BACKGROUND: Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in in vitro human colon cancer models. METHODS: The effect on tumor growth was evaluated in four human colon cancer cell lines (LoVo, LRWZ, WiDr and LoVo Dx) by sulforhodamine B assay, oxidative stress by immunohistochemistry, apoptosis by laddering assay, mitochondrial membrane potential (DeltaPsim) by flow cytometry, and apoptosis- and chemoresistance-related markers by western-blot and real-time method, respectively. Prostaglandin E2 levels were determined by ELISA. RESULTS: NCX 4040 produced a higher cytotoxic effect in all the cell lines than that produced by other NO donors tested. In particular, in LoVo and LRWZ cells, NCX 4040 induced a cytocidal effect and apoptosis through p53 and NAG-1 expression, an early DeltaPsim collapse, and a sequential release of cytoplasmatic cytochrome c and caspase -9 and -3 active forms. 8-hydroxyguanine lesions, indicative of oxidative stress, were also observed. Conversely, in WiDr line, the drug caused a cytocidal effect, albeit not through apoptosis, and a concomitant increase in COX-2 activity. In LoVo Dx line, characterized by high levels drug resistance and DNA repair-related markers, only a cytostatic effect was observed, again in concomitance with the increase in COX-2 enzyme activity. CONCLUSION: This study highlights the multiplicity of mechanisms involved in sensitivity or resistance to NCX 4040 and could provide useful indications for tailored therapy by identifying potentially drug-responsive tumors.