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Aim:To investigate effects of adenosine on cell proliferation and apoptosis inhuman HepG2 cells.Methods:HepG2 cells were incubated in the presence ofadenosine(0.1-5 mmol/L)for 12-48 h,and the effect of adenosine on cell prolifera-tion was evaluated by using 3-(4,5-dimethyl-2-yl)-2,5-diphenyl-2H-tetrazolium bro-mide(MTT)assay.Hoechst 33342 fluorescent staining,dUTP-fluoresceinisothiocyanate(FITC)fluorescence and flow cytometric analysis techniques wereused to observe cell apoptosis.The effects of adenosine receptor(A1,A2a,A3and nonspecific receptor)antagonists(8-cpt,DMPX,MRS 1191,and theophylline)and an adenosine transporter protein inhibitor(dipyridamole)on adenosine-in-duced cell apoptosis were observed.Mitochondrial membrane potential was ana-lyzed using DePsipher fluorescent staining,and caspase activity was detectedusing a Fluorometric assay kit and a fluorescence microplate reader.Results:Adenosine significantly reduced cell viability in a dose-and time-dependentmanner.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 apoptoticcells from 27.3% to 7.1%(P<0.05).At 48 h after treatment,3 mmol/L adenosineincreased caspase-3 activity 3.5-fold;dipyridamole markedly decreased caspase-3 activity 1.6-fold,and decreased apoptotic cell numbers.When HepG2 cells weretreated with 3 mmol/L adenosine,mitochondrial membrane potential and theactivity of caspase-8 or-9 remained unchanged.Conclusion:Our results suggestthat adenosine-induced apoptosis in HepG2 cells is related to intracellular eventsrsther than cell surface receptors,and that a caspase-3 cascade activation isrequired,which is not mediated via a mitochondrial pathway.
Aim: To investigate effects of adenosine on cell proliferation and apoptosis in human HepG2 cells. Methods: HepG2 cells were incubated in presence of adenosine (0.1-5 mmol / L) for 12-48 h, and the effect of adenosine on cell prolifera tion was evaluated by using 3- (4,5-dimethyl-2-yl) -2,5-diphenyl-2H-tetrazolium bro- mide (MTT) assay.Hoechst 33342 fluorescent staining, dUTP- fluoresceinisothiocyanate (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, MRS 1191, and theophylline) and an adenosine transporter protein inhibitor (dipyridamole) Mitochondrial membrane potential was ana-lyzed 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. cytotox icity 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) after treatment, 3 mmol / L adenosineincreased caspase-3 activity 3.5-fold; dipyridamole markedly decreased by caspase-3 activity 1.6-fold, and decreased apoptotic cell numbers.When HepG2 cells weretreated with 3 mmol / L adenosine, mitochondrial membrane potential and theactivity of Caspase-8 or-9 remained unchanged. Conlusion: Our results suggest that adenosine-induced apoptosis in HepG2 cells is related to intracellular eventsrsther than cell surface receptors, and that a caspase-3 cascade activation is required, which is not mediated via a mitochondrial pathway.