FIGURE 3.

Caffeic acid effectively suppresses radiotherapy-induced clonogenicity and pluripotency of colorectal CSCs. Colorectal CSC spheres derived from HCT116 cells were exposed to radiation (2Gy). Radiation significantly increased CSC sphere formation (A). The percentages of CD44⁺ CD133⁺ subpopulations were evaluated by FACS analysis. The exposure of HCT116 cells to radiation (2Gy) increased the percentage of CD44⁺ CD133⁺ cells in the total cell population (B). The mRNA levels of KLFG, NANOG, and SOX2 in both control and radiation (2Gy)-exposed cells were measured using real-time PCR (C). Primary CSC spheres were exposed to radiation (2Gy) and dissociated into single cells. These cells were subsequently replated onto culture dishes with or without additional caffeic acid (0.3 mM) treatment to form secondary CSC spheres. Caffeic acid inhibited radiation-resistant sphere formation in HCT116 cells (D). The treatment of HCT116 cells with caffeic acid for 48 h decreased the percentage of radiation-induced CD44⁺ CD133⁺ cells in the total cell population (E). The sphere sizes greater than 100 μm were enumerated, and a representative image of a tumor sphere is shown. Abbreviations: TSFE, tumor sphere-forming efficiency. The results are presented as the means ± SD of three independent experiments.