Suppression of cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells by chemopreventive agents with a resorcin-type structure

Michihiro Mutoh 0 2 Mami Takahashi 2 Kazunori Fukuda 1 2 Yuko Matsushima-Hibiya 2 Hiroshi Mutoh 0 2 Takashi Sugimura 2 Keiji Wakabayashi 2 0 Department of Gastroenterology, Institute of Clinical Medicine, Tsukuba University , Tennoudai 1-1-1, Ibaraki 305-0006, Japan 1 Department of Oriental Medicine, Gifu University School of Medicine , 40 Tsukasa-machi, Gifu 500-8705, Japan 2 Cancer Prevention Division, National Cancer Center Research Institute , 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan 3To whom correspondence should be addressed Email: Cyclooxygenase-2 (COX-2) is abundantly expressed in colon cancer cells. It has been reported that inhibition of COX-2 enzyme activity is shown to prevent colon carcinogenesis. Thus, suppression of COX-2 expression may also be an effective chemopreventive strategy. In the present study, we constructed a -galactosidase reporter gene system in human colon cancer DLD-1 cells, and measured COX-2 promoter-dependent transcriptional activity in the cells. Interferon suppressed this COX2 promoter activity, while 12-O-tetradecanoylphorbol-13acetate and transforming growth factor (TGF) exerted enhancing effects. We then tested the influence of 14 candidate cancer chemopreventive compounds on COX-2 promoter activity. Chemopreventive agents such as quercetin, kaempferol, genistein, resveratrol and resorcinol, all having a common resorcin moiety, were found to effectively suppress the COX-2 promoter activity with and without TGF-stimulation in DLD-1 cells. Since all these compounds have a resorcin moiety as a common structure, a resorcin-type structure may play an active role in the inhibition of COX-2 expression in colon cancer cells. - Colorectal cancer is currently one of the major causes of death from cancer in developed countries and, thus the search for chemopreventive agents effective in the large bowel has become very important. Many epidemiological and experimental studies have demonstrated that inhibition of cyclooxygenase (COX) is an effective measure in reducing the risk of colon carcinogenesis (1,2). COX catalyzes the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently, the presence of two isoforms of COX has been established (3), a constitutive enzyme, COX-1, present in many cells and tissues, and an inducible enzyme, COX-2, observed in cells in response to growth factors, mitogens and proinflammatory cytokines (3,4). COX-1 is constitutively expressed and has a housekeeping role helping to maintain physiological functions such as cytoprotection and blood flow. Abbreviations: -gal, -galactosidase; COX, cyclooxygenase; DHA, docosahexaenoic acid; FBS, fetal bovine serum; IFN, interferon ; IL, interleukin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NSAIDs, non-steroidal anti-inflammatory drugs; TGF, transforming growth factor ; TPA, 12-O-tetradecanoylphorbol-13-acetate; PTKs, protein-tyrosine kinases. In contrast, COX-2 is not present under normal physiological conditions but is upregulated with inflammation and colorectal tumor formation (57). Recent studies have suggested that overexpression of COX-2 and the resultant overproduction of prostaglandins might be involved in the development of colon cancer (8). It has been demonstrated that COX-2 selective inhibitors suppress spontaneous and chemically induced intestinal tumor formation in animal experiments (911). It has been also reported that inactivation of the COX-2 gene in the Apc knockout mouse, a model of human familial adenomatous polyposis, or treatment of these mice with COX-2 selective inhibitors, dramatically reduces the size and number of intestinal polyps (12). It is thus likely that increased expression of COX-2 is an important contributor to colon tumor formation, and compounds that inhibit the activity and/or expression level of this enzyme are potentially of great interest as candidate chemopreventive agents against colon carcinogenesis. Although major efforts have been made to develop selective inhibitors of COX-2 as chemopreventive agents against colon cancer, efforts to identify agents that can selectively suppress the expression of COX-2 at the gene level appear to be equally important. It is also likely that the combination of suppression of COX-2 gene expression and selective inhibition of its enzyme activity may provide the most effective approach to colon cancer prevention. Therefore, developing a simple screening system, which could detect the suppression of COX-2 gene expression, might be useful for searching novel chemopreventive agents. In the present study, we used a -galactosidase (-gal) reporter gene system to estimate COX-2 promoter activity in human colon cancer cells. The human colon cancer cell line DLD-1 can be stably transfected with a construct harboring the promoter sequence of the COX-2 gene fused to the -gal reporter gene. Using this -gal reporter gene system, we evaluated the effects of various cytokines and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on COX-2 promoter activity, and also tested various potential chemopreventive agents for their effects on COX-2 promoter activity. Materials and methods Chemicals Ascorbic acid, alpha-tocopherol, curcumin, epigallocatechin gallate, resveratrol, tannic acid, transforming growth factor (TGF) and TPA were obtained from Sigma Chemical Co. (St Louis, MO). Beta-carotene, daidzein and kaempferol were from Extrasynthese (Genay, France). Genistin was purchased from Fujicco Co. (Kobe, Japan). Genistein, glutathione (reduced form), quercetin and resorcinol were from Wako Pure Chemical Industries (Osaka, Japan). Docosahexaenoic acid (DHA)-ethyl ester was obtained from Sagami Chemical Research Center (Sagamihara, Japan) and interleukin 1 (IL-1) and interferon (IFN) from Genzyme (Cambridge, MA). Cell culture DLD-1 cells, a human colon adenocarcinoma cell line, were obtained from the Health Science Research Resources Bank (Osaka, Japan) and maintained in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT) and antibiotics (100 g/ ml streptomycin and 100 U/ml penicillin) at 37C in 5% CO2. Cells (2.0 105 cells/ml) were plated in 96-well tissue culture plates and precultured for 24 h before treatment with test agents. Cell viability in each culture was determined by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment, the cells were further incubated in a medium containing 0.5 mg/ml of MTT for 1 h. The MTT formazan produced by living cells was dissolved in dimethyl sulfoxide and absorbance at 595 nm was measured on a microplate Reader (Bio-Rad Laboratories, Hercules, CA). Reporter gene assay for COX-2 promoter-dependent transcriptional activity Human genome DNA was isolated from peripheral lymphocytes obtained from a healthy volunteer. A 2078 nucleotide human COX-2 gene promoter fragment stretching from 2046 to 32 ( (...truncated)