Growth inhibitory effect and apoptosis induced by extracellular ATP and adenosine on human gastric carcinoma cells: involvement of intracellular uptake of adenosine.

AIM

To study the growth inhibitory and apoptotic effects of adenosine triphosphate (ATP) and adenosine (ADO) on human gastric carcinoma (HGC)-27 cells in vitro and the mechanisms related to the actions of ATP and ADO.

METHODS

MTT assay was used to determine the reduction of cell viability. The morphological changes of HGC-27 cells induced by ATP or ADO were observed under fluorescence light microscope by acridine orange/ethidium bromide double-stained cells. The internucleosomal fragmentation of genomic DNA was detected by agarose gel electrophoresis. The apoptotic rate and cell-cycle analysis after treatment with ATP or ADO was determined by flow cytometry.

RESULTS

ATP, ADO and the intermediate metabolites, ADP and AMP, and the agonist of purinergic receptors, reduced cell viability of HGC-27 cells at doses of 0.3 and 1.0 mmol/L. The distribution of cell cycle phase and proliferation index (PI) value of HGC-27 cells changed when exposed to ATP or ADO at the concentrations of 0.1, 0.3 and 1 mmol/L for 48 h. ATP and ADO both altered the distribution of cell cycle phase via G0/G1- phase arrest and significantly decreased PI value. Under light microscope, the tumor cells exposed to 0.3 mmol/L ATP or ADO displayed morphological changes of apoptosis; a ladder-like pattern of DNA fragmentation obtained from HGC-27 cells treated with 0.1-1 mmol/L ATP or ADO appeared in agarose gel electrophoresis; ATP and ADO induced the apoptosis of HGC-27 cells in a dose-dependent manner at concentrations between 0.03-1 mmol/L. The maximum apoptotic rate of HGC-27 cells exposed to ATP or ADO for 48 h was 13.53% or 15.9%, respectively. HGC-27 cell death induced by ATP or ADO was significantly inhibited by dipyridamole (10 mmol/L), an inhibitor of adenosine transporter, but was not affected by aminophylline, a broad inhibitor of P1 receptors and pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetrasodium salt (30 micromol/L), a non-selective antagonist of P2 receptors.

CONCLUSION

Extracellular ATP and ADO reduced the cell viability, arrested cell cycle and induced apoptosis in HGC-27 cell line by intracellular uptake of ADO. One of the main routes of ATP-induced apoptosis in HGC-27 cells is through the breakdown to adenosine.