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

Aliment Pharmacol Ther. 1999 Mar;13(3):421-35. doi: 10.1046/j.1365-2036.1999.00442.x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology*
  • Apoptosis / drug effects*
  • Aspirin / analogs & derivatives
  • Aspirin / pharmacology
  • Caspase Inhibitors*
  • Cell Line
  • Cysteine Proteinase Inhibitors / pharmacology*
  • DNA Fragmentation / drug effects
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Flurbiprofen / analogs & derivatives
  • Flurbiprofen / pharmacology
  • Gastric Mucosa / cytology*
  • Gastric Mucosa / drug effects
  • Gastric Mucosa / enzymology
  • Humans
  • In Situ Nick-End Labeling
  • Male
  • Mice
  • Naproxen / analogs & derivatives
  • Naproxen / pharmacology
  • Nitric Oxide / metabolism*
  • Nitric Oxide / pharmacology
  • Peroxidase / antagonists & inhibitors
  • Peroxidase / metabolism
  • Rats
  • Rats, Wistar


  • Anti-Inflammatory Agents, Non-Steroidal
  • Caspase Inhibitors
  • Cysteine Proteinase Inhibitors
  • naproxen-n-butyl nitrate
  • nitroflurbiprofen
  • Nitric Oxide
  • Naproxen
  • Flurbiprofen
  • Peroxidase
  • nitroaspirin
  • Aspirin