Ouabain Inhibits Wnt/?-catenin Signaling And Induces Cytotoxicity In Cancer Cells

The Wnt/?-catenin signaling pathway(Wnt signaling)is a highly conserved during evolution.The aberrant activation of this pathway is closely related to the occurrence and development of tumor.Wnt signaling is a complex signal transduction system consisting of Wnt molecules and their transmembrane receptor proteins,cytoplasmic signaling proteins,regulatory factors,nuclear transcription factors and downstream target genes.The Wnt/?-catenin signaling cascade is initiated when the secreted Wnt proteins bind to the Wnt coreceptor LRP5/6 and a member of the Frizzled(Fzd)family.Subsequently,the adaptor protein Dishevelled(DVL)is phosphorylated,which triggers the destabilization of the ?-catenin destruction complex and prevents degradation of ?-catenin in the proteasome.Stabilized ?-catenin accumulates in the cytoplasm and translocates into the nucleus to form a complex with transcription factors of the T-cell factor/lymphoid enhancing factor(TCF/LEF)family to activate transcription of Wnt target genes.So far there are no effective therapeutic agents targeting this pathway available for cancer treatment.This study identified digitalis(such as ouabain,digoxin and digitoxin)as the novel inhibitors of the Wnt signaling pathway using a SuperTopflash reporter gene assay.Digitalis are currently used to treat patient with congestive heart failure and arrhythmias,while it can inhibit tumor cell migration,induce apoptosis and autophagy in tumor cells possibly by regulating Na+/K+ pump.However,the detailed mechanism underlying anti-cancer activity of digitalis remains unclear.We demonstrated that ouabain,digoxin and digitalis could effectively inhibit the Wnt pathway.Among them,ouabain had the most potent inhibitory effect on Wnt signaling.In colonrectal cancer HCT116 and non-small cell lung cancer A549 cells,nanomolar concentrations of ouabain significantly decreased the levels of phosphorylated LRP6,total LRP6,active ?-catenin and ?-catenin.In addition,ouabain inhibited the expression of Wnt target genes CD44 and fibronection.Furthermore,ouabain reduced the viability of HCT116 and A549 cells and induced apoptosis in both cell lines.This study reveals a novel anti-cancer mechanism of ouabain.