JS-K, a nitric oxide-releasing prodrug, modulates ÃŸ-catenin/TCF signaling in leukemic Jurkat cells: evidence of an S-nitrosylated mechanism
Nath, Niharika; Chattopadhyay, Mitali; Pospishil, Liliya; Cieciura, Lucyna Z; Goswami, Satindra; Kodela, Ravinder; Saavedra, Joseph E; Keefer, Larry K; Kashfi, Khosrow
Î²-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/Î²-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 14Â±0.7 and 9Â±1.2Î¼M at 24 and 48h, respectively. Treatment of the cells with JS-K for 24h, caused a dose-dependent increase in apoptosis from 16Â±3.3% at 10Î¼M to 74.8Â±2% at 100Î¼M 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 24h. However, between 2 and 8h, LDH release was less than 20% for any indicated JS-K concentration. The Î²-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32Â±8, 63Â±5, and 93Â±2% at 2, 10, and 25Î¼M JS-K, respectively, based on luciferase reporter assays. JS-K reduced nuclear Î²-catenin and cyclin D1 protein levels, but cytosolic Î²-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 Î²-catenin was determined to precede its degradation. A comparison of the S-nitrosylated nuclear Î²-catenin to the total nuclear Î²-catenin showed that Î²-catenin protein levels were degraded at 24h, while S-nitrosylation of Î²-catenin occurred earlier at 0-6h. The NO scavenger PTIO abrogated the JS-K mediated degradation of Î²-catenin demonstrating the need for NO.