Ionomycin downregulates β-catenin/Tcf signaling in colon cancer cell line

Abstract

Functional activation of β-catenin/Tcf signaling plays an important role in the early events in colorectal carcinogenesis. We examined the effect of ionomycin against β-catenin/Tcf signaling in colon cancer cells. Reporter gene assay showed that ionomycin inhibited β-catenin/Tcf signaling efficiently. In addition, the inhibition of β-catenin/Tcf signaling by ionomycin in HEK293 cells transiently transfected with a constitutively mutant β-catenin gene, whose product is not phosphorylated by GSK3β, indicates that its inhibitory mechanism is related to β-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunoprecipitation analysis, western blot and electrophoretic mobility shift assay. As a result, our data reveal that the association of β-catenin and Tcf-4 is disrupted and the amount of β-catenin product in the nucleus is decreased by ionomycin in a concentration-dependent manner. Moreover, ionomycin strongly suppressed the binding of the Tcf complexes to its specific DNA-binding sites. The significance of the current work is that ionomycin is a negative regulator of β-catenin/Tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear β-catenin products and to the suppressed binding of Tcf complexes to consensus DNA. © Oxford University Press 2005; all rights reserved.