Evidence is now available showing that cyclooxygenase (COX)-2, which is involved in prostaglandin production, is overexpressed in many types of tumors including breast. Several reports have indicated that HER-2/neu-positive breast tumors are associated with an increased amount of COX-2 protein. In this study, we evaluated the effectiveness of the select COX-1 and COX-2 inhibitors in preventing mammary tumor development in HER-2/neu transgenic mice. At 4 weeks of age, female HER-2/neu mice were fed a #5020 rodent diet supplemented with 900 ppm celecoxib, a COX-2 inhibitor, 64 ppm of SC560, a COX-1 inhibitor, or the unsupplemented #5001 diet (control). The incidence of mammary tumors was significantly lower in the celecoxib-fed mice (71%; P = 0.001 versus control) than in the control mice (95%) or in the SC560-fed mice (91%). Celecoxib-treated mice also developed fewer tumors (1.3 ± 1.1 SD; P = 0.039 versus control) than the control mice (2.2 ± 1.2) or the SC560 treated mice (2.3 ± 1.3). The median time to tumor development was 266 days in the control group versus 291 days in the celecoxib-treated group (P = 0.003 versus control). Lung metastasis was also reduced by treatment with celecoxib. The COX-1 inhibitor SC560 had no protective effect. The protection offered by celecoxib was associated with significantly lower concentrations of prostacyclin and prostaglandin E2 in mammary tumors and their adjacent mammary glands. Our findings provide additional preclinical evidence to support the clinical studies to investigate the potential effectiveness of COX-2 inhibitors in protecting woman who are at high risk for breast cancer.

Breast cancer is the most common type of cancer and second leading cause of death for women in the United States (1). Although successful treatments are available to prevent estrogen receptor (ER)-positive breast cancer, strategies to prevent ER-negative breast cancer remain obscure. In particular, prevention treatments for ER-negative tumors that overexpress the HER-2/neu/c-erbB2 gene are needed. This tumor phenotype is found in 25–30% of human breast cancers (2). These tumors are associated with increased progression, metastasis, and reduced survival (2).

Breast tumors that have ER-negative status and amplification of HER-2 tend to overexpress cyclooxygenase (COX)-2 (3, 4). The COX enzymes control the conversion of arachidonic acid into prostaglandins (PGs). COX-1 is the constitutive isoform involved in normal cell functioning, whereas COX-2 is induced by cytokines, growth factors, and tumor promoters (5, 6, 7). Increased amounts of COX-2 protein were found recently in HER-2/neu-positive breast tumors (8). Moreover, COX-2 protein was induced in HER-2/neu-transfected colon cancer cells (9). Several laboratories demonstrated that increased expression of COX-2 protein might be a contributing factor in breast cancer development. For example, overexpression of COX-2 in a transgenic mouse model was sufficient to increase the production of PGs in the mammary gland (10). COX-2 is up-regulated in a variety of tumors including breast (11). In a study of human breast tumors, COX-2 tended to be localized to breast cancer cells rather than the surrounding stroma (12), and was associated with metastases and poor survival. COX-2 protein was also increased in women with ductal carcinoma in situ compared with normal tissue, which may suggest that COX-2 is an early event in the development of breast cancer and that COX-2 may be a potential target for breast cancer prevention (13).

Evidence is now available indicating that select COX-2 inhibitors protect against breast cancer. Preclinical studies have shown that the selective COX-2 inhibitor, celecoxib, reduced tumor incidence and growth of 7,12-dimethylbenzanthracene-induced mammary tumors in rats (14, 15). In a mouse orthotopic model, the COX-2 inhibitor SC236 was effective in inhibiting mammary tumor growth and lung metastases (16). These studies have demonstrated that COX-2 inhibitors were protective against ER-positive tumor growth.

In this study, we have chosen the HER-2/neu mouse model to test the effectiveness of COX inhibition in the prevention of ER-negative mammary tumor development and metastasis. These mice overexpress unactivated rat neu in the mammary gland under the transcriptional control of the mouse mammary tumor virus long terminal repeat (17). The neu protein has been detected in normal mammary glands and mammary tumors. However, active neu was found only in tumors, and at least 65% of the tumors carried somatic mutations affecting a highly conserved cysteine-rich region within the extracellular domain of neu (18, 19).

Treatment with the COX-1 and COX-2 inhibitors, SC560 and celecoxib, respectively, were begun when the HER-2/neu mice were 4 weeks of age. We also tested the select COX-2 inhibitor, SC560, because ibuprofen, COX-1, and COX-2 inhibitor was effective, but to a lesser extent than celecoxib, in preventing 7,12-dimethylbenzanthracene-induced mammary tumors in rats (14), and SC560 inhibited the growth of established mammary tumors in mice (20). While we were completing this study a preliminary report was published showing that celecoxib delayed the development of mammary tumors in HER-2/neu mice (21). Mammary tumors were detected at 39.6 weeks of age in 12 of 24 mice treated with celecoxib and at 32.3 weeks of age in 13 of 26 mice fed the control diet (P = 0.003). In the present study, we extend their findings by showing that celecoxib significantly reduced tumor incidence, tumor burden, and metastasis in HER-2/neu mice. The COX-1 inhibitor SC560 did not protect HER-2/neu mice against mammary tumor development. We also show that the chemoprotection of celecoxib was associated with reduced prostacyclin (PGI2) and PGE2 (prostaglandin E2) in the mammary tumors and their adjacent mammary glands in comparison with HER-2/neu mice fed the control or SC560-supplemented diets.

Reagents

The selective COX-2 inhibitor, celecoxib, and the selective COX-1 inhibitor, SC560, were generous gifts from Pharmacia (St. Louis, MO). Antibodies to COX-2 and c-erbB2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All of the reagents except where noted were purchased from Sigma-Aldrich (St. Louis, MO).

General Protocol

Animals.

The original breeding pairs of HER-2/neu, line #202 (mouse mammary tumor virus inactivated neu), were obtained from Sandra Gendler (Mayo Clinic, Scottsdale, AZ). The experimental mice were bred by mating HER-2/neu homozygotes. Offspring were weaned at 4 weeks of age.

Chemoprevention Study.

At 4 weeks of age female HER-2/neu mice were assigned to three groups: a control group (n = 46) fed a standard rodent diet (#5001; Purina, St. Louis, MO), a celecoxib-treated group (n = 46) fed the standard diet supplemented with 900 ppm celecoxib (COX-2 inhibitor); or the SC560-treated group (n = 39) fed the standard diet supplemented with 64 ppm SC560 (COX-1 inhibitor). In a preliminary study, we fed 6 HER-2/neu mice #5001 diets supplemented with 900 or 1500 ppm of celecoxib to observe for signs of toxicity. After 12 weeks of feeding, we observed that mice fed diets supplemented with 1500 ppm lost body weight and reduced their food intake (data not shown). Subsequently, celecoxib-treated mice were fed 900 ppm. Experimental mice were housed 3/cage with water and food provided ad libitum. Food intake was monitored weekly. Body weight was recorded every 2 weeks and at the time the mice were sacrificed. Starting at 3 months of age, the mice were palpated weekly for the appearance of tumors. Tumor diameters was measured with a micrometer caliper and recorded. The mice were sacrificed when their tumors reached 20 mm in diameter or when they were 14 months old. To evaluate expression of COX-2 and HER-2/neu in normal mammary glands, a subset of 6 mice were sacrificed at 4 months of age, which was before tumor development. Mammary tissues and mammary tumors were removed and portioned for evaluation of protein expression of biomarkers and histological analysis. Blood was obtained from the retroorbital plexus, centrifuged at 5000 rpm, and the serum was frozen at −80°C. Final tumor volume was calculated using the formula: V = L×W×D×π/6, where V is the volume, L is the length, W is the width, and D is the depth.

To evaluate metastases, lungs were removed and fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Five standard sections were taken 200 μm apart, and the number of visible metastatic nodules was scored using a dissecting microscope.

Celecoxib Analysis

Serum from the control and celecoxib-treated mice was analyzed for celecoxib concentration by liquid chromatography/mass spectrometry.

PG Composition

Mammary glands and mammary tumors were snap-frozen in liquid nitrogen and stored at −80°C. Frozen tissues were homogenized in 100% methanol, centrifuged, and the supernatants were assayed for PGE2 and PGI2 by RIA as described previously (22).

Western Blot Analysis

Mammary glands and mammary tumors were thawed on ice and homogenized at 4°C in lysis buffer for extracting total protein. The protein was quantitated using a bicinchoninic acid reagent. Protein extracts from the mammary glands and mammary tumors were electrophoresed at constant voltage (100 V) on a 7.5% SDS-PAGE under reducing conditions and transferred to nitrocellulose paper. The blots were incubated overnight with PBS contain 0.1% Tween 20 and 5% powdered milk (blocking solution), and then incubated with rabbit anti-COX-2 antibody, or rabbit anti-c-erbB2 (HER-2/neu) antibody. The membranes were washed six times with blocking solution, incubated with mouse monoclonal antirabbit IgG conjugated to alkaline phosphatase (Sigma-Aldrich), and washed six times with blocking solution. Bands were visualized by chemiluminescence (CDP Star; NEN Life Science Products, Boston, MA).

Histology and Immunohistochemistry

Mammary glands, mammary tumors, and lungs were fixed in 10% neutral-buffered formalin for 24 h, embedded in paraffin, and sectioned.

For immunohistochemistry, paraffin sections of mammary tissue and mammary tumors were deparaffinized and hydrated by successive washes with xylene, 100% ethanol, and a phosphate buffer [10 mm (pH 7.4) and 0.138 M saline containing 2.7 mm KCl). Antigen retrieval was accomplished with diluted antigen retrieval buffer (DAKO Corp.) Endogenous peroxidase was blocked with 3% hydrogen peroxide. Subsequently, slides were washed in PBS/KCl, incubated with 10% normal horse serum followed by the primary antibody (rabbit anti-COX-2 antibody or rabbit anti-c-erbB2; HER-2/neu) and incubated overnight at 4°C. The slides were then incubated with biotinylated secondary antibody for 45 min, followed by ABC reagent (Vector Labs) and diaminobenzidine. Counterstaining was done with hematoxylin. Sections were dehydrated by washing sequentially with 95% ethanol, 100% ethanol, and xylene. Coverslips were mounted on slides using Permount. Digital images of stained and unstained cells were obtained using an Olympus microscope equipped with a SPOT digital camera (Diagnostic Instruments, Syerling Heights, MI).

Statistical Analysis

Body weights, food intake, tumor incidence, tumor latency, tumor burden, tumor volume, and metastasis were compared across the three groups: control, SC560, and CBX. Body weights, food intake, and metastasis were analyzed by ANOVA. The Fisher’s exact probability test was used to compare the three groups with respect to proportion of mice that developed tumors, as well as the number of tumors per mouse. Total and average tumor volumes were analyzed by the Kruskal-Wallis test. Tumor latency (proportion of mice with tumors as a function of time) was estimated by the Kaplan-Meier method and analyzed by the log-rank test. Analyses were conducted with StatXact 4.0 (Cytel Software Corporation, Cambridge, MA) and Stata 7.0 (Stata Corporation, College Station, TX).

Celecoxib Inhibits Mammary Tumor Development.

We evaluated the efficacy of the COX-1 inhibitor, SC560, and the COX-2 inhibitor, celecoxib, in preventing mammary tumorigenesis in transgenic mice that overexpress HER-2/neu. The mice received the various treatments for 14 months or when a tumor grew to 20 mm in diameter. Mammary tumors that develop in these mice are ER-negative (23). Fig. 1 shows that tumor development as a function of time was significantly different for the three groups (P = 0.002). The control and SC560 groups had very similar tumor incidence over time, with median time to appearance of first tumor 266 days for the control mice and 265 days for the mice fed SC560 (P = 0.994). In contrast, the median time for tumor development for the HER-2 mice fed the celecoxib diet was 291 days (P = 0.003 versus control). Additional survival analyses of the appearance of multiple tumors confirmed that the control and SC560 groups were very similar (P = 0.634), whereas the celecoxib group had a consistently better tumor incidence profile throughout the study period (P < 0.001 versus control). For example, the median time between the appearance of the first tumor and that of a second tumor was 19, 30, and 57 days, in the control, SC560, and celecoxib groups, respectively.

The proportion of mice with tumors was significantly different across the three groups (P = 0.011). Almost all of the mice in the control (95%) and SC560 (91%) developed at least one tumor (P = 0.658 versus control), whereas mice fed celecoxib had a lower tumor incidence (71%; P = 0.039 versus control; Table 1).

Celecoxib also produced a remarkable decrease in tumor multiplicity (Table 2). The celecoxib-treated mice developed substantially fewer tumors within the study period (P = 0.039 versus control). Although there were fewer tumors in the celecoxib-treated mice, there were no differences in the size of the tumors among the three groups (Table 2). This is not surprising in view of the fact that the tumors were allowed to grow until they reached 20 mm in diameter, at which point they were sacrificed. Thus, the tumors did eventually reach comparable sizes across all of the groups, although the groups differed substantially in the number and rate of growth of those tumors.

At the time of sacrifice lungs were examined for the presence of metastasis, and tumor colonies were counted. Treatment with celecoxib reduced the incidence of lung metastasis (37% ± 4%; n = 29) in comparison with control (69% ± 6%; n = 27) or SC560-treated mice (83% ± 6%; n = 27). The difference in the incidence of lung metastases between the control and celecoxib-treated was significant (P = 0.01 by ANOVA). The total number of metastases in the lungs from celecoxib-treated mice (1 and 0–49, median and range, respectively) was less than the control mice (4 and 0–59, median and range, respectively) or the SC560-treated mice (7 and 0–52, median and range, respectively), but the differences were not significant.

The protective effect of celecoxib was not associated with reduced food intake or weight loss. Food intake was recorded weekly for each cage with 3 mice/cage and was not altered among the three groups (control = 85.18 ± 2.63 g/cage/week ± SD; celecoxib = 85.7 ± 1.86 g/cage/week ± SD; and SC560 = 83.2 ± 2.34 g/cage/week ± SD). Final body weights were also similar in the three treatments (control = 31.3 ± 3.8 g ± SD; celecoxib = 31.1 ± 2.7 g ± SD; and SC560 = 31.3 ± 3.9 g ± SD).

Serum Concentrations of Celecoxib.

Serum concentrations of celecoxib were measured to evaluate whether the protective effect of celecoxib observed at 900 ppp was associated with serum concentrations observed clinically. The concentration of celecoxib in the serum ranged from 3.5 μm to 8.5 μm, which is attainable clinically and sufficient to inhibit PGE2(24).

COX-2 and HER-2/neu Proteins Are Expressed in Mammary Glands and Mammary Tumors.

Recent evidence demonstrated an association between expression of HER-2/neu and COX-2. Increased expression of COX-2 occurred more frequently in HER-2/neu-positive breast cancers (8). COX-2 protein can also be induced in HER-2/neu-transfected cells (9). Fig. 3,A shows that COX-2 and HER-2/neu proteins were present in mammary glands and tumors from HER-2/neu mice. Immunoreactivity of COX-2 was present in the nucleus and cytoplasm of the ductal epithelium, and in the nuclei of the fat cells in the stroma (Fig. 3,A). Treatment with celecoxib did not alter the staining for COX-2 or HER-2/neu in the mammary gland. COX-2 protein and HER-2/neu was also expressed in mammary tumors and was not altered by celecoxib or SC560 treatment (Fig. 3 B).

Celecoxib Inhibits PG Production in Mammary Glands and Mammary Tumors.

Because COX-2 is up-regulated in many types of tumors including breast (25, 26, 27, 28, 29, 30), and COX-2-derived PGs are thought to promote tumor growth, we evaluated the effect of celecoxib on the PG content of the mammary tumor and adjacent mammary gland. The mammary glands and mammary tumors from HER-2/neu mice produced PGI2 and PGE2; the mammary tumors contained more PGE2 than the adjacent mammary glands (Table 3). Treatment with celecoxib significantly reduced the content of both PGE2 in mammary tumors by 55% and PGI2 by 39% (P < 0.05 versus control). Celecoxib also significantly reduced PGE2 and PGI2 in mammary glands by 48% and 44%, respectively (P < 0.05 versus control). We also measured PG content in mice treated with SC560, the COX-1 inhibitor, and found no differences in PGE2 or PGI2 content in mammary glands or mammary tumors from between the untreated and SC560-treated groups (data not shown).

The purpose of this study was to evaluate whether selective inhibition of COX-2 was protective in a mouse model with HER-2/neu-induced mammary tumors. The HER-2/neu mice provide a good model to test the chemoprotective effects of COX-2 inhibitors. The COX-2 protein is present in the ductal epithelium, stroma, and the mammary tumors that develop in these mice. The HER-2/neu mice develop mammary tumors after a period of growth and development similar to breast cancer in humans, and as in humans, metastases occur in most untreated tumors (17). We have demonstrated that inhibiting COX-2 but not COX-1 will reduce tumor incidence, tumor multiplicity, and metastases. Importantly, the protective effect of celecoxib was not associated with changes in food intake, which was monitored weekly throughout the study. Previous studies have shown that chronic calorie restriction prevents tumor development in rats (31, 32, 33). However, a very recent study has found that chronic calorie restriction has no effect on tumor development in HER-2/neu mice (34).

While we were conducting this study, Howe et al.(21) also reported that celecoxib when fed at a diet concentration of 500 ppm decreased tumor incidence and delayed tumor development in HER-2/neu mice. Mice were sacrificed when tumors reached 10 mm in diameter or when the mice were 12 months old. In contrast, we observed the mice until their tumors reached 20 mm in diameter or when they reached 14 months of age. This longer observation period allowed us to evaluate the effect of celecoxib on tumor multiplicity and metastases, which has not been reported previously. In this study, HER-2/neu mice fed the celecoxib at a concentration of 900 ppm developed significantly few tumors and fewer incidences of lung metastases. A previous report showed that celecoxib was successful in preventing the development of mammary tumors in ER-positive breast cancer induced by dimethylbenzanthracene (14). Our study and that reported by Howe et al.(21) have demonstrated that COX-2 inhibitors are also effective in preventing HER-2/neu-positive, ER-negative mammary tumors.

The protective effects of celecoxib were not associated with changes in protein expression of the erbB-2 transgene. However, celecoxib reduced the content of PGE2 and PGI2 in the mammary tumors and adjacent mammary glands. Celecoxib reduced tumor incidence and metastases at serum concentrations of celecoxib that are clinically attainable and sufficient to reduce PG production. PGs have long been implicated in tumor development and metastasis. Earlier studies reported that breast tumors with high production of PGE2 tended to have increased metastases (35, 36). A recent prospective cohort study showed that survival was significantly worse for those women with breast tumor PGE2 levels >15 ng/g than those with lower levels (37). In a murine model of metastatic breast cancer, higher concentrations of PGE2 were positively correlated with increased metastatic potential (38). Considering the evidence to support the relationship of increased COX-2-derived PGs and aggressive tumor growth, it is highly probable that the antitumor effect of celecoxib observed in this study may be attributed, in part, to the reduction in mammary tumor PGs levels.

COX-2 inhibitors have been shown to decrease cell proliferation (39, 40), stimulate apoptosis (41), and inhibit angiogenesis (42). A current point of discussion is whether these anticancer effects of COX-2 inhibitors are also due to COX-2-independent mechanisms that are not related to the decrease in COX-2-derived PGs. Typically, COX-2-independent effects are observed in vitro at high concentrations (>50 μm celecoxib), which are higher than circulating concentrations attainable in humans (21). Although we cannot rule out non-COX mechanisms in this study, it is very likely that COX-2-derived PGs were responsible for antitumor effect, because protection was evident with circulating concentrations of celecoxib between 3.5 μm and 8.5 μm.

The reduction in lung metastases observed in the celecoxib-treated mice may be related to a direct effect of the COX-2 inhibitor on metastatic activity or a consequence of the fewer number of tumors. Because we evaluated these tumors at an advanced stage in their growth (20 mm in diameter or at 14 months of age), it was not possible to evaluate these separate effects. There are studies that show that the COX inhibitors can have a direct effect on metastasis (16, 20). However, these studies also do not permit an evaluation the relationship of tumor multiplicity to presence of metastasis.

Other chemopreventive agents have also been shown to protect HER-2/neu mice against tumor development. Tamoxifen led to a 50% reduction of tumor incidence when given early before the presence of subclinical tumors (43). Feeding soy isoflavones increased tumor latency in Her-2/neu mice, but had no effect on incidence, number, or size of tumors (44). Treatment of HER-2/neu mice with the retinoid X receptor-selective retinoid LGD1069 significantly reduced tumor incidence (74%) in comparison with untreated mice (100%), and also increased tumor latency and decreased tumor multiplicity (23). The protective effect of LGD1069 on tumor incidence (74%) was comparable with that observed for celecoxib on tumor incidence (71%) in Her-2/neu mice.

In summary, celecoxib was effective in suppressing mammary tumor development, and decreasing tumor incidence, multiplicity, and lung metastasis in HER-2/neu mice. Our results indicate that the COX-1 inhibitor SC560 does not have a protective role in this mouse model. The results of this study along with previous studies in the rat (14) and the HER-2/neu mouse (21) show that celecoxib can provide only partial protection against development of mammary tumors. However, celecoxib may be more effective in preventing HER-2/neu-overexpressing mammary tumors when combined with other agents such as those that target the epidermal growth factor receptor family of which HER-2 is a member. Herceptin, a monoclonal antibody targeting HER-2, has been shown to be effective against HER-2-overexpressing metastatic breast cancers (45). More recently ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, prevented the growth of BT474 breast cancer xenografts, which overexpress HER-2/neu(46). Thus, combined chemoprevention with celecoxib and inhibitors of the epidermal growth factor receptor family has the potential for enhancing their individual protective effects.

Grant support: United States Army Medical Research Material Command under Grant DAMD17-00-1-0299 (S. L-J.).

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.

Requests for reprints: Susan Lanza-Jacoby, Department of Surgery, Jefferson Medical College, 1025 Walnut Street, Philadelphia, PA 19107. Phone: (215) 955-7903; E-mail: [email protected]

Fig. 1.

Kaplan-Meier plot of the incidence of mammary tumor development as a function of time. HER-2/neu mice were fed the control diet (#5001), the #5001 diet supplemented with 900 ppm CBX, or the #5020 diet supplemented with 64 ppm of SC560 (cyclooxygenase-1 inhibitor) from 1 month of age to 14 months of age or when the tumor reached 20 mm in diameter.

Fig. 1.

Kaplan-Meier plot of the incidence of mammary tumor development as a function of time. HER-2/neu mice were fed the control diet (#5001), the #5001 diet supplemented with 900 ppm CBX, or the #5020 diet supplemented with 64 ppm of SC560 (cyclooxygenase-1 inhibitor) from 1 month of age to 14 months of age or when the tumor reached 20 mm in diameter.

Close modal
Fig. 2.

Tumor multiplicity over time. The mean number of tumors per mouse was evaluated in HER-2/neu mice fed the control diet (#5001; Purina), the #5001 diet supplemented with 900 ppm CBX, or the #5020 diet supplemented with 64 ppm of SC560 (cyclooxygenase-1 inhibitor). Starting at 3 months of age, the mice were palpitated weekly for the appearance of tumors.

Fig. 2.

Tumor multiplicity over time. The mean number of tumors per mouse was evaluated in HER-2/neu mice fed the control diet (#5001; Purina), the #5001 diet supplemented with 900 ppm CBX, or the #5020 diet supplemented with 64 ppm of SC560 (cyclooxygenase-1 inhibitor). Starting at 3 months of age, the mice were palpitated weekly for the appearance of tumors.

Close modal
Fig. 3.

Expression of cyclooxygenase (COX) -2 and HER-2/neu in mammary glands and mammary tumors from HER-2/neu mice. A, 6 mice from each group were sacrificed at 4 months of age. Mammary glands were removed, prepared for immunohistochemical staining, and probed with rabbit anti-COX-2 antibody or rabbit anti-c-erbB2 (HER-2/neu) antibody. Representative fields of 6 mammary glands from control and CBX-treated are shown. B, mice were sacrificed at 14 months of age or when the tumors reached 20 mm in diameter. Mammary tumors were removed for analysis of COX-2 and HER-2/neu proteins. A representative Western blot from 6 tumors shows that CBX did not alter expression of COX-2 or HER-2/neu in mammary tumors.

Fig. 3.

Expression of cyclooxygenase (COX) -2 and HER-2/neu in mammary glands and mammary tumors from HER-2/neu mice. A, 6 mice from each group were sacrificed at 4 months of age. Mammary glands were removed, prepared for immunohistochemical staining, and probed with rabbit anti-COX-2 antibody or rabbit anti-c-erbB2 (HER-2/neu) antibody. Representative fields of 6 mammary glands from control and CBX-treated are shown. B, mice were sacrificed at 14 months of age or when the tumors reached 20 mm in diameter. Mammary tumors were removed for analysis of COX-2 and HER-2/neu proteins. A representative Western blot from 6 tumors shows that CBX did not alter expression of COX-2 or HER-2/neu in mammary tumors.

Close modal
Table 1

Effect of COXa inhibitors on the development and incidence of mammary tumors in HER-2/neu mice

Treatment groupNumber of miceNumber of mice with tumorsIncidence (%)Tumor latency (days)
Control 38 36 95 266.0b 
Celecoxib 38 27 71c 291.0d 
SC560 33 30 91 265.0 
Treatment groupNumber of miceNumber of mice with tumorsIncidence (%)Tumor latency (days)
Control 38 36 95 266.0b 
Celecoxib 38 27 71c 291.0d 
SC560 33 30 91 265.0 
a

COX, cyclooxygenase.

b

The data represent the median days.

c

Significantly different from control group, P = 0.001.

d

Significantly different from control group, P = 0.003.

Table 2

Effect of COXa inhibitors on the growth of mammary tumors in HER-2/neu mice

Treatment groupNumber of miceTumor multiplicity (number of tumors/mouse)Tumor volume (mm3)
MedianMean
Control 38 2.2 ± 1.2b 1051 1127 ± 618 
Celecoxib 38 1.3 ± 1.1c 963 1117 ± 579 
SC560 33 2.3 ± 1.3 907 1027 ± 669 
Treatment groupNumber of miceTumor multiplicity (number of tumors/mouse)Tumor volume (mm3)
MedianMean
Control 38 2.2 ± 1.2b 1051 1127 ± 618 
Celecoxib 38 1.3 ± 1.1c 963 1117 ± 579 
SC560 33 2.3 ± 1.3 907 1027 ± 669 
a

COX, cyclooxygenase.

b

Data represent means ± SD.

c

Significantly different from control group (P = 0.039).

Table 3

Effect of celecoxib treatment on prostaglandin content of mammary tumor and mammary glands from HER2 micea

TissuePGI2a (pg/mg protein)PGE2 (pg/mg protein)
ControlCBXControlCBX
Mammary tumor 161 ± 15 98 ± 6b 291 ± 33 131 ± 10c 
Adjacent mammary gland 149 ± 12 78 ± 4c 127 ± 10 71 ± 18c 
TissuePGI2a (pg/mg protein)PGE2 (pg/mg protein)
ControlCBXControlCBX
Mammary tumor 161 ± 15 98 ± 6b 291 ± 33 131 ± 10c 
Adjacent mammary gland 149 ± 12 78 ± 4c 127 ± 10 71 ± 18c 
a

Values are means ± SD for 15 mice.

b

PGI2, prostacyclin; PGE2, prostaglandin E2.

c

P < 0.05 between control and CBX treatments.

1
Jemal A., Murray T., Samuels A., Ghafoor A., Ward E., Thun M. J. Cancer statistics, 2003.
CA A Cancer J. Clin.
,
53
:
5
-26,  
2003
.
2
Slamon D. J., Godolphin W., Jones L. A., Holt J. A., Wong S. G., Keith D. E., Levin W. J., Stuart S. G., Udove J., Ullrich A. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science (Wash. DC)
,
244
:
707
-712,  
1989
.
3
Liu X., Rose D. Differential expression and regulation of cyclooxygenase in two human breast cancer cells lines.
Cancer Res.
,
56
:
5125
-5127,  
1996
.
4
Ristimaki A., Sivula A., Lundin J., Lundin M., Salminen T., Haglund C., Joensuu H., Isola J. Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer.
Cancer Res.
,
62
:
632
-635,  
2002
.
5
Jones D. A., Carlton D. P., McIntyre T. M., Zimmerman G. A., Prescott S. M. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines.
J. Biol. Chem.
,
268
:
9049
-9054,  
1993
.
6
Prescott S. M., White R. L. Self-promotion? Intimate connections between APC and prostaglandin H synthase-2.
Cell
,
8
:
783
-786,  
1996
.
7
Subbaramaiah K., Altorki N., Chung W. J., Mestre J. R., Sampat A., Dannenberg A. J. Inhibition of cyclooxygenase 2 gene expression by p53.
J. Biol. Chem.
,
274
:
10911
-10915,  
1999
.
8
Subbaramaiah K., Norton L., Gerald W., Dannenberg A. J. Cyclooxygenase-2 is overexpressed in HER-2/neu-positive breast cancer.
J. Biol. Chem.
,
277
:
18649
-18657,  
2002
.
9
Vadlamudi R., Mandal M., Adam L., Steinbach G., Mendelsohn J., Kumar R. Regulation of cyclooxygenase-2 pathway by HER2 receptor.
Oncogene
,
18
:
305
-314,  
1999
.
10
Liu C. H., Chang S. H., Narko K., Trifan O. C., Wu M. T., Smith E., Haudenschild C., Lane T. F., Hla T. Over-expression of cyclooxygenase (COX)-2 is sufficient to induce tumorigenesis in transgenic mice.
J. Biol. Chem.
,
276
:
18563
-9,  
2001
.
11
Soslow R., Dannenberg A., Rush D., Woerner B. M., Khan K. N., Masferrer H., Koki A. T. Cox-2 is expressed in human pulmonary, colonic, and mammary tumors.
Cancer (Phila.)
,
89
:
2637
-265,  
2000
.
12
Hwang D., Scollard D., Byrne J., Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer.
J. Natl. Cancer Inst.
,
90
:
455
-460,  
1998
.
13
Half E., Tang X. M., Gwyn K., Sahin A., Wathen K., Sinicrope F. A. Cyclooxygenase-2 expression in human breast cancer and adjacent ductal carcinoma in situ.
Cancer Res.
,
62
:
1676
-1681,  
2002
.
14
Harris R. E., Alshafie G. A., Abou-Issa H. M., Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor.
Cancer Res.
,
60
:
2101
-2103,  
2000
.
15
Alshafie G. A., Abou-Issa H. M., Seibert K., Harris R. E. Chemotherapeutic evaluation of celecoxib, a cyclooxygenase-2 (COX-2) Inhibitor, in rat mammary tumor model.
Oncol. Rep.
,
7
:
1377
-1314,  
2000
.
16
Connolly E. M., Harmey J. H., O’Grady T., Foley Roche-Nagle G., Kay E., et al Cyclo-oxygenase inhibition reduces tumour growth and metastasis in an orthotopic model of breast cancer.
Br. J. Cancer
,
87
:
231
-237,  
2002
.
17
Guy C. T., Webster M. A., Schaller M., Parsons T. J., Cardiff R. D., Muller W. J. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease.
Proc. Natl. Acad. Sci. USA.
,
89
:
10578
-10582,  
1992
.
18
Siegel P. M., Dankort D. L., Hardy W. R., Muller W. J. Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.
Mol. Cell. Biol.
,
14
:
7068
-7077,  
1994
.
19
Siegel P. M., Muller W. J. Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.
Proc. Natl. Acad. Sci. USA
,
93
:
8878
-8883,  
1996
.
20
Kundu N., Fulton A. M. Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease in a murine model of breast cancer.
Cancer Res.
,
62
:
2343
-2346,  
2002
.
21
Howe L. R., Subbaramaiah K., Patel J., Masferrer J. L., Deora A., Hudis C., Thaler H. T., Muller W. J., Du B., Brown A. M. C., Dannenberg A. J. Celecoxib, a selective cyclooxygenase 2 inhibitor, protects against human epidermal growth factor receptor 2 (HER-2)/neu-induced breast cancer.
Cancer Res.
,
62
:
5405
-5407,  
2002
.
22
Lanza-Jacoby S., Flynn J. T., Miller S. Parenteral supplementation with a fish-oil emulsion prolongs survival and improves rat lymphocyte function during sepsis.
Nutrition
,
17
:
112
-116,  
2001
.
23
Wu K., Zhang Y., Xiao-Chun X., Hill J., Celestino J., Hee-Tae K., Mohsin S. K., Hilsenbeck S. G., Lamph W. W., Bissonette R., Brown P. H. The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice.
Cancer Res.
,
62
:
6376
-6380,  
2002
.
24
Niederberger E., Tegeder I., Vetter G., Schmidtko A., Schmidt H., Euchenhofer C., Brautigam L., Grosch S., Geisslinger G. Celecoxib loses its anti-inflammatory efficacy at high doses through activation of NF-kb.
FASEB J.
,
15
:
1622
-16664,  
2001
.
25
Eberhart C. E., Coffey R. J., Radhika A., Giardiello F. M., Ferrenbach S., DuBois R. N. Up-regulation of cyclooxygenase gene expression in human colorectal adenomas and adenocarcinomas.
Gastroenterology
,
107
:
1183
-1188,  
1994
.
26
Chan G., Boyle J. O., Yang E. K., Zhang F., Sacks P. G., Shah J. P., Edelstein D., Soslow R. A., Koki A. T., Woerner B. M., Masferrer J. L., Dannenberg A. J. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.
Cancer Res.
,
59
:
991
-994,  
1999
.
27
Hida T., Yatabe Y., Achiwa H., Muramatsu H., Kozaki K., Nakamura S., Ogawa M., Mitsudomi T., Sugiura T., Takahashi T. Increased expression of cyclooxygenase-2 occurs frequently in human lung cancers, specifically in adenocarcinomas.
Cancer Res.
,
58
:
3761
-3764,  
1998
.
28
Gupta S., Srivastava M., Ahmad N. Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma.
Prostate
,
42
:
73
-78,  
2000
.
29
Tucker O. N., Dannenberg A. J., Yang E. K., Zhang F., Teng L., Daly J. M., Soslow R. A., Masferrer J. L., Woerner B. M., Koki A. T., Fahey T. J., 3rd. Cyclooxygenase-2 expression is up-regulated in human pancreastic cancer.
Cancer Res.
,
59
:
987
-990,  
1999
.
30
Kulkarni S., Rader J. S., Zhang F., Liapis H., Koki A. T., Masferrer J. L., Subbaramaiah K., Dannenberg A. J. Cyclooxygenase-2 is overexpressed in human cervical cancer.
Clin Cancer Res.
,
7
:
2001 Feb.
429
-434,
31
Tucker M. J. The effect of long-term food restriction on tumors in rodents.
Int. J. Cancer
,
23
:
803
-807,  
1979
.
32
Kritchevsky D., Weber M. M., Klurfeld D. M. Dietary fat versus caloric content in initiation and promotion of 7, 12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats.
Cancer Res.
,
44
:
3174
-3177,  
1984
.
33
Klurfeld D. M., Welch C. B., Davis M. J., Kritchevsky D. Determination of degree of energy restriction necessary to reduce DMBA-induced mammary tumorigenesis in rats during the promotion phase.
J. Nutr.
,
119
:
286
-291,  
1989
.
34
Pape-Ansorge K. A., Grande J. P., Christensen T. A., Maihle N. J., Cleary M. P. Effect of moderate calorie restriction and/or weight-cycling on mammary tumor incidence and latency in MMTV-neu female mice.
Nutr. Cancer
,
44
:
162
-168,  
2002
.
35
Bennet A., Charlier E. M., McDonald A. M., Simpson J. S., Stamford I. F. Prostaglandins and breast cancer.
Lancet
,
2
:
624
-626,  
1977
.
36
Bennett A., Berstock D. A., Raja B., Stamford I. F. Survival time after surgery is inversely related to the amounts of prostaglandins extracted from human breast cancers.
Br. J. Pharmacol.
,
66
:
451
-455,  
1970
.
37
Fulton A. M., Gimotty P., Alonsozana E., Dorsey R., Kundu N. Elevated prostaglandin E2 levels in human breast cancer are associated with poor long-term survival.
Proc. Am. Assoc. Cancer Res.
,
41
:
3666A
2000
.
38
Kundu N., Yang Q., Dorsey R., Fulton A. M. Increased cyclooxygenase-2 (COX-2) expression and activity in a murine model of metastatic breast cancer.
Int. J. Cancer
,
93
:
681
-686,  
2001
.
39
Shiff S. J., Koutsos M. I., Oiai L., et al Nonsteroidal antiinflammmatory drugs inhibit the cell proliferation of colon adenocarcinoma cells: effects on cell cycle and apoptosis.
Exp. Cell Res.
,
222
:
179
-188,  
1996
.
40
Husain S. S., Szabo I. L., Pai R., Soreghan B., et al MAPK (ERK2) kinase–a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.
Life Sci.
,
69(25–26)
:
3045
-3054,  
2001
.
41
Liu X. H., Yao S., Kirschenbaum A., et al NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells.
Cancer Res.
,
58
:
4245
-4249,  
1998
.
42
Tsujii M., Kawano S., Tsuiji S., Sawaoka H., Hori M., DuBois R. N. Cyclooxygenase regulates angiogenesis induced by colon cancer cells.
Cell
,
93
:
705
-716,  
1998
.
43
Menard S., Aiello P., Tagliabue E., Rumio C., Lollini P. L., Colnaghi M. I., Balsari A. Tamoxifen chemoprevention of a hormone-independent tumor in the proto-neu transgenic mice model.
Cancer Res.
,
60
:
273
-275,  
2000
.
44
Jin Z., MacDonald R. S. Soy isoflavones increase latency of spontaneous mammary tumors in mice.
J. Nutr.
,
132
:
3186
-3190,  
2002
.
45
Slamon D. J, Leyland-Jones B., Shak S., Fuchs H., Paton V., Bajamonde A., Fleming T., Eiermann W., Wolter J., Pergram M., Baselga J., Norton L. Use of chemotherapy plus a monoclonal antibody against HER-2 for metastatic breast cancer that overexpresses HER-2.
N. Engl. J. Med.
,
344
:
783
-792,  
2001
.
46
Moulder S. L., Yakes F. M., Muthuswamy S. K., Bianco R., Simpson J. F., Arteaga C. L. Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo.
Cancer Res.
,
61
:
8887
-8895,  
2001
.