Figure 3
Effect of caffeic acid and 3,4-dihydroxy-phenylacetic acid (PAA) on the cell cycle and apoptosis after long (two cell cycles) incubation times. Cells were incubated with the indicated phenolic acids for 5 days. The medium, containing 10-7 M of the agents, was changed every other day. At the end of the incubation period, cells were removed from plates by scraping, stained with annexin V and/or propidium iodide (PI), as described in Materials and methods, and analyzed by flow cytometry. Early apoptotic changes were identified as the cell population having a normal (diploid) DNA content and annexin V staining. Late apoptotic cells were those presenting a hypodiploid DNA content identified by PI staining. After permeabilization of cells, staining with PI and flow cytometry revealed cell cycle phases. Necrotic cells were identified by forward and side scatter. (a) Typical flow cytometric analysis of the cell cycle. Cells were stained with PI. (b) Cumulative cell cycle phases of nonapoptotic cells (mean ± standard error of the mean of three measurements). (c) Cumulative effects of phenolic acids on apoptosis (mean ± standard error of the mean of three experiments). Cells were stained with annexin V and PI. (d) Effect of phenolic acids on apoptosis-related proteins. Semiquantitative western blot analysis on cell homogenates from T47D cell cultures treated with 10-7 M various phenolic acids for 5 days. At the end of the incubation period, cells were removed from plates by scraping and apoptosis-related proteins were measured with western blot analysis, as described in Materials and methods. Quantification results are depicted, expressed as the percentage of nontreated control values. Data are mean ± standard error of the mean of three independent experiments.