Extracellular adenosine induces apoptosis in Caco-2 human colonic cancer cells by activating caspase-9/-3 via A(2a) adenosine receptors.
BACKGROUND
Extracellular adenosine has been shown to induce apoptosis in a variety of cells via an intrinsic pathway linked to adenosine uptake into cells and the ensuing signaling cascades and an extrinsic pathway linked to adenosine receptors. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis of Caco-2 human colonic cancer cells.


METHODS
To observe cell viability, an MTT assay was carried out in Caco-2 cells untransfected or transfected with the A(2a) adenosine receptor pcDNA3.1. Apoptotic cell death was assessed with flow cytometry using propidium iodide and annexin V and internucleosomal DNA fragmentation analysis. Activities of caspase-3, -8, and -9 were measured using a caspase fluorometric assay kit. Mitochondrial membrane potentials were monitored using a DePsipher kit. Expression of adenosine receptors was examined with the reverse transcription-polymerase chain reaction (RT-PCR) method.


RESULTS
Extracellular adenosine induced Caco-2 cell apoptosis in a concentration-dependent (1-20 mM) and treatment time-dependent (24-72 h) manner. The adenosine effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors and SQ22536, an inhibitor of adenylate cyclase. CGS21680, an agonist of A(2a) adenosine receptors, and forskolin, an adenylate cyclase activator, mimicked the adenosine action. Caco-2 cell death was still induced by overexpressing A(2a) adenosine receptors, and adenosine further promoted the cell death. Adenosine disrupted mitochondrial membrane potentials and activated caspase-9 and -3, but not caspase-8.


CONCLUSIONS
Extracellular adenosine induces apoptosis in Caco-2 cells by activating caspase-9 and the downstream effector caspase caspase-3 in association with mitochondrial damage via A(2a) adenosine receptors.
Yasuda
Yoshiyuki
Y
Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, 663-8501, Japan.
Saito
Masaru
M
Yamamura
Takehira
T
Yaguchi
Takahiro
T
Nishizaki
Tomoyuki
T
D000241:Adenosine / Q000502:physiology*; D017209:Apoptosis / Q000502:physiology*; D018938:Caco-2 Cells; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism*; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D043705:Receptor, Adenosine A2A; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D000241:Adenosine