Nitric oxide-donating aspirin inhibits β-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear β-catenin–TCF association

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Abstract
Dysregulation of the Wnt pathway and altered β-catenin expression are central early events in colorectal carcinogenesis. We studied the ortho, meta, and para (o-, m-, and p-) positional isomers of NO-donating aspirin (NO-ASA), a chemopreventive agent against colon cancer, for their effect on β-catenin/T cell factor (TCF) signaling. In human SW480 colon carcinoma cells, cell-growth inhibition by NO-ASA [IC50 values for p-, o-, and mwere 48.1 ± 4.3 (mean ± SEM), 60.4 ± 2.1, and 900 ± 50 μM, respectively] was accompanied by significant inhibition of β-catenin signaling. We determined β-catenin-dependent TCF-4 transcriptional activity by measuring the activity of the luciferase gene placed under the control of TCF-4 regulatory sequences. The IC50 values for β-catenin/TCF-4-signaling inhibition by NO-ASA were: o-, 2.6 ± 0.4; m-, 15 ± 5; p-, 1.1 ± 0.1 μM; and for ASA, >5,000 μM. Total or nuclear levels of β-catenin and its distribution in the cell were not altered by NO-ASA, as judged by protein expression levels and semiquantitative immunofluorescence analysis. NO-ASA disrupted the association of β-catenin and TCF-4 in the nucleus, whereas ASA did not affect it. NO-ASA reduced the expression of cyclin D1, a downstream target gene that plays an important role in colon carcinogenesis. In contrast, a structural analog of NO-ASA lacking the —NO2 moiety did not affect TCF-4 transcriptional activity. Thus, NO-ASA inhibits β-catenin-mediated TCF activity by preventing the formation of the β-catenin/TCF-4 complex. This effect, occurring at NO-ASA concentrations far below those required to inhibit cell growth, may be a critical early event in the chemopreventive activity of NO-ASA against colon cancer.