A(1) 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 (0.3-10mM)- and treatment time (24-72h)-dependent manner. In the apoptosis assay using propidium iodide (PI) and annexin V, treatment with adenosine (1mM) for 72h 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 (1mM) for 72h 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(1) 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(1) 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(1) 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.
Department of Physiology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan.
Katsunari
K
Sai
Dongqin
D
Yang
Hideyuki
H
Yamamoto
Hirokazu
H
Fujikawa
Satoshi
S
Yamamoto
Tetsu
T
Nagata
Masaru
M
Saito
Takehira
T
Yamamura
Tomoyuki
T
Nishizaki
D055372:AMP-Activated Protein Kinases; D000241:Adenosine / Q000494:pharmacology / Q000502:physiology*; D000704:Analysis of Variance; D000818:Animals; D001254:Astrocytoma / Q000503:physiopathology*; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D051336:Mitochondrial Membranes / Q000187:drug effects; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000502:physiology*; D013778:Tetrazolium Salts; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D013844:Thiazoles
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D043682:Receptor, Adenosine A1; D013778:Tetrazolium Salts; D013844:Thiazoles; C053907:8-cyclopentyl-1,3-dimethylxanthine; D013806:Theophylline; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D020169:Caspases; C022616:thiazolyl blue; D000241:Adenosine