[CANCER RESEARCH 62, 2343–2346, April 15, 2002] Selective Cyclooxygenase (COX)-1 or COX-2 Inhibitors Control Metastatic Disease in a Murine Model of Breast Cancer 1 Namita Kundu and Amy M. Fulton 2 Department of Pathology [N. K., A. M. F.] and Greenebaum Cancer Center [A. M. F.], University of Maryland School of Medicine, Baltimore, Maryland 21201 ABSTRACT Using a highly metastatic mammary tumor cell line that expresses both cyclooxygenase (COX) isoforms, we now show that oral administration of either a selective COX-2 inhibitor (celecoxib) or a selective COX-1 inhib- itor (SC560) to mice with established tumors results in significant inhibi- tion of tumor growth. Administration of the dual inhibitor, indomethacin, leads to even better growth control. Metastatic capacity is also reduced by treatment of tumor-bearing mice with either COX-1 or COX-2 selective inhibitors. Pretreatment of tumor cells with COX inhibitors also reduces metastatic success, indicating that tumor cells may be a direct target of action by COX inhibitors. Growth of a second cell line, which does not express COX-2 in vivo, is also reduced by celecoxib, implicating both COX-dependent and COX-independent mechanisms. INTRODUCTION The inducible isoform of COX, 3 COX-2, is commonly overex- pressed in solid tumors, suggesting that this enzyme may contribute to malignant behavior (1, 2). Epidemiological studies as well as early clinical trials also suggest that administration of either dual COX-1/ COX-2 or selective COX-2 inhibitors may reduce the risk of cancer development (3). Preclinical studies also indicate that COX inhibition may be useful in models of chemoprevention (4). More limited studies suggest that COX inhibitors may have potential in the treatment of established disease (5). Although these early results are encouraging, most of these studies have been carried out in models of solid tumors in which metastatic disease does not occur. We have now examined the efficacy of COX-2 inhibition in a model of metastatic breast cancer. Much less attention has been paid to a possible role for the COX-1 isoform in cancer. Murine mammary tumors express both COX-1 and COX-2 isoforms, and we have also determined the effect of treatment with a selective COX-1 inhibitor on tumor behavior. MATERIALS AND METHODS Cell Lines and Tumors. Murine mammary tumor cell line 410 was de- rived from a spontaneously arising tumor of a Balb/cfC3H mouse. This line is tumorigenic but rarely metastasizes. Line 410.4 was derived from a metastatic lesion that occurred in a mouse bearing a s.c. implant of line 410. Both cell lines are maintained in DMEM supplemented with 10% FCS (Gemini Bio- Products, Inc., Calabasas, CA), 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 0.1 mM nonessential amino acids. For growth studies in vivo, 5–50  10 5 viable cells were injected s.c. into syngeneic Balb/cByJ female mice (Jackson Laboratories, Bar Harbor, ME). When tumors become palpable, tumor diameters were determined twice weekly by caliper, and tumor size was expressed as the mean of the longest and the perpendicular tumor diameters. To determine the degree of metastatic disease, mice bearing s.c. tumor implants were sacrificed, on an individual basis, when tumors achieved an average diameter of 18 mm; lungs were removed, and surface lung tumor colonies were counted under a dissecting microscope. COX Inhibitor Treatments. The selective COX-2 inhibitor, celecoxib, and the selective COX-1 inhibitor, SC560, were a generous gift of Pharmacia (St. Louis, MO). The dual COX inhibitor indomethacin was purchased from Sigma Chemical Co. (St. Louis, MO); the selective COX-2 inhibitor NS398 was from Cayman Chemical Co. (Ann Arbor, MI). All drugs were dissolved in a solution of methylcellulose (0.5%) and Tween 20 (0.025%) and administered by oral gavage twice/day to achieve a dose of 5 mg/kg/day (celecoxib and SC560) or 1 mg/kg/day (indomethacin). Drugs or vehicle were administered beginning on the day of tumor transplantation or 1 week later, when 410.4 tumors become palpable. Treatment of mice bearing the slower growing tumor 410 was initiated on either day 0 or day 14. To determine the direct effect of COX inhibitors on tumor cells, in the absence of host effects, line 410.4 tumor cells were cultured in the presence of SC560 (0.01 M), celecoxib (0.1 M), NS398 (1.0 M), indomethacin (1.0 M), ethanol (vehicle control for indo- methacin), or DMSO (control for SC560, celecoxib, and NS398). Forty-eight h after drug addition, cells were washed, and 3 or 5  10 5 viable tumor cells were injected into the lateral tail vein of syngeneic mice. No further drug treatments were carried out. On day 20, mice were sacrificed, and pulmonary metastases were quantitated. RESULTS AND DISCUSSION COX-2 is commonly overexpressed in both rodent and human tumors, suggesting that this isoform is an important determinant of tumor behavior (1, 2). Less attention has been focused on the COX-1 protein, which is also commonly detected in tumors. We have shown previously that the highly metastatic murine mammary tumor line 410.4 expresses both the COX-1 and COX-2 isoforms in culture and in tumors arising from transplantation to syngeneic hosts (6). We have also shown that a dual COX-1/COX-2 inhibitor, indomethacin, has potent antitumor activity in a murine model of metastatic breast cancer (7). Those studies could not distinguish a role for COX-1 versus COX-2 inhibition in the therapeutic effect. Using COX inhib- itors that are selective for either COX-1 (SC560) or COX-2 isoforms (celecoxib), we have now examined tumor inhibition and compared the efficacy of these selective drugs to indomethacin. We transplanted 5  10 5 of line 410.4 tumor cells s.c. to syngeneic Balb/cByJ mice. Drugs were administered by oral gavage beginning either on the day of tumor transplantation or at day 7, when all tumors are palpable. Drug treatments were continued on a daily basis until day 28. Fig. 1 shows that all three drugs resulted in statistically significant inhibition of tumor size in comparison with vehicle-treated control animals. We confirmed our previous study showing that indomethacin, initiated on day 0, results in the most marked tumor inhibition of 410.4. In comparison to the dual inhibitor, the selective COX-2 inhibitor celecoxib led to somewhat less tumor inhibition than indomethacin; however, tumors in these mice were still significantly smaller than in vehicle-treated animals. Treatment with either indo- methacin or celecoxib was still effective, even if treatment com- menced in mice with established (day 7) tumors. Interestingly, treat- ment with the selective COX-1 inhibitor, SC560, also resulted in significant tumor growth inhibition comparable with that achieved with the COX-2 inhibitor. Therapeutic activity of a selective COX-1 inhibitor has not been described in vivo previously. Because 410.4 tumors express both COX isoforms, it was possible Received 12/20/01; accepted 2/14/02. 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 Supported by the United States Department of Defense DAMD 17-00-1-0322 (to A. M. F.). 2 To whom requests for reprints should be addressed, at Department of Pathology, University of Maryland, 10 South Pine Street, Baltimore, MD 21201. Phone: (410) 706- 6479; Fax: (410) 706-8414; E-mail:afulton@umaryland.edu. 3 The abbreviations used are: COX, cyclooxygenase; PGE, prostaglandin E. 2343 Research. on September 11, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from