Elena Cattabriga

Elevated Expression of A3 Adenosine Receptors in Human Colorectal Cancer Is Reflected in Peripheral Blood Cells

Purpose: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A3 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 A3 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 A3 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 A3 receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A3 receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A3 mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A3 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 A3 receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer. Conclusions: This study provides the first evidence that A3 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.

Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils

The present study describes the effect of low frequency, low energy, pulsing electromagnetic fields (PEMFs) on A2A adenosine receptors in human neutrophils. Saturation experiments performed using a high affinity adenosine antagonist [3H]‐ZM 241385 revealed a single class of binding sites in control and in PEMF‐treated human neutrophils with similar affinity (KD=1.05±0.10 and 1.08±0.12 nM, respectively). Furthermore, after 1 h of exposure to PEMFs the receptor density was statistically increased (P<0.01) (Bmax =126±10 and 215±15 fmol mg−1 protein, respectively). The effect of PEMFs was specific to the A2A adenosine receptors. This effect was also intensity, time and temperature dependent. In the adenylyl cyclase assays the A2A receptor agonists, HE‐NECA and NECA, increased cyclic AMP accumulation in untreated human neutrophils with an EC50 value of 43 (40 – 47) and 255 (228 – 284) nM, respectively. The capability of HE‐NECA and NECA to stimulate cyclic AMP levels in human neutrophils was increased (P<0.01) after exposure to PEMFs with an EC50 value of 10(8 – 13) and 61(52 – 71) nM, respectively. In the superoxide anion (O2−) production assays HE‐NECA and NECA inhibited the generation of O2− in untreated human neutrophils, with an EC50 value of 3.6(3.1 – 4.2) and of 23(20 – 27) nM, respectively. Moreover, in PEMF‐treated human neutrophils, the same compounds show an EC50 value of 1.6(1.2 – 2.1) and of 6.0(4.7 – 7.5) nM respectively. These results indicate the presence of significant alterations in the expression and in the functionality of adenosine A2A receptors in human neutrophils treated with PEMFs.

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line

The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. Adenosine receptors were detected by RT – PCR experiments. A1 receptors were characterized using [3H]‐DPCPX binding with a KD of 1.9±0.2 nM and Bmax of 23±7 fmol mg−1 of protein. A2A receptors were studied with [3H]‐SCH 58261 binding and revealed a KD of 5.1±0.2 nM and a Bmax of 220±7 fmol mg−1 of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]‐MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3±0.7 nM and Bmax of 291±50 fmol mg−1 of protein. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy‐ and enthalpy‐driven. In functional assays the high affinity A2A agonists HE‐NECA, CGS 21680 and A2A – A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl‐IB‐MECA, IB‐MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

Adenosine receptors in colon carcinoma tissues and colon tumoral cell lines: Focus on the A3 adenosine subtype

Adenosine may affect several pathophysiological processes, including cellular proliferation, through interaction with A1, A2A, A2B, and A3 receptors. In this study we characterized adenosine receptors in human colon cancer tissues and in colon cancer cell lines Caco2, DLD1, HT29. mRNA of all adenosine subtypes was detected in cancer tissues and cell lines. At a protein levels low amount of A1, A2A, and A2B receptors were detected, whilst the A3 was the most abundant subtype in both cancer tissues and cells, with a pharmacological profile typical of the A3 subtype. All the receptors were coupled to stimulation/inhibition of adenylyl‐cyclase in cancer cells, with the exception of A1 subtype. Adenosine increased cell proliferation with an EC50 of 3–12 µM in cancer cells. This effect was not essentially reduced by adenosine receptor antagonists. However dypiridamol, an adenosine transport inhibitor, increased the stimulatory effect induced by adenosine, suggesting an action at the cell surface. Addition of adenosine deaminase makes the A3 agonist 2‐chloro‐N6‐(3‐iodobenzyl)‐N‐methyl‐5′‐carbamoyladenosine (Cl‐IB‐MECA) able to stimulate cell proliferation with an EC50 of 0.5–0.9 nM in cancer cells, suggesting a tonic proliferative effect induced by endogenous adenosine. This effect was antagonized by 5‐N‐(4‐methoxyphenyl‐carbamoyl)amino‐8‐propyl‐2(2furyl)‐pyrazolo‐[4,3e]‐1,2,4‐triazolo [1,5‐c] pyrimidine (MRE 3008F20) 10 nM. Cl‐IB‐MECA‐stimulated cell proliferation involved extracellular‐signal‐regulated‐kinases (ERK1/2) pathway, as demonstrated by reduction of proliferation with 1,4‐diamino‐2,3‐dicyano‐1,4‐bis‐[2‐amino‐phenylthio]‐butadiene (U0126) and by ERK1/2 phosphorylation. In conclusion this study indicates for the first time that in colon cancer cell lines endogenous adenosine, through the interaction with A3 receptors, mediates a tonic proliferative effect. J. Cell. Physiol. 211: 826–836, 2007.

Aberrant A2A receptor function in peripheral blood cells in Huntington's disease

A2A adenosine receptors specifically found on striatal medium spiny neurons play a major role in sensory motor function and may also be involved in neuropsychiatric and neurodegenerative disorders. One hypothesis concerning Huntingtons disease (HD) proposes that an imbalance of the cortico‐striatal pathway, due to the mutation in the HD gene, leads to striatal vulnerability. An A2A receptor dysfunction has been previously demonstrated in striatal cells engineered to express mutant huntingtin. Here we tested whether a similar dysfunction (i.e., the binding and functional parameters of A2A adenosine receptors) is present in peripheral blood cells (platelets, lymphocytes, and neutrophils) of subjects carrying the mutant gene. This study involved 48 heterozygous and three homozygous patients compared with 58 healthy subjects. Moreover, we selected seven at‐risk mutation carriers. A2A receptor density and function are substantially increased in peripheral blood cells from both patients and subjects at the presymptomatic stage. In the neutrophils of the three homozygous HD subjects receptor dysfunction was higher than in heterozygotes. These data indicate the existence of an aberrant A2A receptor phenotype in the peripheral blood cells of subjects carrying the HD mutation. Future studies will assess whether this parameter can be exploited as a peripheral biomarker of Huntingtons disease.

Synthesis by microwave irradiation and binding properties of novel 5-HT1A receptor ligands

This work reports the synthesis by microwave irradiation and the binding tests on the 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors of new substituted piperazines in order to identify selective ligands for 5-HT(1A) subtype receptor. Conventional heating and microwave irradiation of the reactions was compared. Synthesis by microwave irradiation gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. Some resulting active compounds (29 and 39) were characterised by a good selectivity profile for the 5-HT(1A) subtype receptor. The more active compounds were selected and further evaluated for their binding affinities on D(1), D(2) dopaminergic and alpha(1), alpha(2) adrenergic receptors. The compound with higher affinity and selectivity for the 5-HT(1A) over all the considered receptors was the 3-[4-[4-(1,2,3,4-tetrahydronaphthyl)-1-piperazinyl]butan]-benzotriazinone (-)29 (5-HT(1A) K(i)=36 nM, other receptors not active).

A convenient synthesis by microwave heating and pharmacological evaluation of novel benzoyltriazole and saccharine derivatives as 5-HT1A receptor ligands

A series of novel 1,2,3-4-benzoyltriazole and saccharine derivatives were designed and synthesized by microwave heating. They were evaluated on a battery of receptors, including serotonin 5-HT(1A,) 5-HT(2A) and 5-HT(2C), and the most interesting compounds were further evaluated on dopaminergic D(1), D(2) and adrenergic alpha(1), alpha(2) receptors. Conventional and microwave heating of the reactions were compared. Synthesis by microwave heating gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. All compounds displayed moderate affinity for 5-HT(1A) receptor. The most interesting compound 33 showed a high affinity (K(i)=93 nM) which was combined with no affinity on the other receptors considered.

Effects of Doxazosin and Propranolol on A2A Adenosine Receptors in Essential Hypertension

Abstract—A2A adenosine receptors inhibit neutrophil adhesion and superoxide anion generation. The aim of the present study was to evaluate the effect of antihypertensive treatment with doxazosin or propranolol on the binding and functional parameters of A2A adenosine receptors of lymphocytes and neutrophils in essential hypertensive patients. Two groups of previously untreated, essential hypertensive patients were studied. The mean affinity (Kd) and density (Bmax) of adenosine receptors, by the A2A selective radioligand [3H]-ZM-241385 binding assays, and EC50, by cAMP assays, were obtained first on no medication and a second time after treatment for up to 13 weeks with doxazosin (13 patients) or propranolol (8 patients). A third group of 15 healthy normotensive volunteers matched by age, sex, and body mass index was used as a control. Binding and functional parameters of the A2A adenosine receptors were significantly higher in the 2 hypertensive groups than in controls (P always <0.0001), both in lymphocyte and neutrophil membranes. After treatment with propranolol, the binding parameters did not change significantly, whereas after treatment with doxazosin, Kd, Bmax, and EC50 values returned to control levels. In never-treated essential hypertensive patients, lower affinity, higher density, and impaired function of A2A adenosine receptors are present. The binding and functional parameters of A2A adenosine receptors appear to be normalized after treatment with doxazosin but not with propranolol.