Identification of CGK062 as a potent small-molecule inhibitor of Wnt/β-catenin pathway.

<p>(A) Screening of compounds that inhibit the Wnt/β-catenin pathway. Compounds modulating TOPFlash reporter activity were screened using the HEK293 reporter cells. The controls were assayed in the presence or absence of Wnt3a-CM. (B) Chemical structure of CGK062. (C) Dose-dependent inhibition of β-catenin response transcription. HEK293 reporter cells were incubated with indicated concentrations of CGK062 in the presence of Wnt3a-CM. After 15 h, luciferase activities were determined and reported as relative light unit (RLU) normalized to cell titer. The results are the average of three experiments, and the bars indicate standard deviations. *, <i>P</i><0.05, and **, <i>P</i><0.01, compared with the Wnt3a-CM-treated control group. (D) Cytosolic proteins were prepared from HEK293 reporter cells treated with either vehicle (DMSO) or indicated concentrations of CGK062 in the presence of Wnt3a-CM for 15 h and then subjected to Western blotting with β-catenin antibody. (E) Semi-quantitative RT-PCR for β-catenin, and GAPDH was performed with total RNA prepared from HEK293 reporter cells either vehicle (DMSO) or indicated concentrations of CGK062 in the presence of Wnt3a-CM for 15 h. (F) Cytosolic proteins prepared from HEK293 reporter cells, which were incubated with vehicle (DMSO) or CGK062 (25 µM) in the presence or absence of Wnt3a-CM, exposed to MG-132 (10 µM) for 8 h, were subjected to Western blotting with anti-β-catenin antibody. In (D) amd (F), to confirm equal loading, the blot was reprobed with anti-actin antibody.