Figure 11

Co-treatment with decaffeinated coffee, caffeine and tamoxifen induces apoptosis in MCF-7 cells. MCF-7 cells were treated with decaffeinated coffee (2.5, 5 v/v%) with/without caffeine (2 mM) in the presence and absence of tamoxifen (2.5, 5 μM) for 24 h. (A) Cell viability was measured by the trypan blue exclusion method. Results represent the mean ± SD of three independent experiments. *p < 0.05 significantly different from control cells. # and ## indicates p < 0.05 and p < 0.01, respectively. (B) The proliferation rate was determined by BrdU incorporation assay. Results represent the mean ± SD of four independent experiments. *p < 0.05, ***p < 0.001 significantly different from control cells. #, ## and ### indicate p < 0.05, p < 0.01 and p < 0.001, respectively. (C) Whole cell lysates were immunoblotted with an anti-cyclin D1 antibody or anti-β-actin antibody. The relative expression levels of cyclin D1 are shown in the graph. Results represent the mean ± SD of three independent experiments. ## indicates p < 0.01. (D) Cells were fixed, permeabilized and treated with propidium iodide, and the cell cycle was examined using a flow cytometric analysis. The ratios of cells in the Sub-G1 phase, G₀/G₁ phase, S phase and G₂/M phase were graphed. Data were expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01 significantly different from control cells. # and ## indicates p < 0.05 and p < 0.01, respectively. (E) MCF-7 cells were treated with decaffeinated coffee (2.5, 5 v/v%) with/without caffeine (2 mM) in the presence and absence of tamoxifen (2.5, 5 μM) for 18 h. Annexin V–FITC and propidium iodide (PI) double staining was performed. The ratios of early-phase apoptotic cells and late-phase apoptotic cells were graphed. Data were expressed as means ± SD (n = 3). # and ## indicates p < 0.05 and p < 0.01, respectively.