Purpose: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A 3 adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work, we addressed the question of the putative relevance of A 3 subtypes in colorectal adenocarcinomas.Experimental Design: Seventy-three paired samples of tumor and surrounding peritumoral normal mucosa at a distance of 2 and 10 cm from the tumor and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A 3 receptors by means of binding, immunocytochemistry, and real-time reverse transcription-polymerase chain reaction studies.Results: As measured by receptor binding assays, the density of A 3 receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A 3 receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A 3 mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A 3 receptors in tumors was reflected in peripheral blood cells, where the density was approximately 3-fold higher compared with healthy subjects (P < 0.01). In a cohort of 10 patients studied longitudinally, expression of A 3 receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer.Conclusions: This study provides the first evidence that A 3 receptor plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

The antagonistic interaction between adenosine and dopamine receptors could have important pathophysiological and therapeutic implications in Parkinson's disease (PD). The primary aim of this study was to investigate the expression, affinity, and density of A(1), A(2A), A(2B), and A(3) adenosine receptors (ARs) and D(2) dopamine receptors (D(2)Rs) in PD. An increase in A(2A)AR density in putamen was found. The presence and functionality of ARs in human lymphocyte and neutrophil membranes from patients with PD revealed a specific A(2A)AR alteration compared with healthy subjects. A statistically significant linear correlation among the A(2A)AR density, functionality, or tumor necrosis factor-alpha (TNF-alpha) levels and Unified Parkinson's Disease Rating Scale (UPDRS) motor score was reported. Adenosine concentration and TNF-alpha levels were increased in plasma of patients with PD. In rat adrenal pheochromocytoma (PC12) cells, a widely useful model, adenosine antagonists decreased dopamine uptake, and an opposite effect was mediated by A(2A) agonists. This is the first report showing the presence of an A(2A)AR alteration in putamen in PD that mirrors a similar up-regulation in human peripheral blood cells. Moreover, the correlation found between A(2A)AR density or A(2A) agonist potency and UPDRS motor score highlights the central role of A(2A)ARs in the pharmacological treatment of PD.

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB 1 receptors in some isolated intact tissues. We found that the basal incorporation of [35 S]-GTPgS in Chinese hamster ovary cells expressing human recombinant CB 1 and CB 2 receptors was inhibited by SR141716A (mean pEC 50 s 8.26 and 6.00, respectively), whereas cannabinol (10 mM) had no signi®cant eect at hCB 1 receptors but inhibited the binding at hCB 2 receptors. As cannabinol had no eect on basal [35 S]-GTPgS binding at hCB 1 at a concentration 100 fold higher than its binding anity (K i =0.1 mM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB 1 receptor, and the actions of SR141716A and cannabinol at the hCB 2 receptor, are due to inverse agonism.

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Frequent coffee consumption has been associated with a reduced risk of colorectal cancer in a number of case-control studies. Coffee is a leading source of methylxanthines, such as caffeine. The induction of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) is an essential feature of tumor angiogenesis, and the hypoxia-inducible factor-1 (HIF-1) transcription factor is known to be a key regulator of this process. In this study, we investigated the effects of caffeine on HIF-1 protein accumulation and on VEGF and IL-8 expression in the human colon cancer cell line HT29 under hypoxic conditions. Our results show that caffeine significantly inhibits adenosineinduced HIF-1␣ protein accumulation in cancer cells. We show that HIF-1␣ and VEGF are increased through A 3 adenosine receptor stimulation, whereas the effects on IL-8 are mediated via the A 2B subtype. Pretreatment of cells with caffeine significantly reduces adenosine-induced VEGF promoter activity and VEGF and IL-8 expression. The mechanism of caffeine seems to involve the inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and Akt, leading to a marked decrease in adenosine-induced HIF-1␣ accumulation, VEGF transcriptional activation, and VEGF and IL-8 protein accumulation. From a functional perspective, we observe that caffeine also significantly inhibits the A 3 receptor-stimulated cell migration of colon cancer cells. Conditioned media prepared from colon cells treated with an adenosine analog increased human umbilical vein endothelial cell migration. These data provide evidence that adenosine could modulate the migration of colon cancer cells by an HIF-1␣/VEGF/IL-8-dependent mechanism and that caffeine has the potential to inhibit colon cancer cell growth.Coffee and tea are the most commonly consumed beverages in the world (Fredholm, 1999). Results of epidemiological studies have not resolved whether coffee consumption is related to colorectal cancer risk. A report by the World Cancer Research Fund concluded that the available evidence was not sufficient to draw any firm conclusions about a decreased risk of colorectal cancer associated with coffee consumption (World Cancer Research Fund/American Institute for Cancer Research, 1997). However, some researchers contend that a link between high consumption of coffee and a low incidence Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.032920.ABBREVIATIONS: IL-8, interleukin-8; Cl-IB-MECA, N 6 (3-iodobenzyl)2-chloroadenosine-5ЈN-methyluronamide; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; MRE 2029F20, N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy

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This is the first report showing the presence of adenosine receptors in lung parenchyma in subjects with COPD compared with control smokers. These novel findings strengthen the hypothesis of a potential role played by adenosine receptors in the pathogenesis of COPD.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% O2), adenosine upregulates HIF-1alpha protein expression in a dose-dependent and time-dependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2) protein accumulation through the induction of HIF-1alpha. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1alpha and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.