Abstract A35: JS-K, a nitric oxide-donating prodrug, inhibits the growth of leukemic jurkat cells and modulates β-catenin/TCF signaling
Authors: Nath Niharika , Chattopadhyay Mitali , Pospishil Liliya , Cieciura Lucyna , Saavedra Joseph , Keefer Larry , Kashfi Khosrow 
Published: May 2012

A35

Introduction

Leukemia is the most common form of cancer in children and acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality. The deregulation of the Wnt signaling cascade and its components has been implicated in T-cell ALL as well as in B-cell chronic lymphocytic leukemia (CLL). The protein β-catenin is a central player of the Wnt signaling pathway that regulates cell-cell adhesion and may promote leukemia cell proliferation. The stabilization and accumulation of β-catenin have a powerful regulatory role in proliferation and differentiation. β-catenin is expressed in T- ALL cells, tumor lines of hematopoietic origin and primary leukemia cells but is undetectable in normal peripheral blood T cells. Among the leukemic cell lines, β-catenin is expressed in high levels in Jurkat T-cells. Currently, the role of β-catenin and its regulation in non-adherent cells are not very clear. JS-K [O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1yl]diazen-1-ium-1,2-diolate] is a prodrug of the diazeniumdiolate class that releases NO through a reaction catalyzed by GST utilizing glutathione. This agent has been shown to inhibit the growth of various cancer cell lines, and in HL-60 cells (leukemia cell line) it induced apoptosis, and oxidative stress through the induction of the intrinsic pathway. However, the effect of this agent on the Wnt/ β-catenin pathway is essentially unknown. This report highlights the effects of JSK on the growth properties of human Jurkat T-acute lymphoblastic leukemia cells and on the β-catenin signaling pathway.

Methods

JS-K: synthesized by us. Cell cycle phase distribution: flow cytometry; apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms. Transient transfection: 400 ng of luciferase reporter constructs TOPflash or FOPflash and 200 ng of pSV- βgal vector as internal control. Luciferase activity: per manufacturer’s instructions; β-galactosidase activity: standard protocols. β-catenin protein levels: immunoblotting.

Results

JS-K inhibited the growth of Jurkat cells, IC50s of 14 ± 2 µM and 9 ± 1 µM at 24 h and 48 hr, respectively. This effect was due, in part, to a dose-dependent induction of apoptosis as observed by flow cytometry of annexin V stained JS-K treated cells. Jurkat cells constitutively express high levels of β-catenin, which translocates to the nucleus, interacting with TCF-4, modulating transcription of specific genes. In the cell line SW480, which also expresses high levels of β-catenin and is an adherent cell line of colorectal cancer type that transfects with higher efficiency than Jurkat cells, we found that JS-K dose-dependently inhibited β-catenin/TCF-4 signaling by reporter assays. In Jurkat cells, JS-K reduced β-catenin levels in the nucleus and cytoplasm in a dose-dependent manner. JS-K, dose-dependently altered the distribution of the cells in the cell cycle, causing an arrest in G0/G1.

Conclusions

These findings establish a strong inhibitory effect of JS-K in human Jurkat T-acute lymphoblastic leukemia cells, an effect that is modulated through inhibition of Wnt/ β-catenin/TCF-4 signaling and through induction of apoptosis. Our findings underscore the potential therapeutic application of JS-K to leukemias.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):A35.