The Adenosine A3 Receptor Agonist Cl-IB-MECA Induces Cell Death Through Ca2+/ROS-Dependent Down Regulation of ERK and Akt in A172 Human Glioma Cells

Abstract

Adenosine A(3) receptor (A3AR) is coupled to G proteins that are involved in a variety of intracellular signaling pathways and physiological functions. 2-Chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (Cl-IB-MECA), an agonist of A3AR, has been reported to induce cell death in various cancer cells. However, the effect of CI-IB-MECA on glioma cell growth is not clear. This study was undertaken to examine the effect of CI-IB-MECA on glioma cell viability and to determine its molecular mechanism. CI-IB-MECA inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Treatment of CI-IB-MECA resulted in an increase in intracellular Ca(2+) followed by enhanced reactive oxygen species (ROS) generation. EGTA and N-acetylcysteine (NAC) blocked the cell death induced by CI-IB-MECA, suggesting that Ca(2+) and ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis showed that CI-IB-MECA induced the down-regulation of extracellular signal-regulated kinases (ERK) and Akt, which was prevented by EGTA, NAC, and the A3AR antagonist MRS1191. Transfection of constitutively active forms of MEK, the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA induced caspase-3 activation and the CI-IB-MECA-induced cell death was blocked by the caspase inhibitors DEVD-CHO and z-VAD-FMK. In addition, expression of XIAP and Survivin were decreased in cells treated with Cl-IB-MECA. Collectively, these findings demonstrate that CI-IB-MECA induce a caspase-dependent cell death through suppression of ERK and Akt mediated by an increase in intracellular Ca(2+) and ROS generation in human glioma cells. These suggest that A3AR agonists may be a potential therapeutic agent for induction of apoptosis in human glioma cells.

Methotrexate up-regulates ecto-5′-nucleotidase/CD73 and reduces the frequency of T lymphocytes in the glioblastoma microenvironment

“…GBM is also characterized by an impaired immune response that results in tumor progression [30]. The purinergic system, which is known to be involved in cancer development, has a direct influence on cancer cells and mediates the modulation of immune cells through P1 adenosine receptors [9,31]. Although the supra-physiological concentration of adenosine in the tumor microenvironment has been studied extensively, there is no final consensus regarding the pro-or anti-tumoral role of the adenosinergic system.…”

“…Although the supra-physiological concentration of adenosine in the tumor microenvironment has been studied extensively, there is no final consensus regarding the pro-or anti-tumoral role of the adenosinergic system. Most studies have shown the immunosuppressive effects of adenosine (pro-tumoral) [7], whereas other studies have shown that adenosine may lead to tumor cell death (antitumoral) [13,31]. In fact, targeting the CD73 enzyme, the main extracellular factor that produces adenosine, has been discussed as an alternative treatment for solid tumors.…”

The A3 Adenosine Receptor: History and Perspectives

“…In addition, in prostate cancer cells, A 3 AR inhibits ERK1/2 activity through the reduction of AC and protein kinase A (Jajoo et al, 2009). In glioma, Cl-IB-MECA mediates suppression of ERK1/2, thus inducing caspase-dependent cell death (Kim et al, 2012). Inhibition of ERK1/2 is also associated with the A 3 AR inhibition of LPS-stimulated TNF-a release in mouse RAW 264.7 cells (Martin et al, 2006).…”

“…In transfected CHO cells, the ability of both recombinant hA 3 ARs to inhibit cAMP accumulation and endogenous A 3 ARs in RBL-2H3 to stimulate PLC is abolished by pretreatment with pertussis toxin, suggesting a functional coupling of this G i protein receptor (Ali et al, 1990;Zhou et al, 1992;Varani et al, 2000). Furthermore, A 3 ARs signaling could increase phosphatidylinositol-specific PLC activity (Ali et al, 1990;Ramkumar et al, 1993;Abbracchio et al, 1995;Zheng et al, 2007) and cause the release of Ca 2+ from intracellular stores in different cellular models Merighi et al, 2001;Englert et al, 2002;Fossetta et al, 2003;Shneyvays et al, 2004Shneyvays et al, , 2005Kim et al, 2012). In a broad study of site-directed mutagenesis of the A 3 AR, the mutation of the highly conserved tryptophan (W6.48) in the transmembrane domain 6 of GPCRs was first characterized .…”

Purinergic signalling and cancer

“…Pulsed electromagnetic field exposure significantly increased the anti-tumour effect mediated by A 3 receptors in a human glioblastoma cell line [631]. Data have been presented to suggest that A 3 receptor agonists may be potential therapeutic agents for the induction of apoptosis in human glioma cells [632].…”