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 protein kinase (PKG) by exisulind and analogs and test the hypothesis that PKG is involved in the induction of apoptosis in colon tumor cells. Exisulind and analogs are proapoptotic drugs developed as inhibitors of cGMP phosphodiesterase gene families 5 and 2 that have been shown to sustain increased cGMP 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 HCT116. The guanylyl cyclase activators YC-1 and guanylin increased PKG activity secondary to increased cellular cGMP and induced apoptosis in colon tumor cells. Exisulind and CP461 had no direct effect on purified PKG activity or on basal and stimulated PKG activity from cell supernatants. An additional effect of exisulind after 8 h of drug treatment was a dose-dependent increase of PKG Ibeta protein expression. beta-Catenin, a potential new substrate for PKG, whose regulation influences apoptosis, was phosphorylated by PKG in vitro. 32P-labeled cells treated with exisulind showed increased phosphorylation of beta-catenin. These data indicate that exisulind and analogs activate and induce PKG, resulting in increased phosphorylation of beta-catenin and enhanced apoptosis to promote colon tumor cell death.
Liu
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Cell Pathways, Inc., Horsham, Pennsylvania 19044, USA. lliu@cellpathways.com
Li
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H
Underwood
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T
Lloyd
M
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David
M
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Sperl
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Pamukcu
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Thompson
W J
WJ
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D003598:Cytoskeletal Proteins / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology*; D005768:Gastrointestinal Hormones; D006801:Humans; D009154:Mutation; D045265:Natriuretic Peptides; D010455:Peptides / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis; D010766:Phosphorylation; D011863:Radioimmunoassay; D013467:Sulindac / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015534:Trans-Activators; D014407:Tumor Cells, Cultured; D051176:beta Catenin
C439285:(5-fluoro-2-methyl-1-(4-pyridyl)methylene-3-(N-benzyl)-indene)-acetamide hydrochloride; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D020536:Enzyme Activators; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010455:Peptides; D015534:Trans-Activators; D051176:beta Catenin; C072685:guanylin; D013467:Sulindac; D000242:Cyclic AMP; D017869:Cyclic GMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP; C025463:sulindac sulfone