FIGURE 5.

Activation or inhibition of Akt signaling modulates the caffeic acid-induced effects on clonogenicity and pluripotency of colorectal CSCs. Western blotting results reveal the changes in Akt activity in both vehicle- and caffeic acid-treated groups (A). Colorectal CSCs were pretreated with the Akt activator SC79 (10 μM) for 24 h prior to treatment with 0.3 mM caffeic acid for 48 h, and the changes in the activities of PI3K and Akt were determined by western blotting (B). The attenuating effects of Akt activation on the caffeic acid-induced sphere-forming ability and the percentage of CD44⁺CD133⁺ subpopulations were assessed by sphere-forming culture (C) and FACS analysis (D), respectively. Colorectal CSCs were pretreated with the Akt inhibitor V (10 μM) for 24 h prior to treatment with 0.3 mM caffeic acid for 48 h, and the changes in the activities of PI3K and Akt were determined by western blotting (E). The synergistic effects of Akt inhibition on the caffeic acid-induced sphere-forming ability and the percentage of CD44⁺ CD133⁺ subpopulations were assessed by sphere-forming culture (F) and FACS analysis (G), respectively. 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. β-actin was used as the internal control. The data represent the mean ± SD of three independent experiments.