The H2S releasing naproxen derivative, ATB-346, reduces human colon and lung cancer cell viability more potently than its parent compound.: implications for improved efficacy of cyclo-oxygensae inhibitors as chemo preventative agents.

C Cheadle1, NS Kirkby1, M Chan2,3, HH Gashaw1, DM Reed1, JL Wallace2, JA Mitchell1, MJ Paul-Clark1. 1Dept. of Cardiothoracic Pharmacology, National Heart and Lung Institute, Imperial College London, London, UK, 2Dept of Pharmacology, University of Toronto, Toronto, Canada, 3William Harvey Research Institute, Queen Mary University of London, London, UK

Introduction: Colorectal cancer (CRC) is a major cause of mortality, responsible for 600,000 deaths worldwide. The process of tumourgenesis for this carcinoma is well understood but improved therapies are increasingly required. Mutations in the adenomatous polyposis coli (APC) gene are major driving factors in tumour development. Cyclo-oxygenase (COX)2 is also associated with cancer, with 86% of the carcinomas expressing elevated levels of COX 2 mRNA (1). In line with this COX-2 inhibitors reduce the advancement of colorectal cancer and protect against its reoccurrence (2). However, long-term use of COX inhibitors is associated with gastrointestinal (GI) and cardiovascular side effects. To over come this problem the COX inhibitor naproxen, which has a relatively low cardiovascular risk but serious GI toxicity, has been modified to include a H2S releasing moiety (ATB-346) in order to protect the gut (3). However the potential chemo preventive properties of naproxen or ATB-346 are not fully understood. Here we investigated the effects of ATB-346 versus authentic naproxen on viability of two human cancer cell lines.

Methods: Caco-2, a human CRC cell line with an APC mutation, and A549, a human lung epithelial carcinoma cell line, were used in these experiments. Caco-2 cells were seeded at 3x103 cells/well and A549 cells were seeded at 10x103 cells/well in 96 well plates. Cells were grown for 24 to 72h in the presence and absence of naproxen, ATB-346 or vehicle (0.003% DMSO; 0.012% ethanol; 0.0075% chremaphor in water). Cell viability was assessed using an AlamarBlue reagent. COX-2 activity was induced in A549 cells by co-treatment with IL-1β (1ng/ml) and subsequent PGE2 measured using ELISA.

Results: ATB-346 was more effective at reducing cell viability of Caco-2 or A549 cells than the parent compound naproxen (Figure 1). However, whilst both compounds inhibited COX-2 activity (PGE2 production) in a concentration dependent manner, naproxen was more potent than ATB-346 (Figure 1).

Figure 1. (A) Effects naproxen versus ATB-346 on viability of Caco-2 (A) and A549 (B) cells and on COX-2 activity in A549 cells (C). Data are mean± SEM for (n=3). *p≤ 0.05 one-way ANOVA with Dunnett’ s post-hoc test; #p≤0.05 two-way ANOVA.

Conclusion: Naproxen and ATB-346 inhibit COX-2 and reduce viability of human colon cancer and lung cancer cell lines. ATB-346 is more potent than naproxen at inhibiting viability but less potent at inhibiting COX-2. These observations suggest that ATB-346 may have anti-cancer properties that are independent of its ability to inhibit COX-2, possibly via a H2S mediated mechanism.

References:

(1) Eberhart CE et al. (1994). Gasteroenterology, 107: 1183-88.

(2) Baron JA et al. (2006). Gasteroenterology, 113: 1674-82.

(3) Wallace JL et al (2007). Gasteroenterology, 115: 101-9.