Cyclic GMP-dependent protein kinase activation and induction by exisulind and CP461 in colon tumor cells

These studies report on the activation and induction of cGMP-dependent prot ein kinase (PKG) by exisulind and analogs and test the hypothesis that PKG is involved in the induction of apoptosis in colon tumor cells. Exisulind a nd analogs are proapoptotic drugs developed as inhibitors of cGMP phosphodi esterase gene families 5 and 2 that have been shown to sustain increased cG MP in SW480 and HT29 cells. At concentrations that induced apoptosis, both exisulind and CP461 increased PKG activity in SW480 cell supernatants. PKG activation was dose-dependent and sustained. Activation of PKG by exisulind and analogs was also seen in the colon tumor cell lines HT29, T84, and HCT 116. The guanylyl cyclase activators YC-1 and guanylin increased PKG activi ty secondary to increased cellular cGMP and induced apoptosis in colon tumo r cells. Exisulind and CP461 had no direct effect on purified PKG activity or on basal and stimulated PKG activity from cell supernatants. An addition al effect of exisulind after 8 h of drug treatment was a dose-dependent inc rease of PKG I beta protein expression. beta -Catenin, a potential new subs trate for PKG, whose regulation influences apoptosis, was phosphorylated by PKG in vitro. P-32-labeled cells treated with exisulind showed increased p hosphorylation of beta -catenin. These data indicate that exisulind and ana logs activate and induce PKG, resulting in increased phosphorylation of bet a -catenin and enhanced apoptosis to promote colon tumor cell death.