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

Abstract

β-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 IC(50)s for cell growth inhibition were 14±0.7 and 9±1.2μM at 24 and 48h, respectively. Treatment of the cells with JS-K for 24h, caused a dose-dependent increase in apoptosis from 16±3.3% at 10μM to 74.8±2% at 100μM 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 β-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32±8, 63±5, and 93±2% at 2, 10, and 25μM 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 24h, while S-nitrosylation of β-catenin occurred earlier at 0-6h. The NO scavenger PTIO abrogated the JS-K mediated degradation of β-catenin demonstrating the need for NO.

Fig. 1.

Structure of JS-K (O²-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate).

Fig. 2.

Concentration- and time-dependent inhibition of Jurkat T cell growth by JS-K and its cytotoxicity. Cells were treated with various concentrations of JS-K as described in Section 2. (A) Cell numbers were determined at 24 and 48 h from which IC₅₀s for growth inhibition were determined. (B) Cells were treated for 2, 4, 8, and 24 h with various concentrations of JS-K including Triton X-100 as positive control. LDH released from the cells was quantified and cytotoxicity was plotted as a percentage of total LDH released from Triton X-100. Results represent means ± SEM of three different experiments performed in triplicate.

Fig. 3.

JS-K induces apoptosis and inhibits proliferation of Jurkat T cells. Cells were treated with JS-K at the concentration indicated for 24 h after which the cells were stained with annexin and propidium iodide and subjected to flow cytometric analysis as described in Section 2 (Panel A). The percentage of apoptotic cells increased in a concentration-dependent manner (Panel B). Panel C, cells were treated with JS-K at the concentrations indicated for 24 h after which PCNA expression was determined by flow cytometry and expressed as percentage positive cells as described in Section 2. Results are mean ± SEM of three different experiments. *P < 0.05; ^†P < 0.01 compared with untreated cells.

Fig. 4.

Effect of JS-K on cell cycle in Jurkat T cells. Cells were treated for 24 h with various concentrations of JS-K, and their cell cycle phase distribution was determined by flow cytometry, as described in Section 2. Results are representative of two different experiments. This study was repeated twice generating results within 10% of those presented here.

Fig. 5.

Kinetics of NO release and apoptosis. Cells were treated with either 20 or 50 μM JS-K after which apoptosis and NO levels were measured as a function of time. JS-K induced apoptosis in a time- and concentration-dependent manner. The release of NO also increased at 20 μM and 50 μM JS-K as a function of time. Results are mean ± SEM of three different experiments. *P < 0.05 compared to respective untreated cells.

Fig. 6.

Concentration-dependent inhibition of TCF-4 responsive reporter gene by JS-K. Jurkat T cells, cotransfected with luciferase reporter plasmids (TOP or FOP) and the pSV-ßgal as in Section 2 were treated with various concentrations of JS-K for 24 h. Relative TCF activity (fold) of treated cells is shown (DMSO control was set as 1); values are mean ± SEM of three different experiments performed in triplicate. *P < 0.05 compared to DMSO treated control cells.

Fig. 7.

JS-K reduces nuclear β-catenin and cyclin D1 levels. Jurkat T cells treated with increasing concentrations of JS-K for 24 h were analyzed for total, cytosolic, and nuclear β-catenin expression by immunoblot of lysates (panel A, left side). Densitometry evaluation of levels of nuclear β-catenin and α-actin performed from three such immunoblots are shown in panel A, on the right side. *P < 0.01 compared to untreated controls. The protein levels of cyclin D1 were also reduced by JS-K (panel B, left) and as shown for three immunoblot evaluations in panel B, right. ^†P < 0.05 compared to untreated controls. Panel C shows that the reduction in nuclear β-catenin by JS-K was mediated by NO.

Fig. 8.

JS-K induces S-nitrosylation of nuclear proteins detected by the biotin switch assay (BSA). Panel A: In absence of ascorbate, the BSA leads to loss of biotinylation, resulting in no visible bands in the control samples (lane 1). In presence of ascorbate, total lysates from the untreated cells showed basal levels of S-nitrosylation of some proteins (lane 2). Increasing concentrations of JS-K (lanes 3–5) S-nitrosylated several cellular proteins in a dose-dependent manner. Panel B: Increasing concentrations of JS-K increased the total S-nitrosylated content of the nuclear proteins in a dose-dependent manner, at 50 μM JS-K, the % increase was 180 ± 12% compared to control cells (no JS-K in presence of ascorbate). Results are representative of two different experiments.

Fig. 9.

JS-K degrades nuclear β-catenin via NO release. JS-K degraded S-nitrosylated nuclear β-catenin in a concentration-dependent manner (panel A). The cytoplasmic levels of S-nitrosylated α-actin (Fig. 8A lower panel) were used as an internal control for the biotin switch assay, and were not altered by JS-K. Nuclear β-catenin was S-nitrosylated between 0 and 6 h (panel B), where as total nuclear β-catenin protein levels was degraded at 24 h (panel C). Pretreatment with carboxy-PTIO, reversed the effects of JS-K (panel D). Results are representative of two different experiments ± range.