Celecoxib but Not Rofecoxib Inhibits the Growth of Transformed Cells in Vitro

Vol. 10, 267–271, January 1, 2004 Clinical Cancer Research 267 Celecoxib But Not Rofecoxib Inhibits the Growth of Transformed Cells in Vitro Diana Kazanov,1 Hadas Dvory-Sobol,1,3 Conclusions: Celecoxib may prove to be a very efficient Marjorie Pick,2 Eliezer Liberman,1,3 component in the prevention and treatment of gastrointes- Ludmila Strier,1 Efrat Choen-Noyman,1,3 tinal tumors because it inhibits the growth of cancerous cells without affecting the growth of normal cells. Varda Deutsch,2,3 Talya Kunik,1 and Nadir Arber1,3 Departments of 1Cancer Prevention and 2Hematology, Tel Aviv INTRODUCTION Medical Center, and 3Sackler Faculty of Medicine, Tel Aviv Colorectal cancer (CRC), with an estimated lifetime risk of University, Tel Aviv, Israel 5–6%, is a major health concern in the Western world (1, 2). The goal of achieving effective cancer prevention is driven by ABSTRACT the prediction that cancer will become the leading cause of death (surpassing heart disease) during this decade, with an estimated Purpose: Nonsteroidal anti-inflammatory drugs reduce 1,000,000 new cases and Ͼ500,000 deaths/year, worldwide the risk of colorectal cancer. The cyclooxygenase (COX) (1–3). Despite continuing advances in diagnosis and therapy, pathway of arachidonic acid metabolism is an important long-term survival has not improved significantly over the last target for nonsteroidal anti-inflammatory drugs. Increased four decades. Nearly 50% of all CRC patients will eventually expression of COX-2 was recently shown to be an important die of their disease (1, 2). step in the multistep process of colorectal cancer carcino- The association between nonsteroidal anti-inflammatory genesis. The new COX-2-specific inhibitors offer the benefit drugs (NSAIDs) and CRC is an intriguing one and has been of cancer protection without the gastrointestinal toxicity studied extensively. There are several lines of evidence suggest- reported for the old drugs. The purpose of this study was to ing that NSAIDs reduce CRC incidence and mortality (for compare the growth effects of two specific COX-2 inhibitors, review, see Refs. 4–6). However, long-term usage of NSAIDs celecoxib (Pfizer, Inc., New York, NY), and rofecoxib is limited because of the high incidence of side effects and the (Merck, White House Station, NJ) in normal and trans- significant cost. In 1997, 107,000 patients were hospitalized and formed enterocytes. 16,500 patients died, in the United States alone, as a direct Experimental Design: Cultures of normal rat intestinal consequence of NSAID usage (7). This mortality rate, of 50 epithelial cell line, IEC-18, vector control cells, c-K-ras, patients/day, is equal to the mortality rate from AIDS or leuke- c-K-ras-bak, and antisense-bak derivatives were treated with mia (7). different dosages of celecoxib (0–60 ␮M) and rofecoxib There are at least two isoforms of the cyclooxygenase (0–20 ␮M). Cell cycle analysis and apoptosis were assessed (COX) enzyme. COX-1 is found in the normal gastrointesti- by fluorescence-activated cell sorting analysis. Protein ex- nal mucosa and is usually constitutively expressed. It serves pression was assessed by Western blot analysis and caspases as the housekeeping protein. The COX-2 gene was discov- 3 and 8 activities by ELISA. ered about a decade ago (8). Although it is usually undetect- Results: Celecoxib inhibited cell growth and induced able in the normal gastrointestinal mucosa, its expression can apoptosis in a time- and dose-dependent manner. IEC18 be induced by inflammatory and neoplastic stimuli (9). Up- parental cells were two to four times more resistant to regulation of COX-2 expression occurs in 40–50% of colo- celecoxib than ras, ras-bak, and antisense bak transformed rectal polyps and in up to 85% of CRC (9). The lack of cells that overexpress the COX-2 protein. The induction of COX-2 expression in the normal colonic mucosa, together apoptosis by celecoxib involved the caspase pathways. Ro- with its increased expression in colonic neoplasm, constitute fecoxib, up to its maximal concentration of 20 ␮M, did not the rationale for the selective action of COX-2 inhibitors on inhibit cell growth or induce apoptosis. neoplastic colonic mucosa, without major biological effects on the normal colonic mucosa. Reddy et al. (10) showed that celecoxib had chemopreven- tive activity in the rat aberrant crypt focus model induced by Received 3/10/03; revised 9/24/03; accepted 9/24/03. azoxymethane. In a landmark study, Oshima et al. (11) demon- Grant support: Pfizer, Inc. (to N. A.), General Apostrophes of the State strated that crossing COX-2 knockout mice with APC mutant of Israel (to N. A.), and the Shapira Foundation (to D. K.). The costs of publication of this article were defrayed in part by the Min-mice resulted in a marked reduction in the number of payment of page charges. This article must therefore be hereby marked intestinal adenomas. Both celecoxib and rofecoxib have suc- advertisement in accordance with 18 U.S.C. Section 1734 solely to cessfully been shown, in this model, to inhibit polyp number and indicate this fact. multiplicity in a dose-dependent manner (12, 13). Indeed, a Requests for reprints: Dr. Nadir Arber, Head–Department of Cancer Prevention, Tel Aviv Medical Center, 6 Weizmann Street, Tel Aviv controlled trial of Celebrex (Celecoxib, Pfizer, Inc., New York, 64239, Israel. Phone: 972-3-6974968, ext. 280; Fax: 972-3-6950339; NY) in familial polyposis patients demonstrated a 30% reduc- E-mail: [email protected] or [email protected]. tion in tumor burden (14). Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. 268 Coxibs and Growth Inhibition Our group, in an open labeled study, demonstrated that Flow Cytometric Analysis. Cells were plated at a den- rofecoxib (25 mg qd) prevented the growth of 80% of adenomas sity of 7 ϫ 106/10-cm dish in complete medium. The adherent in familial polyposis patients for up to 30 months (15). and nonadherent cells were collected during exponential growth In recent years, our group has shown that transfection of of the cells and counted. A total of 1–2 ϫ 106 cells was washed normal enterocytes (IEC18 cells) by a variety of oncogenes in PBS, and the pellet was fixed in 3 ml of ethanol for1hat4°C. resulted in malignant cell transformation (16–18). These cells Cells were pelleted and resuspended in 1 ml of PBS and incu- proliferate faster, form colonies in soft agar, and have higher bated for 30 min with 0.64 mg/ml RNase at 37°C. Cells were saturation density and plating efficiency. Most importantly, stained with 45 ␮g/ml propidium iodide at least 1 h before these cells form tumors when injected s.c. in nude mice (16–18). analysis by flow cytometry, using a standard protocol for cell These sets of normal and transformed cell lines can serve as a cycle distribution and cell size (17). Necrotic cells were counted unique in vitro model to assess the effects of drugs on cell lines using trypan blue before fixation. All experiments were repeated that differ in only one oncogene. three times with similar results. Data acquisition was performed In the present study, the growth inhibition of the two most on a FACS calibur and analyzed using CellQuest software important coxibs, celecoxib and rofecoxib, was evaluated in (Becton Dickinson Immunocytometry Systems, San Jose, CA). normal and transformed intestinal cells. The growth inhibitory All fluorescence and laser light scatter measurements were effects produced two unanticipated findings. First, celecoxib made with linear signal processing electronics. Data for 20,000 and rofecoxib, having similar COX-2-selectivity and clinical cells were collected for each data file. efficacy for inflammatory indications, but differed significantly Protein Extraction and Western Blotting. Exponen- tially growing cells were collected with a rubber policeman and in their in vitro antiproliferative effects on cancer cell lines. washed three times in ice-cold PBS. Cell pellets were resus- Second, the antiproliferative effect of celecoxib was noted to pended in lysis buffer [20 mM Tris-HCI (pH 7.4), 2 mM EDTA, particularly inhibit the growth of the transformed cells but not 6mM 6-mercaptoethanol, 1% NP40, 0.1% SDS and 10 mM NaF, the growth of the normal cells. plus the protease inhibitors 10 ␮g/ml leupeptin, 10 ␮g/ml ap- We conclude that in this in vitro model, the antitumor rotonin, and 0.1 mM phenylmethylsulfonyl fluoride). For West- effect of rofecoxib was much lower than the antitumor effect of ern blotting, samples containing 50 ␮g of total cell lysate were celecoxib, an equally powerful COX-2 inhibitor. This difference loaded onto a 10% SDS-polyacrylamide gel and subjected to implies that the antitumor effects of these drugs may be distinct electrophoresis. Proteins were transferred to Hybond-C mem- from their effects on COX-2 inhibition. branes (Amersham, Arlington Heights, IL) in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol), using a Trans MATERIALS AND METHODS Blot transfer apparatus at 70 mA for 12–18 h at room temper- Reagents and Chemicals. Celecoxib was provided by ature. Membranes were blocked with blocking buffer (PBS/ 0.2% Tween 20/0.5% gelatin) for1hatroom temperature and Pfizer, Inc. Merck Research and Development (White House were subsequently washed three times for 5 min in a washing Station, NJ) supplied rofecoxib. All other reagents, with the buffer (PBS/0.05% Tween 20). The membranes were incubated highest purity, were purchased from Sigma Chemical Co. (St. with a 1:1000 diluted monoclonal human anti-COX-2 antibody Louis, MO). (Santa Cruz Biotechnology, Santa Cruz, CA) for1hatroom Cell Growth.

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