A1 adenosine receptor signal and AMPK involving caspase-9/-3 activation are responsible for adenosine-induced RCR-1 astrocytoma cell death

Extracellular adenosine reduced viability of RCR-1 rat astrocytoma cells in a dose (0.3-10 mM)- and treatment time (24-72 h)-dependent manner. In the apoptosis assay using propidium iodide (PI) and annexin V, treatment with adenosine (I mM) for 72 h increased the population of PI-negative/annexin V-positive cells, that is related to early apoptosis, and that of PI-positive/annexin V-positive cells, that is related to late apoptosis/secondary necrosis. In addition, nuclei of cells treated with adenosine (1 mM) for 72 h were reactive to an antibody against single-stranded DNA. Adenosine activated caspase-3, -8 and -9, but mitochondrial membrane potentials were not affected. Adenosine-induced RCR-1 cell death was significantly inhibited by 8-CPT, an antagonist of A, adenosine receptors, and forskolin, an adenylate cyclase activator. SQ22536, an adenylate cyclase inhibitor, alternatively, exhibited an effect similar to adenosine. CHA, an agonist of A I adenosine receptors, activated caspase-3 and -9, but not caspase-8. Adenosine-induced cytotoxicity of RCR-1 cells was also significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, and AMDA, an inhibitor of adenosine kinase. AICAR, an activator of AMP-activated protein kinase (AMPK), reduced RCR-1 cell viability, but synergistic effect was not obtained with co-treatment with adenosine and AICAR. AICAR activated caspase-3 and -9, but not caspase-8. An additive inhibition was found in the co-presence of 8-CPT and dipyridamole. Extracellular adenosine, thus, appears to activate caspase-9 followed by the effector caspase, caspase-3, at least via two independent pathways linked to A, adenosine receptor-mediated adenylate cyclase inhibition and adenosine uptake into cells/conversion to AMP/activation of AMPK, possibly regardless of mitochondrial damage, thereby leading to RCR-1 cell death, dominantly by apoptosis. Moreover, caspase-8 activation could again contribute to adenosine-induced cytotoxicity, although the underlying mechanism is currently unknown. Collectively, the results of the present study may represent a new pathway for caspase activation relevant to diverse adenosine signals in cell death. (c) 2006 Elsevier Inc. All rights reserved.