Introduction. The majority of colorectal cancers (CRC) have mutationally-driven constitutive activation of the Wnt pathway. Despite this, tumor cells remain responsive to Wnt signals arising at the cell membrane through the interaction of extracellular Wnt ligands and cell surface frizzled receptors; the strength of the Wnt signal can be modified by Wnt pathway inhibitors (sFRP) in the tumor microenvironment. Genistein, a natural product from soy, is known to remove epigenetic blockage of sFRP promoters and decrease nuclear beta-catenin expression, a marker for Wnt pathway activation. In this study we have examined the effect of genistein on Wnt throughput in CRC cell lines and on proliferation in tissue culture and growth in soft agar.
Methods. Human CRC cell lines HT29, DLD1 and RKO were transfected with Wnt/beta-catenin reporter plasmids and treated with different concentrations of genistein. Pathway activity was measured using a super TOP-flash luminescence reporter construct. Proliferation was estimated with an MTT assay. A soft agar assay was utilized to define non-adherent colony formation.
Results. Genistein reduced proliferation of HT29 and DLD1 at 50uM, with an inhibitory P50 at 100uM. RKO cells were inhibited at lower concentrations. Inhibition of soft agar colony growth occurred at similar concentrations. Wnt/beta-catenin activity was significantly inhibited at 75uM for HT29 (p=0.025) and at 50uM for DLD1. The reduction in proliferation and colony growth correlated with reduced Wnt signaling, with the latter most strongly correlated (R2=0.81 vs. 0.47 for the former). Cells growing independently of anchorage were more sensitive to genistein treatment than those attached to the plastic : colony counts decreased for RKO 8-fold at 50 uM while proliferation on plastic decreased less than 4x; colony counts decreased for HT29 7-fold for at 100 uM while proliferation on plastic decreased less than 2x.
Conclusions. Genistein reduces Wnt/beta-catenin activity in CRC cell lines; this reduction correlates with cell proliferation in vitro and with soft agar colony growth. Cells growing in an anchorage-independent fashion appear more sensitive to genistein treatment. Further investigation into the mechanisms of this effect in CRC is ongoing. These data suggest that genistein may be a useful adjunctive therapy for patients with CRC.
Citation Format: Sofya Pintova, Kestutis Planoutis, Marina Planutiene, Randall F. Holcombe. Genistein-induced reduction of Wnt/beta-catenin pathway activity in colorectal cancer cell lines correlates with inhibition of proliferation and colony growth in soft agar . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4000. doi:10.1158/1538-7445.AM2013-4000