Effect of 7-methylxanthine on human retinal pigment epithelium cells cultured in vitro.

ABSTRACT

Purpose: To evaluate the effects of 7-methylxanthine (7-MX) on the growth of human retinal pigment epithelium (RPE) cells and to observe the changes in the expression of adenosine receptors (ADORs) in RPE cells upon 7-MX treatment. Methods: Human RPE cells (monolayer at about 80% confluence) were cultured in vitro in the presence or absence of 7-MX. Cell proliferation was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell cycle distribution and apoptosis level were analyzed with flow cytometry. Quantitative PCR and immunofluorescence assay were used to examine the mRNA and protein expression of ADORs. Results: 7-MX at low concentrations had no effect on the proliferation of RPE cells, whereas 100 µmol/l 7-MX slightly decreased cell proliferation at 48 h but without a statistically significant difference. The 7-MX treatment was performed at the low concentration of 10 µmol/l in the following experiments. The proportion of RPE cells in the G1 stage was slightly increased at 24 h (p=0.035) but decreased at 48 h (p=0.0045) upon 7-MX treatment; and the proportion was restored to normal at 72 h. No statistically significant change in apoptosis levels was found in RPE cells cultured with 7-MX. The expression of ADORA1, ADORA2A, and ADORA2B in RPE cells was inhibited by 7-MX treatment at 48 h, while the expression levels appeared to rebound at 72 h. Conclusions: 7-MX has little effect on the proliferation or apoptosis level of human RPE cells; however, in short-term treatment, 7-MX disturbs the proportion of cells in the G1 stage and inhibits the expression of ADORA1, ADORA2A, and ADORA2B.