Cell experiment:

Cells are seeded at 20,000 cells/well into 96-well plates. After overnight incubation, cells are treated with DMSO or each Wnt inhibitor (iCRT-3, 75 μM; iCRT-5, 200 μM; iCRT-14, 50 μM; IWP-4, 5 μM and XAV-939, 10 μM) for 48 hours. Cell viability is determined using the Cell Titer-Glo luminescent cell viability assay kit. Luminescence is measured using FLUOstar microplate reader. All treatments are performed in triplicate, and each experiment is repeated three times.

iCRT 14 is a potent and specific inhibitor of β-catenin responsive transcription (CRT) with IC50 value of 40.3 nM [1].

iCRT 14 belongs to the thiazolidinedione class and is screened out by the dTF12-luciferase reporter assay. It inhibits the direct interaction between β-catenin and TCF4 and does not affect the interaction of β-catenin with other cognate protein partners. iCRT 14 also inhibits the mammalian Wnt responsive STF16 luciferase reporter in HEK293 cells with IC50 value of 40.3 nM. Besides that, iCRT 14 is specific against CRT over other conserved cell signaling pathways. It had no effect on the phosphorylation of Dvl induced by Wnt [1].

iCRT 14 can also interfere with TCF binding to DNA. Moreover, the inhibition of CRT resulted in the modulation of CRT-induced molecular and morphological changes including cell transformation phenotype and cell invasion. Furthermore, the treatment of HCT116 and HT29 cells with iCRT 14 exhibited cell cycle arrest in the G0/G1 phase. In the xenograft models of HCT116 and HT29, administration of iCRT 14 at dose of 50 mg/kg caused significant decrease in CycD1 and the subsequent suppression of tumor growth [1].

References:
[1] Gonsalves F C, Klein K, Carson B B, et al. An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway. Proceedings of the National Academy of Sciences, 2011, 108(15): 5954-5963.