Abstract 4610: NO-NSAIDs inhibit colon tumor cell growth by a cGMP-independent mechanism

Abstract

Abstract Nitric oxide-releasing NSAIDs (NO-NSAIDs) were originally developed as prodrugs to reduce the gastrointestinal toxicity of conventional NSAIDs and were found to have improved potency to inhibit tumor growth compared with the parent NSAID. Although different mechanisms have been proposed for their potent anticancer properties, the role of the NO-moiety has not been clearly defined. We have investigated whether the NO group contributes to the anticancer properties of NO-NSAIDs by inducing soluble guanylyl cyclase and elevating intracellular cGMP levels, effects that are associated with the activity of NO donors. We confirmed the increased potency of the NO-derivatives of sulindac, aspirin (o-, p- and m- isomers), exisulind and naproxen with respect to the parent compounds for inhibiting growth of HCT116, SW480 and COLO741 human colon tumor cells. The two derivatives with the lowest IC50 values, NO-sulindac (16.50 µM) and NO-ASA(o) (8 µM) were also tested for cGMP elevation using a novel cGMP biosensor assay in live cells. In brief, HEK293 cells were transfected with a biosensor construct containing cGMP binding domains fused to the modified firefly luciferase gene (Promega) which produces bioluminescence in the cGMP-bound or ‘activated’ state. Cells were treated with NO-Sulindac, NO-ASA(o) and known NO-donors, NOR-3 and SNP as positive controls. Chemiluminescence was measured over a period of 1h. A strong and sustained signal was obtained with the NO-donors, SNP and NOR-3 (50 µM), although no induction was observed with NO-NSAID treatment (50 µM). In addition, SW480 colon tumor cells were treated with 50 µM NO-Sulindac, NO-ASA(o) or NOR-3 and levels of phosphorylated vasoactivator stimulated phosphoproteins (p-VASP), an intracellular marker of cGMP signaling activity, were measured by Western Blotting. NOR-3 caused a time-dependent increase in p-VASP expression, whereas the NO-NSAIDs had no such effect. Since sulindac and certain derivatives have previously been reported to inhibit cGMP-phosphodiesterase (PDE) and inhibit tumor cell growth by a mechanism involving cGMP elevation, we also determined if NO-NSAIDS can inhibit cGMP PDE activity. NO-NSAIDs did not inhibit cGMP hydrolysis by either recombinant PDE5 or in lysates from colon tumor cells, although sulindac sulfide was an effective inhibitor as previously reported. These results suggest that mechanisms other than cGMP elevation explain the anticancer properties of NO-NSAIDs. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2011-4610