSCC migration and invasion through adenosine A3R stimulation and the activation of EGF/EGFR signaling. Moreover, in vivo xenograft studies demonstrated that CD73 promotes tumorigenesis. In conclusion, our study highlights a role for CD73 as a poor prognostic marker of patient survival and also as a candidate therapeutic target in HNSCCs.

Cardiovascular complications are one of the major factors for early mortality in the present worldwide scenario and have become a major challenge in both developing and developed nations. It has thus become of immense importance to look for different therapeutic possibilities and treatments for the growing burden of cardiovascular diseases. Recent advancements in research have opened various means for better understanding of the complication and treatment of the disease. Adenosine receptors have become tool of choice in understanding the signaling mechanism which might lead to the cardiovascular complications. Adenosine A3 receptor is one of the important receptor which is extensively studied as a therapeutic target in cardiovascular disorder. Recent studies have shown that A3AR is involved in the amelioration of cardiovascular complications by altering the expression of A3AR. This review focuses towards the therapeutic potential of A3AR involved in cardiovascular disease and it might help in better understanding of mechanism by which this receptor may prove useful in improving the complications arising due to various cardiovascular diseases. Understanding of A3AR signaling may also help to develop newer agonists and antagonists which might be prove helpful in the treatment of cardiovascular disorder.

Background Lipid transfer protein (LTP), an abundant protein in fruits, vegetables, and nuts, is a common food allergen in Mediterranean areas causing diverse allergic reactions. Approximately 40% of food-related anaphylaxis induced by LTPs requires nonsteroidal anti-inflammatory drugs (NSAIDs) as a triggering cofactor. Objective We sought to better understand the determinants of NSAID-dependent and NSAID-independent LTP-induced anaphylaxis (LTP-A). Methods Selection of patients was based on a proved clinical history of NSAID-dependent or NSAID-independent anaphylaxis to LTPs, positive skin prick test response to LTPs, and serum LTP IgE. Whole-transcriptome (RNA sequencing) analysis of blood cells from 14 patients with NSAID-related LTP-A (NSAID-LTP-A), 7 patients with LTP-A, and 13 healthy control subjects was performed to identify distinct gene expression signatures. Results Expression of genes regulating gastrointestinal epithelial renewal was altered in both patient sets, particularly in those with LTP-A, who also presented with gene expression profiles characteristic of an inflammatory syndrome. These included altered B-cell pathways, increased neutrophil activation markers, and increased reactive oxygen species levels. Increased expression of the IgG receptor (CD64) in patients with LTP-A was mirrored by the presence of LTP-specific IgG 1 and IgG 3 . Conversely, patients with NSAID-LTP-A were characterized by reduced expression of IFN-γ–regulated genes and IFN-γ levels, as well as upregulated expression of adenosine receptor 3 (ADORA3) and genes related to adenosine metabolism. Conclusions Gene ontology analysis suggests disturbances in gut epithelial homeostasis in both groups with LTP-A, with potential integrity breaches in patients with LTP-A that might explain their distinct inflammatory signatures. Differential regulation in patients with LTP-A and those with NSAID-LTP-A of the IFN-γ pathway, IgG receptors, and ADORA3 might provide the pathogenic basis of their distinct responses.