Low dose resveratrol inhibits Wnt signaling proximal to β-catenin

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BACKGROUND: Resveratrol (RES) is a bioflavonoid found in the skin of grapes and red wine. It has been shown to have colon cancer prevention activity and other beneficial health effects in laboratory animals, though, despite reports of activity in humans, the dosages utilized to achieve these effects, and the dosages which induce apoptosis of cancer cells in vitro, are not realistically attainable in humans. We have examined the effects of RES on Wnt signaling, which is activated in over 85% of colon cancers, at dosages lower than those that inhibit cell growth in vitro. METHODS: Three cell lines were utilized: HT29, a colon cancer cell line with mutationally activated Wnt signaling; RKO, an HNPCC colon cancer cell line without basal Wnt pathway activation; NCM460, a normal mucosa-derived cell line lacking Wnt pathway activation. Cell proliferation was measured by MTT assay; confocal microscopy and fluorescence immunohistochemistry were utilized to observe β-catenin localization; quantitative real-time PCR was utilized to study intracellular levels of mRNA of 3 genes whose protein products modulate β-catenin activity, legless (lgs) and pygopus I and II (pygoI, pygoII); a reporter construct containing LEF/TCF binding sites driving secreted alkaline phosphatase production was utilized to quantitate Wnt signal throughput. RESULTS: Cell proliferation was not affected by RES at <40uM for NCM460 and HT29 and at <20uM for RKO. Wnt signal throughput was reduced in RKO and in NCM460 at concentrations as low as 10uM (p<0.001). This effect was greatest following Wnt pathway stimulation with Wnt3a conditioned medium (CM) and LEF1 transfection and was abrogated by transfection with β-catenin. RES had no effect on Wnt throughput in HT29. 10uM and 20uM RES significantly reduced the proportion of nuclear β-catenin in RKO but not in NCM460 or HT29, with the effect most apparent following pathway stimulation with Wnt3a CM (p=0.002). 5uM and 10uM RES reduced lgs in all cell lines (p<0.0001 to p=0.047) and decreased pygoI in HT29 and NCM460 (p<0.0001 to p=0.033) but not in RKO. PygoII message levels were unaffected. CONCLUSIONS: At dosages lower than those that result in reduced cell proliferation, RES has significant inhibitory effects on the Wnt signaling pathway. The effects were seen primarily in cells without basally activated Wnt signaling. While RES also had significant effects on nuclear β-catenin localization and on the expression of lgs and pygoI, these effects did not correlate with the Wnt pathway inhibitory activity. The eradication of Wnt inhibition following β-catenin transfection suggests that RES affects the Wnt pathway proximal of β-catenin. Wnt pathway inhibition, at lower dosages than previously reported, may contribute to RES’s activity as a cancer prevention agent in humans.