A3 Adenosine Receptor Activation in Melanoma Cells

Abstract

The incidence of melanoma in humans has increased steadily over the past years and is one of the more difficult neoplasias to clinically manage. Due to the limited response of malignant melanoma to conventional chemotherapy and the poor prognosis of patients with metastatic melanoma, new therapies for this disease are needed.Our earlier studies demonstrated that IB-MECA, 1 a stable agonist to A3AR, inhibits the proliferation of neoplastic cells including metastatic melanoma (1-3). A3AR belongs to the family of the G i protein-associated cell surface receptors. Receptor activation leads to inhibition of adenylyl cyclase activity, cAMP formation, and PKA expression. PKA contains a catalytic subunit, PKAc, which dissociates from the parent molecule upon activation with cAMP, resulting in the initiation of various signaling pathways (4, 5). Recent studies have demonstrated that PKAc phosphorylates and inactivates GSK-3␤ (6). We showed that IB-MECA alters the expression of GSK-3␤ and ␤-catenin, key components of the Wnt signaling pathway. Consequently it led to inhibition in the expression of the cell cycle progression genes, c-Myc and cyclin D1 (2). This is an important observation as the Wnt pathway has been linked to the development of malignant melanoma (7-9). It is well established that G i protein receptors are internalized to early endosomes upon agonist binding. Early endosomes serve as the major site of receptor recycling, whereas the late endosomes are involved in the delivery of the internalized receptor to the lysosomes (10). One point to consider while targeting chronically a G i protein receptor is that desensitization may lead to loss of a functional receptor from the cell surface. Interestingly, although A3AR expression level was found to be low in most body tissues, it is highly expressed in tumor cell lines (11)(12)(13). Given that IB-MECA inhibits the growth of B16-F10 melanoma cells, it was hypothesized that these cells exhibit high receptor levels, which may serve as a target for tumor growth inhibition. We thus sought to explore the fate of A3AR upon IB-MECA activation and the consequences on the downstream molecular mechanisms leading to tumor growth inhibition both in vitro and in vivo.Here we show that melanoma cells highly express A3AR, which upon IB-MECA stimulation rapidly internalizes to the cytosol and sorts to endosomes and lysosomes. Resynthesis and externalization of the receptor to the cell surface then occurs. Receptor functionality was demonstrated by the initiation of signal transduction pathways, which resulted in down-regulation of c-Myc and cyclin D1, leading to tumor growth suppression. EXPERIMENTAL PROCEDURESReagents-IB-MECA and MRS 1523 were purchased from RBI/ Sigma. For both reagents, a stock solution of 10 mM was prepared in Me 2 SO, and further dilutions in RPMI medium were performed. RPMI, fetal bovine serum, and antibiotics for cell cultures were obtained from Beit Haemek, Haifa, Israel.

Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

“…Assessment by analytical HPLC of the stability of the monomeric arms containing AR ligands (14,18) in the presence of either CHO or human embryonic kidney 293 cell membranes Assay solutions containing a monomeric arm (diluted from a 5-mM DMSO stock solution of either 14 or 18, 10 μM final concentration) and either Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cell membrane suspensions (100 μg/mL) were prepared in 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl 2 buffer. The solutions in glass culture tubes (20×75 mm) were incubated at 25°C for 1 h in a shaking water bath.…”

“…There are four known subtypes of adenosine receptors (ARs), which belong to the superfamily of GPCRs, referred to as A 1 , A 2A , A 2B , and A 3 -each of which has a unique pharmacological profile, tissue distribution, and combination of effects [12,13]. An agonist of the A 3 AR is currently in clinical trials for hepatocellular carcinoma [13,14]. Fishman et al reported that growth of melanoma cells can be inhibited by activation of A 3 AR via deregulation of the NFκB and Wnt pathways [14].…”

“…An agonist of the A 3 AR is currently in clinical trials for hepatocellular carcinoma [13,14]. Fishman et al reported that growth of melanoma cells can be inhibited by activation of A 3 AR via deregulation of the NFκB and Wnt pathways [14]. They demonstrated that the small molecule nucleoside agonists IB-MECA 1 and Cl-IB-MECA 2 ( Fig.…”

“…They demonstrated that the small molecule nucleoside agonists IB-MECA 1 and Cl-IB-MECA 2 ( Fig. 1), both invented in our laboratory, inhibit the growth of B16-F10 melanoma cells via activation of A 3 AR both in vitro and in vivo, and among these, 2 has already entered phase II clinical trials for hepatocellular carcinoma [14,43]. Additionally, A 2A AR agonists have anti-inflammatory properties and are used in cardiac imaging [13,25].…”

“…Before testing the AuNP conjugates, we ascertained that low MW components of significant biological activity (i.e., that passed through ultrafiltration with 10-30 kDa cutoff) were not present. Also, the stability of the monomeric ligands with attached tether (14,18) in the presence of HEK293 cell membranes was demonstrated (Supporting Information).…”