FIGURE 2.

The inhibitory effects of caffeic acid on the clonogenicity and pluripotency of colorectal CSCs. Colorectal CSCs are more resistant to radiation than bulk tumor cells. Therefore, primary CSC spheres were exposed to radiation (2Gy) and dissociated into single cells. These cells were subsequently replated onto culture dishes without additional radiation exposure to form secondary CSC spheres. Caffeic acid treatment inhibited sphere formation of colorectal CSCs (A). The percentage of CD44⁺ CD133⁺ subpopulations was evaluated by FACS analysis. The treatment of HCT116 cells with caffeic acid (0.3 mM) for 48 h decreased the percentage of CD44⁺ CD133⁺ cells in the total cell population (B). Real-time PCR results reveal the changes in the expression of KLFG, NANOG, and SOX2 in both the vehicle-and caffeic acid-treated groups (C). HCT116 cells were treated with caffeic acid for 24 h, after which the effect of caffeic acid on cell migration ability was evaluated using a transwell migration assay (D). The relative expression levels of key positive regulators of cell migration (MMP-2/9) were assessed using western blotting (E). The inhibition of cell viability by caffeic acid treatment for 72 h was determined by an MTT assay. Cell viability (%) was calculated as a percent of the vehicle control (F). Caffeic acid-induced cytotoxicity was evaluated by flow cytometry using PE-labeled Annexin-V (G). β-actin was used as the internal control. The data represent the mean ± SD of three independent experiments.