JS-K; a nitric oxide-releasing prodrug, modulates beta-catenin/TCF signaling in leukemic Jurkat cells: Evidence of an S-nitrosylated mechanism

beta-Catenin is a central player of the Wnt signaling pathway that regulates cell-cell adhesion and may promote leukemia cell proliferation We examined whether JS-K, an NO-donating prodrug, modulates the Wnt/beta-catenin/TCF-4 signaling pathway in Jurkat T-Acute Lymphoblastic Leukemia cells JS-K inhibited Jurkat T cell growth in a concentration and time-dependent manner. The IC(50)s for cell growth inhibition were 14 +/- 0.7 and 9 +/- 1.2 mu M at 24 and 48 h, respectively. Treatment of the cells with JS-K for 24 h, caused a dose-dependent increase in apoptosis from 16 +/- 3.3% at 10 mu M to 74.8 +/- 2% at 100 mu M and a decrease in proliferation. This growth inhibition was also due, in part, to alterations in the different phases of the cell cycle JS-K exhibited a dose-dependent cytotoxicity as measured by LDH release at 24 h. However, between 2 and 811, LDH release was less than 20% for any indicated JS-K concentration. The beta-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32 +/- 8, 63 +/- 5, and 93 +/- 2% at 2, 10, and 25 mu M JS-K. respectively, based on luciferase reporter assays. JS-K reduced nuclear beta-catenin and cyclin D1 protein levels, but cytosolic beta-catenin expression did not change. Based on a time-course assay of S-nitrosylation of proteins by a biotin switch assay. S-nitrsolyation of nuclear beta-catenin was determined to precede its degradation A comparison of the S-nitrosylated nuclear beta-catenin to the total nuclear beta-catenin showed that beta-catenin protein levels were degraded at 24 h. while S-nitrosylation of beta-catenin occurred earlier at 0-6 h. The NO scavenger PTIO abrogated the JS-K mediated degradation of beta-catenin demonstrating the need for NO (C) 2010 Elsevier Inc. All rights reserved

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