Resveratrol is a bioflavonoid which is known to inhibit cell proliferation and induce apoptosis in cancer cell lines at concentrations above 50uM. It also has colon cancer prevention activity in mouse models and possibly in humans. We have examined the effects of low concentrations of resveratrol on a specific signaling pathway, the Wnt pathway, which is activated in over 85% of sporadic colon cancers. Two colon cancer (HT29 and RKO) and one normal mucosa-derived (NCM460) cell lines were utilized. Cell proliferation was not affected by resveratrol at ≤40uM for HT29 and NCM460 and <20uM for RKO though Wnt signal throughput, as measured by a reporter construct, was reduced in RKO and NCM460 at concentrations as low as 10uM (p<0.001). This effect was most easily appreciated following Wnt pathway stimulation with Wnt3a conditioned medium and LEF1 or LEF1/ β-catenin transfection. Resveratrol did not inhibit Wnt throughput in mutationally activated HT29. Low concentrations of resveratrol significantly decreased the amount and proportion of β-catenin in the nucleus in RKO (p=0.002) and reduced the expression of lgs and pygoI, regulators of β-catenin localization in all cells lines. Thus, at low concentrations, in the absence of effects on cell proliferation, resveratrol significantly inhibits Wnt signaling in colon-derived cells which do not have a basally activated Wnt pathway. This inhibitory effect may be due in part to regulation of intracellular β-catenin localization.
BackgroundWnt signaling is activated in many types of cancer and normal physiological processes. Various Wnt-related secreted factors may influence angiogenesis both in the tumor microenvironment and in normal tissues by direct action on endothelial cells. The mechanism of this Wnt action in angiogenesis is not well defined. We hypothesize that endothelial cells are responsive to Wnt signals and that Lef1, a member of the vertebrate-specific Wnt/beta-catenin throughput-inducing transcription factors' sub-family Lef1/Tcf1, mediates this responsiveness and promotes endothelial cell invasion.MethodsA human endothelial cell line, EAhy926 was exposed to Wnt3a or directly transfected with Lef1. Readouts included assessment of nuclear beta-catenin, Wnt throughput with a SuperTOPflash reporter assay, induction of Lef1 transcription, induction of matrix metalloproteinase (MMP)-2 transcription, cell proliferation and cell invasion through a matrix in vitro. The effects on MMP2 were also evaluated in the presence of Lef1 silencing siRNA.ResultsWnt3a increased nuclear beta-catenin and up-regulated Wnt/beta-catenin throughput. Wnt3a increased Lef1 transcription and activity of the Lef1 promoter. Both Wnt3a treatment and Lef1 overexpression induced MMP2 transcription but this effect was completely abrogated in the presence of Lef1 siRNA. Inhibition of Lef1 also reduced basal MMP2 levels suggesting that Lef1 regulates MMP2 expression even in the absence of exogenous Wnt pathway activation. Lef1 slightly increased proliferation of EAhy926 cells and increased invasion by more than two-fold.ConclusionsEAhy926 cells activate canonical Wnt signaling in response to Wnt3a ligand. The Wnt target Lef1 specifically regulates MMP2 expression in these cells and promotes endothelial cell invasion. The EAhy926 cell line provides a convenient alternative to primary human umbilical vein endothelial cells (HUVEC) in the study of angiogenesis and the role of Wnt signaling on endothelial cell function.
Background: Norrin is a potent Wnt pathway ligand. Aberrant activation of this signaling pathway can result in colon tumors but the role of norrin-based signaling in the genesis of colon cancer, and its relationship to activation of the pathway by traditional Wnt ligands, is not defined.
Norrin binds to the frizzled-4 receptor, stimulating canonical Wnt signaling. We investigate here the role of colorectal cancer (CRC) produced Norrin in endothelial cell growth, motility, and blood vessel formation, as well as the expression of the Norrin signaling pathway components in the CRC tumor microenvironment. Norrin conditioned medium produced by CRC cell line CaCO2 transfected with Norrin expression construct increased endothelial cell motility. Blocking Norrin signaling reduced endothelial cell motility, branch point number (1/mm2), and the network length (mm/mm2) during in vitro angiogenesis. Colorectal tumors express Norrin protein. Endothelial cells in the colorectal tumor microenvironment contain all of the components of the Norrin signaling pathway needed to respond to Norrin protein. This study presents data that Norrin may play a role in the regulation of angiogenesis in the colorectal cancer tumor microenvironment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.