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


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Abstract

Graphical abstractβ-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/β-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 IC50s for cell growth inhibition were 14 ± 0.7 and 9 ± 1.2 μM 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 μM to 74.8 ± 2% at 100 μM 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 8 h, LDH release was less than 20% for any indicated JS-K concentration. The β-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32 ± 8, 63 ± 5, and 93 ± 2% at 2, 10, and 25 μM JS-K, respectively, based on luciferase reporter assays. JS-K reduced nuclear β-catenin and cyclin D1 protein levels, but cytosolic β-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 β-catenin was determined to precede its degradation. A comparison of the S-nitrosylated nuclear β-catenin to the total nuclear β-catenin showed that β-catenin protein levels were degraded at 24 h, while S-nitrosylation of β-catenin occurred earlier at 0–6 h. The NO scavenger PTIO abrogated the JS-K mediated degradation of β-catenin demonstrating the need for NO.

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