PNU 74654 binds to β-catenin (Kd = 450 nM), inhibiting its interaction with the transcription factor T cell factor 4 (Tcf4) resulting in disruption of the Wnt signaling pathway.

In NCI-H295 cells, PNU 74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU 74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU 74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU 74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability[1].

Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU 74654 (5-200 μM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot)[1].