Inosine monophosphate and inosine differentially regulate endotoxemia and bacterial sepsis.

Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. IMP can also serve as an extracellular signaling molecule, and can regulate diverse processes such as taste sensation, neutrophil function, and ischemia-reperfusion injury. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. In this study, we demonstrate that IMP suppressed tumor necrosis factor (TNF)-α production and augmented IL-10 production in endotoxemic mice. IMP exerted its effects through metabolism to inosine, as IMP only suppressed TNF-α following its CD73-mediated degradation to inosine in lipopolysaccharide-activated macrophages. Studies with gene targeted mice and pharmacological antagonism indicated that A2A , A2B, and A3 adenosine receptors are not required for the inosine suppression of TNF-α production. The inosine suppression of TNF-α production did not require its metabolism to hypoxanthine through purine nucleoside phosphorylase or its uptake into cells through concentrative nucleoside transporters indicating a role for alternative metabolic/uptake pathways. Inosine augmented IL-ß production by macrophages in which inflammasome was activated by lipopolysaccharide and ATP. In contrast to its effects in endotoxemia, IMP failed to affect the inflammatory response to abdominal sepsis and pneumonia. We conclude that extracellular IMP and inosine differentially regulate the inflammatory response.

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists.

The A3 adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A3AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A3AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A3AR antagonist for all three species tested, albeit a little less selective for mouse and rat A3AR in comparison to the human A3AR. DPTN's Ki values at respective A1, A2A, A2B, and A3 receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A3ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A3AR antagonists in rats and mice, we also re-examined other commonly used and selective A3AR antagonists under the same experimental conditions. The Ki values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A3ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [125I]I-AB-MECA binding to mouse and rat A3ARs, while potent human A3AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A3AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A3AR-related mechanism or function.

Indirect Medium Spiny Neurons in the Dorsomedial Striatum Regulate Ethanol-Containing Conditioned Reward Seeking.

Adenosine 2A receptor (A2AR)-containing indirect medium spiny neurons (iMSNs) in the dorsomedial striatum (DMS) contribute to reward-seeking behaviors. However, those roles for ethanol-seeking behaviors remain unknown. To investigate ethanol-seeking behaviors, we used an ethanol-containing reward (10% ethanol and 10% sucrose solution; 10E10S). Upon conditioning with 10E10S, mice that initially only preferred 10% sucrose, not 10E10S, showed a stronger preference for 10E10S. Then, we investigated whether the manipulation of the DMS-external globus pallidus (GPe) iMSNs circuit alters the ethanol-containing reward (10E10S) seeking behaviors using the combination of pharmacologic and optogenetic approaches. DMS A2AR activation dampened operant conditioning-induced ethanol-containing reward, whereas A2AR antagonist abolished the effects of the A2AR agonist and restored ethanol-containing reward-seeking. Moreover, pre-ethanol exposure potentiated the A2AR-dependent reward-seeking. Interestingly, mice exhibiting ethanol-containing reward-seeking showed the reduction of the DMS iMSNs activity, suggesting that disinhibiting iMSNs decreases reward-seeking behaviors. In addition, we found that A2AR activation reversed iMSNs neural activity in the DMS. Similarly, optogenetic stimulation of the DMS-GPe iMSNs reduced ethanol-containing reward-seeking, whereas optogenetic inhibition of the DMS-GPe iMSNs reversed this change. Together, our study demonstrates that DMS A2AR and iMSNs regulate ethanol-containing reward-seeking behaviors.SIGNIFICANCE STATEMENT Our findings highlight the mechanisms of how operant conditioning develops the preference of ethanol-containing conditioned reward. Mice exhibiting ethanol-containing reward-seeking showed a reduction of the indirect medium spiny neuronal activity in the dorsomedial striatum. Pharmacological activation of adenosine A2A receptor (A2AR) or optogenetic activation of indirect medium spiny neurons dampened operant conditioned ethanol-containing reward-seeking, whereas inhibiting this neuronal activity restored ethanol-containing reward-seeking. Furthermore, repeated intermittent ethanol exposure potentiated A2AR-dependent reward-seeking. Therefore, our finding suggests that A2AR-containing indirect medium spiny neuronal activation reduces ethanol-containing reward-seeking, which may provide a potential therapeutic target for alcohol use disorder.

Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis.

Adenosine A3 receptor (A3R) is a promising drug target cancer and for a number of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no experimentally determined structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calculations of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helices (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calculations distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32 in the low region and the remote L2647.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.

Blockade of the Adenosine A3 Receptor Attenuates Caspase 1 Activation in Renal Tubule Epithelial Cells and Decreases Interleukins IL-1ß and IL-18 in Diabetic Rats.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1ß and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1ß, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1ß and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

Adenosine and adenosine receptors in the immunopathogenesis and treatment of cancer.

Tumor cells overcome anti-tumor responses in part through immunosuppressive mechanisms. There are several immune modulatory mechanisms. Among them, adenosine is an important factor which is generated by both cancer and immune cells in tumor microenvironment to suppress anti-tumor responses. Two cell surface expressed molecules including CD73 and CD39 catalyze the generation of adenosine from adenosine triphosphate (ATP). The generation of adenosine can be enhanced under metabolic stress like tumor hypoxic conditions. Adenosine exerts its immune regulatory functions through four different adenosine receptors (ARs) including A1, A2A, A2B, and A3 which are expressed on various immune cells. Several studies have indicated the overexpression of adenosine generating enzymes and ARs in various cancers which was correlated with tumor progression. Since the signaling of ARs enhances tumor progression, their manipulation can be promising therapeutic approach in cancer therapy. Accordingly, several agonists and antagonists against ARs have been designed for cancer therapy. In this review, we will try to clarify the role of different ARs in the immunopathogenesis, as well as their role in the treatment of cancer.

The Adenosine A3 Receptor Regulates Differentiation of Glioblastoma Stem-Like Cells to Endothelial Cells under Hypoxia.

Glioblastoma (GBM) is a neoplasm characterized by an extensive blood vessel network. Hypoxic niches of GBM can induce tumorigenic properties of a small cell subpopulation called Glioblastoma stem-like cells (GSCs) and can also increase extracellular adenosine generation which activates the A3 adenosine receptor (A3AR). Moreover, GSCs potentiates the persistent neovascularization in GBM. The aim of this study was to determine if A3AR blockade can reduce the vasculogenesis mediated by the differentiation of GSCs to Endothelial Cells (ECs) under hypoxia. We evaluated the expression of endothelial cell markers (CD31, CD34, CD144, and vWF) by fluorescence-activated cell sorting (FACS), and vascular endothelial growth factor (VEGF) secretion by ELISA using MRS1220 (A3AR antagonist) under hypoxia. We validate our results using U87MG-GSCs A3AR knockout (GSCsA3-KO). The effect of MRS1220 on blood vessel formation was evaluated in vivo using a subcutaneous GSCs-tumor model. GSCs increased extracellular adenosine production and A3AR expression under hypoxia. Hypoxia also increased the percentage of GSCs positive for endothelial cell markers and VEGF secretion, which was in turn prevented when using MRS1220 and in GSCsA3-KO. Finally, in vivo treatment with MRS1220 reduced tumor size and blood vessel formation. Blockade of A3AR decreases the differentiation of GSCs to ECs under hypoxia and in vivo blood vessel formation.

Retinal A2A and A3 adenosine receptors modulate the components of the rat electroretinogram.

Adenosine is a neuromodulator present in various areas of the central nervous system, including the retina. Adenosine may serve a neuroprotective role in the retina, based on electroretinogram (ERG) recordings from the rat retina. Our purpose was to assess the role of A2A and A3 adenosine receptors in the generation and modulation of the rat ERG. The flash ERG was recorded with corneal electrodes from Sprague Dawley rats. Agonists and antagonists for A2A and A3 receptors, and adenosine were injected (5 µl) into the vitreous. The effects on the components of the single flash scotopic and photopic ERGs were examined, and ERG flicker. Adenosine (0.5 mM) increased the mean amplitudes of the scotopic ERG a-waves (68 ± 8 to 97 ± 14 µV, P = 0.042), and b-waves (236 ± 38 µV to 305 ± 42 µV). A2A agonist CGS21680 (2 mM) reduced the mean amplitude of the ERG b-wave, from 298 ± 21 µV in response to the brightest stimulus to 212 ± 19 µV (P = 0.005), and mean scotopic oscillatory potentials (OPs) from 100 ± 9 µV to 47 ± 11 µV (P = 0.023). ZM241385 [4 mM], an A2A antagonist, decreased the scotopic b-wave of the ERG. A3 agonist 2-CI-IB-MECA (0.5 mM) increased the a-wave, while decreasing the scotopic and photopic ERG b-waves, and the scotopic OPs. A3 antagonist VUF5574 (1 mM) increased the mean amplitude of the scotopic a-wave (66 ± 8 to 140 ± 29 µV, P = 0.046) and b-wave (224 ± 20 to 312 ± 39 µV, P = 0.0037). No significant effects on ERG flicker were found. We conclude that retinal neurons containing A2A and/or A3 adenosine receptors contribute to the generation of the ERG a- and b-waves and OPs.

Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding.

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

Topological sub-structural molecular design (TOPS-MODE): a useful tool to explore key fragments of human A3 adenosine receptor ligands.

Adenosine regulates tissue function by activating four G-protein-coupled adenosine receptors (ARs). Selective agonists and antagonists for A3 ARs have been investigated for the treatment of a variety of immune disorders, cancer, brain, and heart ischemic conditions. We herein present a QSAR study based on a Topological sub-structural molecular design (TOPS-MODE) approach, intended to predict the A3 ARs of a diverse dataset of 124 (94 training set/ 30 prediction set) adenosine derivatives. The final model showed good fit and predictive capability, displaying 85.1 % of the experimental variance. The TOPS-MODE approach afforded a better understanding and interpretation of the developed model based on the useful information extracted from the analysis of the contribution of different molecular fragments to the affinity.

Renoprotective Effects of a Highly Selective A3 Adenosine Receptor Antagonist in a Mouse Model of Adriamycin-induced Nephropathy.

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-ß1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1ß, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1ß1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2ß3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1ß1, α2ß1, and α3ß1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH2 substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.

Hide and seek: a comparative autoradiographic in vitro investigation of the adenosine A3 receptor.

PURPOSE: Since the adenosine A3 receptor (A3R) is considered to be of high clinical importance in the diagnosis and treatment of ischaemic conditions (heart and brain), glaucoma, asthma, arthritis, cancer and inflammation, a suitable and selective A3R PET tracer such as [(18)F]FE@SUPPY would be of high clinical value for clinicians as well as patients. A3R was discovered in the late 1990s, but there is still little known regarding its distribution in the CNS and periphery. Hence, in autoradiographic experiments the distribution of A3R in human brain and rat tissues was investigated and the specific binding of the A3R antagonist FE@SUPPY and MRS1523 compared. Immunohistochemical staining (IHC) experiments were also performed to validate the autoradiographic findings. METHODS: For autoradiographic competition experiments human post-mortem brain and rat tissues were incubated with [(125)I]AB-MECA and highly selective compounds to block the other adenosine receptor subtypes. Additionally, IHC was performed with an A3 antibody. RESULTS: Specific A3R binding of MRS1523 and FE@SUPPY was found in all rat peripheral tissues examined with the highest amounts in the spleen (44.0% and 46.4%), lung (44.5% and 45.0%), heart (39.9% and 42.9%) and testes (27.4% and 29.5%, respectively). Low amounts of A3R were found in rat brain tissues (5.9% and 5.6%, respectively) and human brain tissues (thalamus 8.0% and 9.1%, putamen 7.8% and 8.2%, cerebellum 6.0% and 7.8%, hippocampus 5.7% and 5.6%, caudate nucleus 4.9% and 6.4%, cortex 4.9% and 6.3%, respectively). The outcome of the A3 antibody staining experiments complemented the results of the autoradiographic experiments. CONCLUSION: The presence of A3R protein was verified in central and peripheral tissues by autoradiography and IHC. The specificity and selectivity of FE@SUPPY was confirmed by direct comparison with MRS1523, providing further evidence that [(18)F]FE@SUPPY may be a suitable A3 PET tracer for use in humans.

Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 µM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism.

In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 µm(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 µm(2)/s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors.

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X7 receptors. GSC exposure for 48 h to 10-150 µM ATP, P2R ligand, or to ADPßS or MRS2365, P2Y1R agonists, enhanced cell expansion. This effect was counteracted by the PY1R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y2/4R, or 2'(3')-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X7R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A3R or P2X7R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A3R, P2Y1R, and P2X7R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.

Anticancer and antimetastatic effects of cordycepin, an active component of Cordyceps sinensis.

Cordyceps sinensis, a fungus that parasitizes on the larva of Lepidoptera, has been used as a valued traditional Chinese medicine. We investigated the effects of water extracts of Cordyceps sinensis (WECS), and particularly focused on its anticancer and antimetastatic actions. Based on in vitro studies, we report that WECS showed an anticancer action, and this action was antagonized by an adenosine A3 receptor antagonist. Moreover, this anticancer action of WECS was promoted by an adenosine deaminase inhibitor. These results suggest that one of the components of WECS with an anticancer action might be an adenosine or its derivatives. Therefore, we focused on cordycepin (3'-deoxyadenosine) as one of the active ingredients of WECS. According to our experiments, cordycepin showed an anticancer effect through the stimulation of adenosine A3 receptor, followed by glycogen synthase kinase (GSK)-3ß activation and cyclin D1 suppression. Cordycepin also showed an antimetastatic action through inhibiting platelet aggregation induced by cancer cells and suppressing the invasiveness of cancer cells via inhibiting the activity of matrix metalloproteinase (MMP)-2 and MMP-9, and accelerating the secretion of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 from cancer cells. In conclusion, cordycepin, an active component of WECS, might be a candidate anticancer and antimetastatic agent.

The selective A3AR antagonist LJ-1888 ameliorates UUO-induced tubulointerstitial fibrosis.

Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A3 adenosine receptor (A3AR) is up-regulated under stress, but the therapeutic effects of A3AR antagonists on chronic kidney disease are not fully understood. The present study examined the effect of LJ-1888 [(2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A3AR antagonist, on unilateral ureteral obstruction (UUO)-induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equivalent therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A3AR transfection effectively inhibited transforming growth factor-ß1 (TGF-ß1)-induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A3AR blockade. UUO- or TGF-ß1-induced c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A3AR blockade reduced UUO- or TGF-ß1-induced up-regulation of lysyl oxidase, which induces cross-linking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A3AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.

Mitochondrial and caspase pathways are involved in the induction of apoptosis by IB-MECA in ovarian cancer cell lines.

A3 adenosine receptor agonist (IB-MECA) has been shown to play important roles in cell proliferation and apoptosis in a variety of cancer cell lines. The present study was designed to understand the mechanism underlying IB-MECA-induced apoptosis in human ovarian cancer cell lines. The messenger RNA (mRNA) and protein expression levels of A3 adenosine receptor were detected in OVCAR-3 and Caov-4 ovarian cancer cells. IB-MECA was capable of decreasing intracellular cyclic adenosine monophosphate (cAMP) that was the reason for the presence of functional A3 adenosine receptor on the cell lines. IB-MECA significantly reduced cell viability in a dose-dependent manner. Cytotoxicity of IB-MECA was suppressed by MRS1220, an A3 adenosine receptor antagonist. The growth inhibition effect of IB-MECA was related to the induction of cell apoptosis, which was manifested by annexin V-FITC staining, activation of caspase-3 and caspase-9, and loss of mitochondrial membrane potentials (ΔΨm). In addition, downregulation of the regulatory protein Bcl-2 and upregulation of Bax protein by IB-MECA were also observed. These findings demonstrated that IB-MECA induces apoptosis via the mitochondrial signaling pathway. These suggest that A3 adenosine receptor agonists may be a potential agent for induction of apoptosis in human ovarian cancer cells.