Research

Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

Anna Tesei¹ , Marco Rosetti¹ , Paola Ulivi¹ , Francesco Fabbri¹ , Laura Medri² , Ivan Vannini¹ , Manlio Bolla³ , Dino Amadori¹ and Wainer Zoli¹

¹  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy

²  Pathology Unit, Morgagni-Pierantoni Hospital, Forlì, Italy

³  NicOx SA, Sophia-Antipolis, France

author email corresponding author email

Journal of Translational Medicine 2007, 5:52doi:10.1186/1479-5876-5-52

Published:	30 October 2007

Abstract

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 (ΔΨ_m) 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 ΔΨ_m 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.