title: Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53.
abstract: Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O(2)-(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 IC(50) of approximately 2 microM, 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.
authors.affiliation: Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD, USA.
authors.forename: J
authors.initials: J
authors.lastname: Kitagaki
authors.forename: Y
authors.initials: Y
authors.lastname: Yang
authors.forename: J E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: N H
authors.initials: NH
authors.lastname: Colburn
authors.forename: L K
authors.initials: LK
authors.lastname: Keefer
authors.forename: A O
authors.initials: AO
authors.lastname: Perantoni
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001391:Azo Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology* / Q000627:therapeutic use; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D001391:Azo Compounds; C474958:O(2)-(2,4-dinitrophenyl) 1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate; D010879:Piperazines; D011355:Prodrugs; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D009569:Nitric Oxide; D044764:Ubiquitin-Activating Enzymes