Fig. 5.

StAx peptides inhibit β-catenin–mediated transcriptional activity. (A) fStAx peptides inhibit TOPflash luciferase reporter activity in Wnt3a-stimulated HeLa cells. (B) fStAx-35 and -35R suppress luciferase activity in a dose-dependent manner. Negative control fStAx-41 is inactive. (C and D) fStAx peptides do not affect Notch, Hedgehog, BMP, and TGF-β signaling as examined in corresponding luciferase reporter assays in cells treated with 15 μM fStAx (DAPT). (E) β-Catenin and Axin1 protein levels in SW480 cell line were revealed by Western blot. Unlike the tankyrase inhibitor XAV939 (5 μM) or GSK3-β inhibitor LiCl (10 mM), aStAx peptides (10 μM) did not affect protein levels of Axin1 or β-catenin (CM: Wnt3a conditioned medium). (F) fStAx peptides (10 μM) inhibit the mRNA level of Wnt/β-catenin target genes in DLD1 and SW480 cells. Relative mRNA level was normalized with the housekeeping gene β-actin (all treatments for 24 h with 10% serum). (G) Proliferation of DLD1 and SW480 cells was blocked by aStAx-35R in a dose-dependent manner (5-d treatments). (H) Proliferation tendency of DLD1 and SW480 cells was significantly repressed after treatment with 10 μM aStAx-35R for 5 d. (I and J) aStAx-35R showed inhibitory effects on cell proliferation only in β-catenin–dependent cancer cell lines DLD1, SW480, and HCT116 but not on β-catenin–independent cell lines A549 and RKO (all treatments with 10 μM aStAx for 5 d, cell titer determined via cellular ATP level). Data points, mean ± SD.