Figure 7
From: Coffee decoction enhances tamoxifen proapoptotic activity on MCF-7 cells
U0126 and LY294002 induce apoptosis in the presence of tamoxifen in MCF-7 cells. (A) MCF-7 cells were treated with DMSO (0.1%), U0126 (10, 20, 30 μM), or LY294002 (10, 20, 30 μM) for 24 h. Whole cell lysates were immunoblotted with anti-phospho-ERK, anti-ERK, anti-phospho-Akt, or anti-Akt. The relative phosphorylation levels of ERK and Akt are shown in the graphs. Results represent the mean ± SD of three independent experiments. ***p < 0.001 significantly different from control cells. (B-E) MCF-7 cells were treated with DMSO (0.1%), U0126 (10, 20 μM), or LY294002 (20, 30 μM) in the absence or presence of tamoxifen (2.5, 5 μM) for 24 h. (B) 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. #p < 0.05, ##p < 0.01, ###p < 0.001 significantly different from cells treated with 2.5 μM tamoxifen. §p < 0.05, §§p < 0.01 significantly different from cells treated with 5 μM tamoxifen. (C) The proliferation rate was determined by BrdU incorporation assay. Results represent the mean ± SD of four independent experiments. *p < 0.05, **p < 0.01 significantly different from control cells. #p < 0.05, ###p < 0.001 significantly different from cells treated with 2.5 μM tamoxifen. §p < 0.05, §§p < 0.01, §§§p < 0.001 significantly different from cells treated with 5 μM tamoxifen. (D) 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 graphs. Results represent the mean ± SD of three independent experiments. *p < 0.05 significantly different from control cells. #p < 0.05, ##p < 0.01, ###p < 0.001 significantly different from cells treated with 2.5 μM tamoxifen. §p < 0.05, §§p < 0.01, §§§p < 0.001 significantly different from cells treated with 5 μM tamoxifen. (E) Cells were fixed, permeabilized and treated with propidium iodide fixed, 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 of three independent experiments. *p < 0.05, **p < 0.01 significantly different from control cells. #p < 0.01, ##p < 0.05, ###p < 0.001 significantly different from cells treated with 2.5 μM tamoxifen. §p < 0.05, §§p < 0.01, §§§p < 0.001 significantly different from cells treated with 5 μM tamoxifen. (F) MCF-7 cells were treated with DMSO (0.1%), U0126 (10, 20 μM), or LY294002 (20, 30 μM) in the absence or presence 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 of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 significantly different from control cells. #p < 0.05, ##p < 0.01, ###p < 0.001 significantly different from cells treated with 2.5 μM tamoxifen. §p < 0.05, §§p < 0.01, §§§p < 0.001 significantly different from cells treated with 5 μM tamoxifen.
