In addition, experiments performed to elucidate the mechanism of APF activity indicate that this frizzled 8-related glycopeptide induces altered expression or phosphorylation of certain proteins that differ in some aspects from those seen in canonical Wnt/frizzled signaling. Downstream signal transducers for Wnt/frizzled signaling include Akt, GSK3β, and β-catenin [39]. The serine threonine kinase Akt, also known as protein kinase B (PKB), is a central regulator of cell proliferation, motility and survival whose activity is often

altered in human malignancies [40]. Akt mediates its downstream effects via phosphorylation/inactivation of GSK3β ser9, with subsequently decreased phosphorylation of the GSK3β target selleck inhibitor β-catenin, AG-881 chemical structure resulting in increased β-catenin nuclear translocation, binding to T-cell factor, and stimulation of gene expression related to cell proliferation and survival [30, 41]. In addition to its association with malignant cell proliferation, increased Akt phosphorylation/activation has also been linked to the invasive properties of bladder cancer cells [40]. The inhibition of Akt ser473 and thr308 phosphorylation

by APF suggests that APF may profoundly inhibit bladder epithelial cell Akt activity, and therefore decrease bladder carcinoma cell invasive potential, as well. GSK3β activity is reduced by phosphorylation of ser9 PTK6 but stimulated by phosphorylation on tyr216 [42], and the downstream effects of Akt activation/phosphorylation during Wnt/frizzled signaling include increased ser9 phosphorylation with decreased activity of GSK3β, decreased GSK3β-inducedβ-catenin ser33,37 phosphorylation, and subsequently decreased β-catenin ubiquitination and

degradation. If as -APF mediated its activity in T24 cells purely by inhibiting canonical Wnt/frizzled signaling (like other secreted frizzled-related cell growth inhibitors), GSK3β ser9 phosphorylation should have been decreased substantially, while tyr216 phosphorylation (which may be mediated by mitogen-activated protein kinase kinase (MEK) 1/2) [43] should not have been affected. Our results, which showed only a very minimal decrease in GSK3β ser9 phosphorylation, but a substantial decrease in GSK3β yr216 phosphorylation, indicate that as -APF: 1) does not mediate its activity purely by regulating Wnt/frizzled canonical signaling; 2) may inhibit GSK3β and additional kinases (such as MEK 1/2); and 3) may mediate its antiproliferative effects in T24 cells via inhibition of Akt, GSK3β, and/or MEK1/2 involving downstream effects on selleck targets in addition to β-catenin.