NCX 4040, a Nitric Oxide-Donating Aspirin, Exerts Anti-Inflammatory Effects through Inhibition of IκB-α Degradation in Human Monocytes
Emanuela Ricciotti*†,1,2, Melania Dovizio*†,1, Luigia Di Francesco*†, Paola Anzellotti*†, Tania Salvatore*†, Andrea Di Francesco*†, Maria G. Sciulli*†, Giuseppa Pistritto‡, Angela Monopoli§ and Paola Patrignani*†
+ Author Affiliations
*Department of Medicine and Center of Excellence on Aging, School of Medicine, “G. d’Annunzio” University and
†“G. d’Annunzio” University Foundation, Centro Scienze dell’Invecchiamento, Chieti;
‡Department of Neuroscience, University of Rome Tor Vergata Medical School, Rome; and
§NicOx Research Institute, Bresso, Milan, Italy
Address correspondence and reprint requests to Prof. Paola Patrignani, Department of Medicine, Center of Excellence on Aging, School of Medicine, Centro Scienze dell’Invecchiamento, “G. d’Annunzio” University, Via dei Vestini, 31, 66100 Chieti, Italy. E-mail address: firstname.lastname@example.org
↵2 Current address: Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA.
↵1 E.R. and M.D. contributed equally to this work.
NO-donating aspirins consist of aspirin to which a NO-donating group is covalently linked via a spacer molecule. NCX 4040 and NCX 4016 are positional isomers with respect to the ‑CH2ONO2 group (para and meta, respectively) on the benzene ring of the spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins, we aimed to compare their anti-inflammatory effects with those of aspirin in vitro. Thus, we assessed their impacts on cyclooxygenase-2 activity (by measuring PGE2 levels), protein expression, and cytokine generation(IL-1β, IL-18, TNF-α, and IL-10) in human whole blood and isolated human monocytes stimulated with LPS. Interestingly, we found that micromolar concentrations of NCX 4040, but not NCX 4016 or aspirin, affected cyclooxygenase-2 expression and cytokine generation. We compared the effects of NCX 4040 with those of NCX 4016 or aspirin on IκB-α stabilization and proteasome activity in the LPS-stimulated human monocytic cell line THP1. Differently from aspirin and NCX 4016, NCX 4040, at a micromolar concentration range, inhibited IκB-α degradation. In fact, NCX 4040 caused concentration-dependent accumulation of IκB-α and its phosphorylated form. This effect was not reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent cGMP generation. In contrast, IκB-α accumulation by NCX 4040 may involve an inhibitory effect on proteasome functions. Indeed, NCX 4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate supernatants, thus suggesting an indirect inhibitory effect. In conclusion, NCX 4040 is an inhibitor of IκB-α degradation and proteasome function, and it should be taken into consideration for the development of novel anti-inflammatory and chemopreventive agents.
This work was supported by grants from NicOx and the European Community’s Sixth Framework Program (Eicosanox, LSMH-CT-2004-00503) to P.P.