[CANCER RESEARCH 60, 6045– 6051, November 1, 2000] Celecoxib Prevents Tumor Growth in Vivo without Toxicity to Normal Gut: Lack of Correlation between in Vitro and in Vivo Models 1 Christopher S. Williams, Alastair J. M. Watson, Hongmiao Sheng, Rania Helou, Jinyi Shao, and Raymond N. DuBois 2 Departments of Medicine [C. S. W., H. S., R. H., J. S., R. N. D.], and Cell Biology, [C. S. W., R. N. D.], The Vanderbilt Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2279; Veterans Administration Medical Center [R. N. D.], Nashville, Tennessee 37232-2279; and Department of Medicine, University of Liverpool, Liverpool, United Kingdom [A. J. M. W.] ABSTRACT Nonsteroidal anti-inflammatory drugs have potential for use in the prevention and/or treatment of colorectal cancer. We have studied the cytotoxic effect of a specific COX-2 inhibitor, celecoxib, against LLC, HCA-7, and HCT-15 cells grown in cell culture and have compared these results with its effect on HCA-7 cells grown as xenografts in nude mice. “High-dose” celecoxib (>20 mM) reduced the viability of all three cell lines in vitro as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay. Flow cytometric analysis demon- strated that this loss of viability was attributable to the induction of apoptosis. Significantly, concentrations of the drug <10 mM had no effect on cell viability in vitro. The cytotoxic effects of high-dose celecoxib were independent of COX-2 inhibition because similar effects were observed in cox-2 (1/1), cox-2 (1/2) and cox-2 (2/2) fibro- blasts. A plasma concentration of 2.3 6 0.7 mM was achieved when celecoxib (1250 mg/kg of chow) was fed to animals ad libitum. Despite a lack of toxicity at 2–3 mM celecoxib in vitro, there was attenuation of HCA-7 xenograft growth in vivo. Celecoxib had no effect on apoptosis, cell division, or the epithelial architecture of the normal gut in treated mice. These results support the need for additional clinical evaluation of celecoxib for treatment and/or prevention of colorectal cancer in humans. INTRODUCTION Colorectal cancer remains a significant health concern for much of the industrialized world. Diagnosis often occurs at a late stage in the progression of this disease, which reduces the likelihood of effective treatment. Current treatment strategies often involve a combination of surgical resection and adjuvant chemotherapy. Because of the unsat- isfactory outcome of present treatment methods, especially with ad- vanced disease, much emphasis has been placed on developing better treatment and prevention measures. Numerous epidemiological studies indicate that chronic use of NSAIDs 3 lowers the mortality rate from colorectal cancer (1, 2). NSAIDs are effective at inducing regression of existing polyps in familial adenomatous polyposis patients (3) and in reducing the tumor burden in three animal models of colorectal cancer: the multiple intestinal neoplasia mouse (4, 5), the azoxymethane-treated rat model (6), and the nude mouse xenograft model (7–9). NSAIDs inhibit the activity of the cyclooxygenases, which are key enzymes in the conversion of arachidonate to PGH 2 , the immediate substrate for a number of specific prostaglandin synthases. Unfortu- nately, prolonged use of NSAIDs can result in gastrointestinal ulcer- ation and bleeding. It is widely believed that this ulcerogenic activity of NSAIDs is attributable to the chronic inhibition of prostaglandin production in the gastric mucosa. Accordingly, researchers have tried to identify NSAID derivatives that retain anti-neoplastic activity but do not affect prostaglandin production in gastric mucosa. There are two isoforms of cyclooxygenase, COX-1 and COX-2, which differ in their expression pattern and function within the orga- nism. COX-1 is constitutively expressed in many tissues and is thought to be responsible for maintaining gastric mucosal integrity. COX-2 is induced by a variety of stimuli and plays an important role in wound healing, ovulation, fertilization, and in mediating inflam- mation (for review, see Ref. 10). COX-2 expression levels are in- creased in colorectal cancer tissues (11–14). Overexpression of COX-2 has been shown to mediate cell cycle progression and to contribute to such diverse processes such as apoptosis, angiogenesis (15), and tissue invasion (16). On the basis of these effects, we have investigated the role of specific COX-2 inhibitors in the treatment of advanced colorectal cancer. We have studied previously the effects of a selective COX-2 inhibitor, SC-58125, on the growth and viability of colorectal carci- noma cells grown in vitro and in vivo (7). The precise mechanism for growth inhibition of tumors is under evaluation, but does not seem to involve the induction of apoptosis in vivo (17). It has been reported that nonselective COX inhibitors induce a G 2 -M cell cycle arrest resulting in a corresponding reduction of p34 cdc2 levels and activity (18). We have observed a similar effect after SC-58125 treatment of colorectal carcinoma cells. 4 These results suggest that SC-58125 may have therapeutic potential for treatment of colorectal cancer. How- ever, SC-58125 will not be developed for clinical use in humans. Furthermore, although SC-58125 reduces tumor growth, it does not cause tumor regression (7, 9). Therefore, we evaluated other COX-2 inhibitors for their effect on the growth of colorectal carcinoma cells in vitro and in vivo. Here we report that celecoxib (Celebrex), at concentrations .10 mM, potently induces apoptosis and inhibits cell cycle progression in colorectal carcinoma cells grown in culture by mechanisms independ- ent of COX-2 inhibition. Lower concentrations of the drug have no discernible effect on cells grown in vitro. We also found that when celecoxib was administered to mice by inclusion in the diet (1250 mg/kg of chow), serum concentrations of ;2.3 mM were achieved. Nevertheless, in contrast to the results from cell culture experiments, celecoxib significantly reduced the growth rate of colorectal carci- noma cells grown as xenografts, without toxicity to the normal intes- tine. These data highlight the point that the biological effects of celecoxib against cells grown in culture do not predict its effects in Received 5/31/00; accepted 9/1/00. 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 This work was supported in part by USPHS Grants RO1DK-47297 (to R. N. D.), P30CA-68485 (to R. N. D.) and PO1CA-77839 (to R. N. D.). A. J. M. W. is supported by Grant 97/21 from the Association of International Cancer Research. R. N. D. is a recipient of a Veterans Affairs Research Merit Grant and the Mina C. Wallace Professor of Medicine. 2 To whom requests for reprints should be addressed, at Department of Medicine/GI; MCN C-2104, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232-2279. Phone: (615) 343-4747; Fax: (615) 343-6229; E-mail: raymond.dubois@ mcmail.vanderbilt.edu. 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; LLC, Lewis lung carcinoma; MEF, mouse embryo fibroblast; TUNEL, terminal deoxynucle- otidyl transferase-mediated nick end labeling; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; FBS, fetal bovine serum; PGE 2 , prostaglandin E 2 . 4 Unpublished data. 6045 Research. on December 1, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from