Figure 2
Effect of equol and 4-OHT on cell death (A), apoptosis (B) and cell cycle distribution (C). For the determination of cell death (A), MCF-7 cells were seeded in 96-well plates (3 × 103 cells/well). Upon attachment cells were treated with equol (100 μM) and/or 4-OHT (100 μM). After 72 hours, cell death was evaluated using the Cell Death ELISA. The OD reading at 405 nm was proportional to the number of nucleosomes released in the cell lysates of the cells. The data are expressed as OD (405 nm) in comparison to the vehicle- treated control group. Each group was repeated in quadruplicates.* P Equol vs control = 0.023; ** P 4-OHT vs control = 0.032; *** P [Equol+4-OHT] vs control = 0.016. (B) Effect of equol and 4-OHT on MCF-7 cell apoptosis using annexin-V Alexa Fluor® 488/PI staining. Cells were plated in 60-mm plates and treated with equol (100 μM) and 4-OHT (10 μM) for 72 h. Cell viability, death and apoptosis were evaluated using the Tali™ apoptosis kit and the Tali™ Image-based Cytometer. Each experimental group was repeated in triplicate. Bars correspond to the standard error of mean (SEM). * P Equol vs control =0.032; ** P 4-OHT vs control =0.011; *** P [Equol + 4-OHT] vs control = 0.013. (C) Effect of equol and 4-OHT on cell cycle distribution using PI staining. MCF-7 cells were treated with equol (100 μM) and 4-OHT (10 μM) for 72 h. Cell cycle distribution was evaluated using PI staining for 15 min at 37°C. Sample analysis was performed using the Guava EasyCyte™ flow cytometer and the GuavaSoft analysis software. Each experimental group was repeated in triplicate. Bars correspond to the standard error of mean (SEM). * P Equol vs control = 0.0025; ** P 4-OHT vs control = 0.026; *** P [Equol+4-OHT] vs control = 0.0037.