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 chemopreventive 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).

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. The growth inhibition of rofecoxib was temperature. Membranes were washed as described above and tested on the following cell lines: normal enterocytes derived incubated with horseradish peroxidase-conjugated secondary from the rat ileum; IEC-18 cells (19); normal enterocytes trans- antibodies (1:2000) for1hatroom temperature. Additional formed by c-K-ras (IEC-18-ras; Ref. 17); IEC-18-ras cells washes were carried out as described previously and immune overexpressing bak (IEC-18-ras-bak; Ref. 16); and IEC-18 cells detection was performed using the enhanced chemilumines- transformed by antisense-bak (IEC-18-AS-bak; Ref. 18). The cence Western blotting detection system (Amersham). All ex- different cell lines were grown and maintained in DMEM (Bi- periments were repeated at least three times and yielded similar ological Industries, Kibbutz Beit-Haemek, Israel) supplemented results. with 10% FCS, penicillin, and streptomycin at 37°Cinan Fluorogenic Assay of Caspases 3 and 8 Activities. The ␮ atmosphere of 95% oxygen and 5% CO2. A total of 200 g/ml different cells were preincubated in buffer in the presence of G418 (Haemek) served as the selectable marker for IEC-18-ras ␮ ␮ 0–20 M docosahexaenoic acid and exposed to 0–200 M H2O2 and IEC-18-AS-bak cells. Hygromycin served as the selectable for 0–8 h. After incubation, cells were collected, washed, re- marker for IEC-18-ras-bak cells. suspended in 50 mM Tris-HCI buffer (pH 7.4), 1 mM EDTA, 10 Assays for Growth Inhibition. Cells were plated in du- mM EGTA, and lysed by three successive freeze-thaw at dry 4 plicate at a density of 3 ϫ 10 /35-mm well plates, containing 3 ice/37°C. Cell lysates were centrifuged at 20,000 ϫ g for 5 min, ml of DMEM plus 10% FCS. Rofecoxib, celecoxib, or 0.1% and the supernatants were stored at Ϫ70°C. The protein con- DMSO (the drug vehicle) were added to the culture medium at centration of each sample was estimated using the Bio-Rad time 0, at the indicated final concentrations. The number of protein assay. For caspase-3 and caspase-8 activities, a total of viable cells after incubation with these compounds was counted 50 ␮g of protein was incubated with 50 ␮M DEVD-AMC and every 24 h for 14 days, in duplicate, using a Coulter counter. All IEDT-AMP, respectively, at 37°C, for 30 min, and the release of experiments were repeated at least three times and produced 7-amino-4-methylcoumarine was monitored by a spectroflu- similar results. orometer using an excitation wavelength of 360 mm.

RESULTS COX-2 Protein Expression. A low level of COX-2 protein was seen in the parental cells. Increased levels of COX-2 protein (2–5-fold) were seen in IEC 18 cells transformed by ras, ras-bak, and antisense-bak cells. Effect on Cell Growth. We assessed the effects of celecoxib (0–60 ␮M) on cell growth for 72 h. Celecoxib inhibited the growth of the transformed cells in a dose-dependent manner. The normal cells were significantly more resistant to the drug than the transformed cells (Fig. 1). At the same time rofecoxib, up to its maximal solubility concentration of 20 ␮M, did not demonstrate any cell growth inhibition in all of the different clones (Fig. 2). Cell Cycle Changes and Induction of Apoptosis. Cele- Fig. 3 Changes in the level of apoptosis after exposure to COX-2 inhibitor. The parental (IEC 18) cells, vector control cells, and the coxib induced apoptosis for 72 h, in a dose-dependent manner, transformed clones IEC18-ras, IEC18-ras-bak, IEC 18-AS-bak were in all of the transformed cell lines, (Fig. 3). At the same time, no plated in 10-cm dish in complete medium. Cells at 50% confluence were exposed for 72 h to celecoxib (10 ␮M). Cells were analyzed by flow cytometry. Apoptosis was determined as the percentage of subdiploid cells. Similar data were obtained in three separate experiments.

Fig. 4 Changes in the level of caspase 3 after exposure to celecoxib. A total of 50 ␮g protein was incubated with 50 ␮M DEVD-AMC at 37°C for 30 min, and the release of 7 amino-4 methylcoumarine was Fig. 1 Growth curve of the parental and transformed cells after cele- monitored by a spectrofluorometer using an excitation wavelength of coxib treatment. IEC-18, vector control cells (VCs), and the transformed 360 mm. clones were plated in triplicate in 6-well plates in complete medium. The medium was changed after 24 h. The cells were exposed for 72 h to increase concentrations of celecoxib. The number of cells was determined using a Coulter counter. Similar data were obtained in three separate experiments. apoptotic cells were identified in the normal cells (Fig. 3). Exposure of all of the cells to rofecoxib neither altered their cell cycle nor demonstrated an effect on the subdiploid DNA peak; which represents apoptosis (data not shown). Level of Caspases 3 and 8 after 72 h of Exposure to the Coxibs. The induction of apoptosis after celecoxib treatment was associated with a parallel increase in the level of caspase 3 (Fig. 4) and caspase 8 (Fig. 5) in the ras-transformed cells, with a slight but constant decrease in the level of these caspases in the normal cells (Figs. 4 and 5). Rofecoxib did not alter the level of these caspases in any of the cell types.

DISCUSSION Celecoxib and rofecoxib belong to a new class of NSAIDs Fig. 2 Growth curve of the parental and transformed cells after rofe- that specifically inhibits COX-2. They have a significant anti- coxib treatment. IEC-18, vector control cells (VCs), and the transformed inflammatory and analgesic properties but are far less toxic than clones were plated in triplicate in 6-well plates in complete medium. traditional NSAIDs, which inhibit both COX-1 and COX-2 (20, The medium was changed after 24 h. The cells were exposed for 72 h to increase concentrations of rofecoxib. The number of cells was deter- 21). However, not all COX-2 inhibitors share the same antican- mined using a Coulter counter. Similar data were obtained in three cer effects. The predictive discrepancy between the in vitro separate experiments. growth inhibitions of celecoxib and rofecoxib may be indicative

treatment simply because it does not cause inhibition of cell growth. The efficiency of rofecoxib in vivo, including its significant effectiveness in preventing polyp formation in familial polyposis patients, cannot be ignored. It is suggested that the in vivo growth inhibition of rofecoxib may be attributable to direct inhibition of COX-2 expression in stromal cells, inhibition of the angiogenesis process, or other still unknown targets. In a recent study, Zhu et al. (27) examined the structural differences between celecoxib and rofecoxib. The structures of both celecoxib and rofecoxib were modified to produce 50 compounds, and the compounds were then tested for their ability Fig. 5 Changes in the level of caspase 8 after exposure to celecoxib. A total of 50 ␮g protein was incubated with 50 ␮M IEDT-AMP at 37°C for to induce apoptosis in human prostate cancer cells. Zhu’s study 30 min, and the release of AMP was monitored by a spectrofluorometer confirmed that the structural requirements for the induction of using an excitation wavelength of 360 mm. apoptosis are distinct from the structural requirements for the mediation of COX-2 inhibition (22, 23). We have previously shown that sulindac sulfide, which exerts nonspecific activity against COX-1 and COX-2 isoen- of the difference in their mechanism of action. The present study zymes, inhibits the growth of normal cells significantly more provides the first direct comparison of the in vitro anticancer than the growth of neoplastic cells (13, 16, 28). The current effects of the two clinically available COX-2 inhibitors. study suggests that transformed cells, with oncogenic ras or The antiproliferative effect of celecoxib was noted to par- down-regulation of Bak protein (frequent and important events ticularly inhibit the growth of the transformed but not the in the multistep process of CRC carcinogenesis), are more growth of the normal cells. Exposure to 10 ␮M celecoxib, for sensitive than normal cells to celecoxib. Hence, the ultimate 72 h, inhibited transformed cell growth by 50% but had very chemopreventive drug may be a specific COX-2 inhibitor rather little effect on the growth of normal cells (Fig. 1). The IC50sof than a NSAID that offers the benefits of protection against celecoxib ranges between 5 and 20 ␮M across this entire panel cancer without the side effects associated with traditional of cell lines. This concentration is very similar to the concen- NSAIDs. tration that can be achieved in the serum of humans with a In summary, inhibiting the growth of precancerous and standard anti-inflammatory dose (200 mg bid) of celecoxib. cancerous cells without affecting normal cells is the ultimate Celecoxib induced growth inhibition by the induction of aim of cancer treatment in general and chemoprevention studies apoptosis (Fig. 3), most probably by the activation of the in particular because such studies, which are often long term, caspase pathway (Figs. 4 and 5). Exposure to celecoxib induced involve healthy subjects and have strict adverse event require- the characteristic features of apoptosis, including morphological ments. From a clinical perspective, celecoxib might be the changes, subdiploid DNA pick, and caspases activation. More- desired drug that we are looking for because it inhibits the over, the potency of celecoxib in induction of apoptosis is growth of malignant cells without affecting the growth of nor- significantly higher than that of rofecoxib. mal cells. The current focus of this laboratory is the separation Thus, celecoxib can be a unique pharmacological tool in of the apoptosis-inducing effect of celecoxib from its COX-2 the study of apoptosis regulation in colon cancer cells consid- inhibitory activity, which may ultimately lead to the design of a ering its ability to interact with several targets. In addition, new class of therapeutic agents against colon cancer. evidence is accumulating that the apoptosis-inducing effect of the drug may be different from its COX-2 inhibitory activity. It should be noted that similar results were reported in normal and REFERENCES transformed human prostate epithelial cells (22, 23). 1. Parkin, D. M., Pisani, P., and Ferlay, J. Global cancer statistics. 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Diana Kazanov, Hadas Dvory-Sobol, Marjorie Pick, et al.

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