[CANCER RESEARCH 61, 1733–1740, February 15, 2001] Chemoprevention of Intestinal Polyposis in the Apc 716 Mouse by Rofecoxib, a Specific Cyclooxygenase-2 Inhibitor Masanobu Oshima, 1 Naomi Murai(Hata), 1 Stacia Kargman, 1 Meztli Arguello, Pauline Luk, Elizabeth Kwong, Makoto M. Taketo, and Jilly F. Evans 2 Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd. (Merck), Tsukuba 300-2611, Japan [M. O., N. M. H.]; Merck Frosst Center for Therapeutic Research, Pointe-Claire Dorval, Quebec, Canada H9R 4P8 [S. K., M. A., P. L., E. K.]; University of Tokyo, Graduate School of Pharmaceutical Sciences, Laboratory of Biomedical Genetics, Tokyo 113-0033 Japan [M. M. T.]; and Merck & Co., Inc., West Point, Pennsylvania 19486 [J. F. E.] ABSTRACT Mutations in the human adenomatous polyposis (APC) gene are causa- tive for familial adenomatous polyposis (FAP), a rare condition in which numerous colonic polyps arise during puberty and, if left untreated, lead to colon cancer. The APC gene is a tumor suppressor that has been termed the “gatekeeper gene” for colon cancer. In addition to the 100% mutation rate in FAP patients, the APC gene is mutated in >80% of sporadic colon and intestinal cancers. The Apc gene in mice has been mutated either by chemical carcinogenesis, resulting in the Min mouse Apc 850 , or by het- erologous recombination, resulting in the Apc 716 or Apc 1368 mice (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482– 4486, 1995). Although homozygote Apc / mice are embryonically lethal, the heterozygotes are viable but develop numerous intestinal polyps with loss of Apc heterozy- gosity within the polyps (M. Oshima et al., Proc. Natl. Acad. Sci. USA, 92: 4482– 4486, 1995). The proinflammatory, prooncogenic protein cyclooxy- genase (COX)-2 has been shown to be markedly induced in the Apc 716 polyps at an early stage of polyp development (M. Oshima et al., Cell, 87: 803– 809, 1996). We demonstrate here that treatment with the specific COX-2 inhibitor rofecoxib results in a dose-dependent reduction in the number and size of intestinal and colonic polyps in the Apc 716 mouse. The plasma concentration of rofecoxib that resulted in a 55% inhibition of polyp number and an 80% inhibition of polyps >1 mm in size is compa- rable with the human clinical steady-state concentration of 25 mg rofe- coxib (Vioxx) taken once daily (A. Porras et al., Clin. Pharm. Ther., 67: 137, 2000). Polyps from both untreated and rofecoxib- or sulindac-treated Apc 716 mice expressed COX-1 and -2, whereas normal epithelium from all mice expressed COX-1 but minimal amounts of COX-2. Polyps from either rofecoxib- or sulindac-treated mice had lower rates of DNA repli- cation, expressed less proangiogenic vascular endothelial-derived growth factor and more membrane-bound -catenin, but showed unchanged nuclear localization of this transcription factor. This study showing the inhibition of polyposis in the Apc 716 mouse suggests that the specific COX-2 inhibitor rofecoxib (Vioxx) has potential as a chemopreventive agent in human intestinal and colon cancer. INTRODUCTION The majority of epidemiological studies that included the use of NSAIDs 3 as a risk factor have demonstrated that constant use of NSAIDs is associated with a significantly reduced risk of colon cancer (1). Although each NSAID has unique physical properties and phar- macokinetics, the mechanism of action common to all at clinically achievable drug concentrations is the inhibition of COX enzymatic conversion of the polyunsaturated fatty acid arachidonic acid to PGG 2 (2). PGG 2 is converted to protaglandin H 2 by the peroxidase activity of the COX enzyme, and then PGG 2 may be converted to one of several of the five biologically active prostanoids, PGE 2 , prosta- glandin D 2 , prostaglandin F 2 , prostacyclin, or thromboxane (3). Elevated PGE 2 has been measured in rodent and human colonic tumors, and the inhibition of prostaglandin synthesis by NSAID treatment has been shown to inhibit tumor growth in animal models (4 – 6). On the basis of such observations, the NSAID sulindac was studied in FAP patients for prevention of polyp growth (7). This clinical trial showed that treatment with sulindac decreased polyp number and size, and that when sulindac treatment was stopped, polyp growth recurred (7). In the early 1990s, a second form of COX, termed prostaglandin G/H synthase-2 or, more commonly, COX-2, was identified that was 60% identical to the original COX-1 (8 –10). COX-2 mRNA and protein were highly inducible by inflammatory and growth factors, whereas COX-1 expression was constitutive in most tissues, including the GI tract (8 –11). The discovery of the second COX isoform led to the hypothesis that COX-2-specific inhibitors would be as efficacious as nonspecific COX-1/COX-2-inhibitor NSAIDs with respect to pros- taglandin-mediated pain and inflammation in arthritis, but with a much-improved GI safety margin (12). Two specific COX-2 inhibi- tors, i.e., rofecoxib (Vioxx) and celecoxib (Celebrex), have been shown preclinically and clinically to have comparable efficacy to NSAIDs for relief of pain and inflammation in osteoarthritis, but to have decreased risk of GI damage (13–18). Given the epidemiology of NSAID protection for colon cancer, we and others investigated whether this chemopreventive effect might be specifically through the inhibition of COX-2-produced prostaglan- dins. COX-2 mRNA and protein were shown to be markedly elevated in human colon tumor tissue, whereas COX-1 expression remained the same or decreased (19, 20). COX-2 is also overexpressed in human colonic polyps and in macrophages in intimate contact with these sporadic polyps (21, 22). The growth of human colon tumor cells expressing COX-2 can be inhibited in vitro and in vivo by treatment with COX-2 inhibitors (23, 24). Mechanistic studies have revealed that this growth inhibition results from antiproliferative, proapoptopic, and antiangiogenic effects (23–27). Elevated concen- trations of COX-2 mRNA and protein have now been associated with esophageal, head and neck, breast, lung, prostate, and other cancers, and it has been suggested that COX-2 inhibitors may have benefit in malignancies other than colon cancer (28). A relevant animal model in which to test COX-2 inhibitors for prevention of the polyp precursors of adenocarcinomas is the Apc 716 mouse, which develops hundreds of intestinal polyps from birth through the first 3 months of development (29). Both the genetic deletion of COX-2 expression and pharmacological inhibition with the specific COX-2 inhibitor, MF-tricyclic, have been shown to mark- edly attenuate the number and size of polyps in the Apc 716 mouse (30). The specific COX-2 inhibitor celecoxib (Celebrex) has been shown to decrease polyp number and size in the chemically induced Apc mutant Min mouse (31). In clinical trials in FAP patients, cele- coxib has also shown moderate efficacy, at twice the approved ar- Received 8/8/00; accepted 12/13/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Joint first authors; these authors contributed equally to this work. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Merck & Co., Inc., WP26A-3000, 770 Sumneytown Pike, West Point, PA 19486. Phone: (215) 652-1254; Fax: (215) 993-4007; E-mail: jilly_evans@merck.com. 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; PGG 2 , prostaglandin G 2 ; PGE 2, prostaglandin E 2 ; FAP, familial ade- nomatous polyposis; GI, gastrointestinal; VEGF, vascular endothelial growth factor; HPLC, high-performance liquid chromatography; BrdUrd, bromodeoxyuridine. 1733 Research. on February 21, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from