Cyclooxygenase-2-dependent and -independent inhibition of proliferation of colon cancer cells by 5-aminosalicylic acid

doi:10.1016/j.bcp.2007.09.020

Biochemical Pharmacology

Volume 75, Issue 3, 1 February 2008, Pages 668-676

https://doi.org/10.1016/j.bcp.2007.09.020 Get rights and content

Abstract

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway may have a pathogenic role in colorectal cancer (CRC). Recent studies suggest that 5-aminosalycilic acid (5-ASA) reduces the risk of inflammatory bowel disease-related CRC, but the mechanism by which 5-ASA interferes with CRC cell growth remains unknown. In this study, we have examined whether the negative effect of 5-ASA on CRC cells is dependent on COX-2/PGE2 axis inhibition. We show that 5-ASA down-regulates both constitutive and TNF-α or IL-1β-induced COX-2 in HT-115 and HT-29 cells. Inhibition of COX-2 by 5-ASA occurs at the RNA and protein level, and is associated with a significant decrease in PGE2 synthesis, arrest of growth and enhanced death of CRC cells. However, exogenous PGE2 does not revert the 5-ASA-mediated CRC cell proliferation block. 5-ASA also inhibits the growth of DLD-1, a COX-deficient CRC cell line, thus suggesting that the anti-proliferative effect of 5-ASA on CRC cells is not strictly dependent on the inhibition of COX-2/PGE2. Taken together our data indicate that 5-ASA causes both a COX-2-dependent and -independent inhibition of CRC cell growth.

Introduction

Patients with inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) have an increased risk for the development of colorectal cancer (CRC), that is influenced in part by the duration and anatomical extent of the disease, as well as by the severity of the ongoing inflammation [1]. Present CRC management techniques in IBD patients include surveillance colonoscopy and/or colectomy, but such strategies have not yet been proven to reduce mortality [1]. The development of safe and effective chemopreventive measures for reducing the risk of CRC would thus be of substantial benefit to IBD patients.

Several experimental animal studies, large retrospective and prospective population-based studies indicate that regular intake of non-steroidal anti-inflammatory drugs (NSAIDs) may reduce the risk of developing polyps and sporadic CRC, and may induce the regression of adenomas in familial adenomatous polyposis (FAP) [2], [3], [4], [5], [6], [7], [8]. A major molecular target for CRC chemoprevention by these agents is the cyclooxygenase (COX) [2], [3]. COX transforms arachidonic acid into prostaglandin (PG)G2, an unstable intermediate which is rapidly converted to PGH2. Subsequently, PGH2 is metabolized into different structurally related PG, including PGE2, PGD2, PGF2, PGI2 and thromboxane A2 [9]. COX-1 is the constitutive isoform of this enzyme, and is expressed in a wide range of mammalian tissues. It functions as a housekeeping gene that controls the production of prostacyclins, PG and tromboxane, which are essential for physiological functions, such as protection of gastric mucosa, platelet aggregation and dynamics of the renal microvasculature. In contrast, COX-2 is constitutively expressed in human kidney and brain, and it is inducible by inflammatory cytokines, growth factors, oncogenes, serum and tumour promoters in several cell types [9], [10]. COX-1 expression remains unaltered in CRC, whereas increased levels of COX-2 have been seen in 50% of colorectal adenomas and in up to 85% of sporadic CRC [10], [11]. Evidence also suggests that COX-2 could play a role in the pathogenesis of CRC complicating the natural history of patients with IBD [12], [13].

The clinical relevance of these observations relates to the demonstration that specific pharmacological inhibition or genetic ablation of COX-2 can prevent the development of CRC and can even cause regression of existing adenomatous polyps in both humans and rodents [3], [14]. Moreover, blockade of COX-2 associates with a reduced frequency of colitis-driven CRC in mice [13]. It has been postulated that the antitumor effect of NSAIDs, including COX-2 inhibitors, is mediated through reduction in PG production, most notably PGE2, which is frequently overexpressed in CRC tissues [2], [3]. Nonetheless, more recent studies suggest that the role of COX-2 in the pathogenesis of CRC may be more complex than that indicated by initial studies. In fact, various COX-independent targets of NSAIDs have been described, as well as there is evidence that some agents that induce COX-2 are chemopreventive [15], [16]. Unfortunately, the wide spread use of NSAIDs and COX-2 inhibitors in the chemoprevention of CRC has been limited by their frequent and often severe side effects [17], [18]. Moreover, because NSAIDs may aggravate the symptoms of colitis, their sustained use for the purpose of cancer chemoprevention is relatively contraindicated in IBD patients [19]. Thus, there is a need to identify alternative chemopreventive agents that are more appropriate for use in IBD patients.

Mesalazine is a 5-aminosalicylic acid (5-ASA) compound largely used for maintaining remission, as well as in the treatment of mildly flare-ups in IBD. 5-ASA is safe and free of serious adverse effects. Recent epidemiological studies suggest that long-term consumption of 5-ASA reduces the risk of CRC developing in patients with IBD [12], [20], [21]. Additionally, several experimental studies have shown that 5-ASA markedly reduces the growth and survival of CRC cells [22], [23], [24], [25]. In this context, we have previously shown that 5-ASA inhibits epidermal growth factor receptor (EGFR) activation, a transmembrane tyrosine kinase which triggers mitogenic signalling in CRC cells [26]. Moreover, Bos et al. showed that 5-ASA affects the Wnt/β-catenin pathway in CRC cells via the inhibition of the phosphatase 2A, and degradation of β-catenin [27]. Finally, Rousseaux et al. demonstrated that 5-ASA interacts with and enhances the expression and activation of PPAR-γ, a negative regulator of colonic inflammation and cancer [28]. Despite these advances, the precise mechanisms by which 5-ASA inhibits CRC cell growth have not been fully established. In this study, we have analyzed if 5-ASA inhibits COX-2/PGE2 axis in CRC cells and evaluated whether the regulatory effects of 5-ASA on CRC cells are strictly dependent on the control of this pathway.

Section snippets

Cell culture

5-ASA was kindly provided by Giuliani S.p.A. (Milan, Italy) and dissolved as a 100 mM stock solution in culture medium. The pH of the drug solution was adjusted to 7.4 with NaOH, and experiments carried out protected from light. All reagents were from Sigma–Aldrich (Milan, Italy) unless specified. The human CRC cell lines, HT-29 and DLD1, were maintained in McCoy's 5A and RPMI 1640 medium, respectively, both supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 50 μg/ml

5-ASA dose-dependently inhibits the growth and induces apoptosis in HT-115

COX-2 is over-expressed in the vast majority of CRC, and inhibition of COX-2 in CRC cells results in a decreased growth and survival [2], [6], [7], [8]. Therefore, COX-2 may represent an important target for CRC chemoprevention. However, there is evidence that various NSAIDs, including selective COX-2 inhibitors, can regulate CRC cell activity by modulating also COX-2-independent pathways [15], [29]. As a starting point for our studies, we evaluated whether 5-ASA regulates the proliferation and

Discussion

This study was undertaken to examine whether the ability of 5-ASA to regulate CRC cell growth and death is strictly dependent on the inhibition of COX-2, the inducible form of COX, whose expression is up-regulated in the inflamed gut of patients with IBD [35] and in the majority of neoplastic lesions of the colon [11]. We first show that 5-ASA inhibits the growth of HT-115, a CRC cell line that expresses a functionally active COX-2, and this anti-mitogenic effect of 5-ASA is associated with a

Acknowledgements

The study was supported by the Fondazione “Umberto Di Mario”, Rome and Associazione Italiana per la Ricerca sul Cancro and Giuliani SpA, Milan, Italy.

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¹: These authors contributed equally to the study.

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Cyclooxygenase-2-dependent and -independent inhibition of proliferation of colon cancer cells by 5-aminosalicylic acid

Abstract

Introduction

Section snippets

Cell culture

5-ASA dose-dependently inhibits the growth and induces apoptosis in HT-115

Discussion

Acknowledgements

Lancet

Semin Oncol

Gastroenterology

Cell

Biochem Pharmacol

Clin Gastroenterol Hepatol

Eur J Cancer

J Biol Chem

Prostaglandins Leukot Essent Fatty Acids

Gastroenterology

Cell Signal

Eur J Pharmacol

Gastroenterology

J Biol Chem

Gastroenterology

Review article: colorectal carcinoma and inflammatory bowel disease

Aliment Pharmacol Ther

Use of nonsteroidal anti-inflammatory drugs and incidence of colorectal cancer: a population-based study

Arch Intern Med

Celecoxib for the prevention of colorectal adenomatous polyps

N Engl J Med

Celecoxib for the prevention of sporadic colorectal adenomas

N Engl J Med

The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis

N Engl J Med

Cyclooxygenases 1 and 2

Annu Rev Pharmacol Toxicol

Cyclooxygenase in biology and disease

FASEB J