Abstract
Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) induces apoptotic cell death in vitro and in vivo, indicating that it is a promising new therapeutic for cancer. However, the mechanism of its tumor-killing activity remains unclear. Ubiquitin plays an important role in the regulation of tumorigenesis and cell apoptosis. Our earlier report has shown that inactivation of the ubiquitin system through blocking E1 (ubiquitin-activating enzyme) activity preferentially induces apoptosis in p53-expressing transformed cells. As E1 has an active cysteine residue that could potentially interact with NO, we hypothesized that JS-K could inactivate E1 activity. E1 activity was evaluated by detecting ubiquitin-E1 conjugates through immunoblotting. JS-K strikingly inhibits the ubiquitin-E1 thioester formation in cells in a dose-dependent manner with an IC50 of approximately 2 μM, whereas a JS-K analog that cannot release NO did not affect these levels in cells. Moreover, JS-K decreases total ubiquitylated proteins and increases p53 levels, which is mainly regulated by ubiquitin and proteasomal degradation. Furthermore, JS-K preferentially induces cell apoptosis in p53-expressing transformed cells. These findings indicate that JS-K inhibits E1 activity and kills transformed cells harboring wild-type p53.
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Abbreviations
- E1:
-
ubiquitin-activating enzyme
- NO:
-
nitric oxide
- RPE:
-
Tert-immortalized human retinal pigment epithelial cells
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Acknowledgements
We are grateful to the NIH Fellows Editorial Board for revisions of this paper. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and by National Cancer Institute Contract No. NOI-CO-12400 to SAIC Inc. JK was a fellow of the Japanese Society for the Promotion of Science.
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Kitagaki, J., Yang, Y., Saavedra, J. et al. Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53. Oncogene 28, 619–624 (2009). https://doi.org/10.1038/onc.2008.401
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DOI: https://doi.org/10.1038/onc.2008.401
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