Molecular mechanisms of adenosine-induced apoptosis in human HepG2 cells.
OBJECTIVE
To investigate effects of adenosine on cell proliferation and apoptosis in human HepG2 cells.


METHODS
HepG2 cells were incubated in the presence of adenosine (0.1-5 mmol/L) for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by using 3-(4,5-dimethyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Hoechst 33342 fluorescent staining, dUTP-fluorescein isothiocyanate (FITC) fluorescence and flow cytometric analysis techniques were used to observe cell apoptosis. The effects of adenosine receptor (A1, A2a, A3 and nonspecific receptor) antagonists (8-cpt, DMPX, MRS1191, and theophylline) and an adenosine transporter protein inhibitor (dipyridamole) on adenosine-induced cell apoptosis were observed. Mitochondrial membrane potential was analyzed using DePsipher fluorescent staining, and caspase activity was detected using a Fluorometric assay kit and a fluorescence microplate reader.


RESULTS
Adenosine significantly reduced cell viability in a dose- and time-dependent manner. The cytotoxicity of adenosine was related to the induction of cell apoptosis. Four adenosine receptor antagonists had no effect on cell apoptosis. However, dipyridamole significantly reduced the percentage of adenosine-induced apoptotic cells from 27.3% to 7.1% (P<0.05). At 48 h after treatment, 3 mmol/L adenosine increased caspase-3 activity 3.5-fold; dipyridamole markedly decreased caspase-3 activity 1.6-fold, and decreased apoptotic cell numbers. When HepG2 cells were treated with 3 mmol/L adenosine, mitochondrial membrane potential and the activity of caspase-8 or -9 remained unchanged.


CONCLUSIONS
Our results suggest that adenosine-induced apoptosis in HepG2 cells is related to intracellular events rather than cell surface receptors, and that a caspase-3 cascade activation is required, which is not mediated via a mitochondrial pathway.
Department of Gastroenterology, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China. lingfeiwu@21cn.com
Ling-Fei
LF
Wu
Guo-Ping
GP
Li
Jia-Lin
JL
Feng
Ze-Jin
ZJ
Pu
D000241:Adenosine / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004176:Dipyridamole / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000502:physiology; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology
C053907:8-cyclopentyl-1,3-dimethylxanthine; D004176:Dipyridamole; D013806:Theophylline; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D000241:Adenosine