Modulation of Key Elements of the Wnt Pathway by Apple Polyphenols

Glycogen synthase kinase-3β (GSK3β) is one of the key elements of the Wnt pathway involved in the regulation of β-catenin homeostasis. The inhibition of GSK3β kinase activity might lead to the onset of β-catenin/TCF/LEF-mediated gene transcription, representing a potentially mitogenic stimulus. Apple polyphenols have been shown to mediate several biological effects that might be of interest with respect to chemoprevention. The objective of the study was to elucidate whether apple polyphenols also modulate key elements of the Wnt pathway, an effect that might limit the usefulness of these compounds for the prevention of carcinogenesis. A polyphenol-rich apple juice extract (AE02) was found to effectively inhibit the kinase activity of GSK3β, immunoprecipitated from HT29 cells. Treatment of HT29 cells with AE02 for 24 h resulted in a concentration-dependent decrease of the cellular GSK3β kinase activity. The inhibition of the kinase activity in HT29 cells was observed at polyphenol concentrations corresponding to the concentration of the constituents in the original apple juice. The apple characteristic dihydrochalcone glycoside phloridzin was found to be inactive up to 500 μM. The free aglycon phloretin as well as the flavonol quercetin effectively inhibited isolated GSK3β, but did not affect the respective kinase activity within HT29 cells. In accordance with the inhibition of GSK3β kinase activity by AE02, treatment of HT29 cells resulted in a significant decrease of phosphorylated β-catenin. However, the total intracellular β-catenin level was also found to be diminished, indicating that the interference of the apple constituents with GSK3β was not associated with a stabilization of β-catenin in HT29 cells. In line with these results, TCF/LEF-mediated gene transcription remained unaffected by treatment with AE02 as shown in a reporter gene approach. We can assume from the results that at consumer-relevant concentrations apple polyphenols do not mediate growth-stimulating effects in HT29 cells via the Wnt pathway. Keywords: Phloretin; phloridzin; quercetin; glycogen synthase kinase-3β; β-catenin; colon carcinoma; HT29