NCX4016 (NO-Aspirin) has multiple inhibitory effects in LPS-stimulated human monocytes.

NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A(2) metabolite TXB(2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 - 1000 micromol l(-1) dose-dependently reduced TXB(2) concentration, measured by RIA in the supernatant of 10 microg ml(-1) LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 micromol l(-1): -86.0+/-10.1%, NCX4016 300 micromol l(-1): -92.2+/-9.0%, ASA 30 micromol l(-1): -92.3+/-7.5%, ASA 300 micromol l(-1): -97.3+/-1.0%, n=6, M+/-s.d.). Most of the activity of NCX4016 up to 100 micromol l(-1) was prevented by 10 micromol l(-1) ODQ, inhibitor of cyclic GMP. NCX4016 100 - 300 micromol l(-1) reduced TNF-alpha (NCX4016 300 micromol l(-1)=-77.2+/-19.9%, n=6) and IL-6 (NCX4016 300 micromol l(-1): -61.9+/-15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 micromol l(-1): 53.7+/-39.9%, n=4) and immunoreactive TF (NCX4016 300 micromol l(-1): -93.9+/-7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA(2) generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

BACKGROUND: Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. AIM: To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. METHODS: Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). RESULTS: In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. CONCLUSIONS: NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.

New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma.

The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer.

IL-1 beta converting enzyme is a target for nitric oxide-releasing aspirin: new insights in the antiinflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs.

Caspase-1, the IL-1beta converting enzyme (ICE), is required for intracellular processing/maturation of IL-1beta and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40-80% inhibition of IL-1beta, IL-8, IL-12, IL-18, IFN-gamma, and TNF-alpha release with an EC(50) of 10-20 microM for IL-1beta and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E(2) generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-D-L-penicillamine, inhibited caspase-1 activity with an EC(50) of approximately 20 microM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60-80% reduction of IL-1beta, IL-8, IFN-gamma, and TNF-alpha release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1beta and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.