Abstract 1487: Nitric oxide-releasing NSAIDs suppress NF-κB signaling and increase caspase-3 enzyme activity in vitro and in vivo through S-nitrosylation
Authors: Chattopadhyay Mitali , Goswami Satindra , Kodela Ravinder , Boring Daniel , Crowell James A. , Kashfi Khosrow 
Published: February 2011

Abstract
Introduction: Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) consisting of a conventional NSAID to which an NO releasing moiety -ONO2 is covalently attached through a spacer have emerged as a new class of pharmaceutical agents. The released NO has proapoptotic effects which may cause DNA damage, leading to p53 activation, proteasome inhibition, and/or cytochrome c release from mitochondria, resulting from activation of the mitochondrial permeability transition pore or damage to mitochondrial membrane phospholipids. NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which has emerged as an important posttranslational protein modification based on prototypic redox mechanisms in signal transduction. The transcription factor, NF-κB is constitutively expressed in many tumors and characterizes all inflammatory responses. As apoptosis is a dominant anti-tumor pathway for chemotherapy-induced cell death, NF-κB may also be involved in resistance to cancer chemotherapy. Caspase-3 activation is intimately involved in apoptosis. In this study we evaluated the actions of meta-NO-aspirin (m-NO-ASA) and NO-naproxen which inhibit cell growth and induce apoptosis on NF-κB expression and caspase-3 activity and determined whether these agents S-nitrosylate these proteins.
Methods: m-NO-ASA and NO-naproxen synthesized by us. Cell line: HT-29 human colon cancer cells. Growth inhibition: MTT. Animal studies: Rats (5 per group) were gavaged with ASA (180 mg/kg) or naproxen (40 mg/kg) or equimolar amounts of NO-ASA or NO-naproxen and killed 6h later after which their stomachs were rapidly removed, gently washed, and frozen in liquid nitrogen. Caspase-3 activity: commercial kit. Protein S-nitrosylation: Biotin switch assay (Jaffrey et al, Nature Cell Biology, 2001, 3:193-197); NF-κB, caspase-3, TNF-α, and biotinylated protein levels: immunoblotting. We also used the NO-donor SNAP as a positive control.
Results: In vitro: m-NO-ASA and NO-naproxen inhibited the growth of HT29 colon cancer cells with IC50s of 187 ± 10 µM and 97 ± 5 µM at 24 hr respectively. NF-κB expression was reduced, however, caspase-3 enzyme activity and protein levels were increased by these agents in a dose- and time-dependent manner. NO-NSAID treatment for 24 hr at 0.5 x IC50, IC50 and 2 x IC50 caused an increase in both the total S-nitrosylated proteins and S-nitrosylated NF-κB (p-65 protein) and caspase-3 expression. These effects were reversed by carboxy-PTIO (an NO scavenger). In vivo: NO-NSAIDs caused an increase in total S-nitrosylated proteins and an increase in S-nitrosylated NF-κB, and TNF-α protein levels.
Conclusions: These data strongly suggest S-nitrosylation as a potential molecular mechanism for the observed effects of NO-NSAIDs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1487.