Introduction Colonic DUOX2 expression produces hydrogen peroxide, a reactive oxygen species (ROS), which is up-regulated in active ulcerative colitis (UC). Overproduction of hydrogen peroxide amplifies ROS-induced genetic damage and causes cellular transformation which may explain the increased colorectal cancer (CRC) risk associated with chronic UC. Mesalazine (5-ASA) has been shown to be chemo-preventative for UC associated CRC and scavenges ROS. Here, we aimed to identify and investigate the effect of 5-ASA on DUOX2 expression using human rectal cancer cell lines and mucosal tissue biopsies.
Methods Mucosal biopsies were taken from 35 patients with UC and 24 patients with normal colons for in vivo experiments, and 24 patients with UC and 14 patients with normal colons for ex vivo experiments. Total RNA was extracted and quantitative real-time PCR used to calculate expression of DUOX2. Cytometric bead array technology was used on ex vivo culture supernatants to measure cytokine profiles. In situ hybridisation for DUOX2 expression was performed on sections from eight matched pairs of non-inflamed/inflamed biopsies and five matched pairs of non-inflamed/inflamed/dysplasia biopsies from UC patients. Human rectal cancer cells were used for quantitative real-time PCR, with multiparameter flow cytometry utilised to measure hydrogen peroxide levels, apoptosis, DNA damage and cell proliferation.
Results DUOX2 is expressed throughout the colonic epithelium, is upregulated in active compared to quiescent ulcerative colitis and also in areas of UC associated dysplasia. In the setting of intestinal inflammation, but not in quiescent disease, 5-ASA enhances DUOX2 expression in vivo and ex vivo. As expected, 5-ASA was found to suppress cytokine (IL-6 and IL-8) production during an inflammatory flare and to maintain low cytokine levels during remission. The addition of 5-ASA in vitro led to upregulation of DUOX2 and elevated levels of hydrogen peroxide, DNA damage and apoptosis. These effects were further enhanced in a setting of hypoxia.
Conclusion We have shown that 5-ASA over stimulates DUOX2 expression in the setting of inflammation and hypoxia, but not in quiescent disease. Importantly, this suggests that during a flare 5-ASA could act as a carcinogen rather than a chemo-preventative drug. Further investigations to confirm the functional relevance of DUOX2 up-regulation in the colonic mucosa of patients with active UC is indicated.
Competing interests T Macfie Grant/Research Support from: SHIRE, A Parker: None declared, A Nijhuis: None declared, T Boitsova: None declared, N Suraweera: None declared, R Jeffery: None declared, R Poulsom: None declared, J Lindsay Grant/Research Support from: SHIRE, A Silver: None declared.
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