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Activation of cyclooxygenase-2 (COX-2) and inhibition of peroxisome proliferators-activated receptor γ (PPARγ) have been observed in human and animal models of breast cancer. Both inhibition of COX-2 and activation of PPARγ can inhibit proliferation of breast cancer cells in vitro. Here we examine the effects of the COX-2 inhibitor celecoxib and the PPARγ agonist F-L-Leu on mouse breast tumor cells in vitro and in vivo.
We created and characterized a mouse mammary adenocarcinoma cell (MMAC1) line from C3 (1)-SV40 T-antigen mice to study COX-2 and PPARγ expression and response to celecoxib and F-L-Leu in vitro. To study the in vivo effects, C3 (1) SV40 T-antigen mice were given either control diet or diets containing three different concentrations of celecoxib and F-L-Leu as well as a combination of both agents. Mice were then followed for tumor formation up to one year.
MMAC1 cells express both COX-2 and PPARγ mRNA and exhibited cooperative growth inhibition with a combination of celecoxib and F-L-Leu. In mice, the median age of death due to mammary tumors was significantly delayed in celecoxib treated animals at all three concentrations, but was not significantly affected by F-L-Leu treatment alone. A combination of celecoxib and F-L-Leu was significantly more effective than celecoxib alone.
Our findings suggest that a combination of a COX-2 inhibitor and PPARγ agonist can delay breast cancer in a mouse model and suggest these agents should be studied in the context of human populations with high breast cancer risk.
Breast cancer is the most frequent cancer and the second leading cause of death in women in the United States with more than 40,000 deaths per year (1). These statistics emphasize the importance of developing new methods to either treat or prevent the disease. Chemoprevention strategies have shown promise in breast cancer with the development of anti-estrogens such as tamoxifen and raloxifene for high-risk individuals (2). However, other chemopreventive strategies may also be effective at lowering risk.
Cyclooxygenase-2 (COX-2) represents one potential chemoprevention target for breast cancer. COX-2 is known to be upregulated in human breast cancer and is a key enzyme in the production of prostaglandins, which stimulate cell proliferation, inhibit apoptosis, promote metastasis and promote angiogenesis in mammary tumor cells (3). Selective COX-2 inhibitors, such as celecoxib, inhibit breast cancer cell growth in vitro and in mouse models (4–6). Epidemiologic studies suggest that both nonsteroidal anti-inflammatories (NSAIDS) and selective COX-2 inhibitors reduce the risk of breast cancer in both primary and secondary prevention settings (7, 8).
Like COX-2, peroxisome proliferator-activated receptor γ (PPARγ) is upregulated in breast cancer cells (9). PPARγ is a transcription factor that heterodimerizes with RXR and binds to specific DNA response elements in gene promoters (10). Targets of PPARγ include genes involved in metabolism and lipid transport, resulting in adipocyte differentiation. In order to activate transcription, PPARγ must bind an activating ligand (11). Synthetic PPARγ agonists known as thiozolidinediones are currently used to treat type 2 diabetes. N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu) as a novel PPARγ agonist that lacks the thiozolidinedione ring, has potent insulin-sensitizing activity, but weaker adipogenic activity (12). Exposure of human breast cancer cell lines to thiozolidinedione has been shown to inhibit cell proliferation in cell culture, as well as in nude mice (13). 15-deoxy-Δ12,14-prostaglandin J2 (15PGJ2) is an endogenous ligand of PPARγ that is a downstream product of COX-2 and thus PPARγ activity may be down-regulated in the presence of COX-2 inhibitors.
As described above, there is good evidence that both COX-2 inhibitors and PPARγ agonists have anti-tumor effects individually. However, since the key endogenous ligand of PPARγ is 15PGJ2, and this ligand is a downstream product of COX-2, it is unlikely that the anti-tumorigenic activity of COX-2 inhibitors is being mediated by upregulation of PPARγ function. In fact, inhibition of COX-2 may actually inhibit the anti-tumor effects of PPARγ because of reduced 15PGJ2 production. If this is the case, then addition of a PPARγ agonist to a COX-2 inhibitor may have a cooperative or synergistic effect because both anti-tumorigenesis pathways would be functional. The central hypothesis for this study is that a combination of both COX-2 inhibitors and PPARγ agonists may be more effective in inhibiting the growth of breast cancer cells than either agent alone.
To examine the effects of celecoxib and F-L-Leu we have chosen to use the C3 (1)-SV40 Tag mouse, originally developed in the lab of Dr. Jeffrey Green at the NCI (14). This mouseexpresses simian virus 40 (SV40) large tumor antigen (Tag) under control of the rat prostatic steroid binding protein promoter. Somewhat unexpectedly, it was found that female C3 (1)-SV40 Tag mice consistently develop ductal adenocarcinoma by six months of age, due to expression of SV40 Tag mRNA in breast tissue (15). Time course studies show that there is a progression observed in the breast tissue of these animals over time, starting with hyperplasia (2 months), progressing to nodular lesions (3 months), and then to adenocarcinomas (4–6 months of age) (16). SV40 large T-Ag is known to inhibit p53 and RB function, inactivation of which is common in human breast cancer (17, 18).This model has also been used to test several other chemopreventive agents for breast cancer including dehydroepiandrosterone, the ornithine decarboxylase inhibitor DFMO, and the anti-angiogenic compound endostatin (19, 20).
Here we have examined the effects of the selective COX-2 inhibitor celecoxib and the PPARγ agonist F-L-Leu on tumor formation in C3 (1)-SV40 Tag mice. Our results show that the combination of celecoxib and PPARγ is particularly effective at delaying breast tumor formation in these animals.
A mouse mammary adenocarcinoma from a C3 (1)-SV40 Tag mouse was aseptically removed, rinsed with cold PBS, minced with a scalpel, and transferred into 10 ml of 0.02% collagenase and rotated in 37°C water bath for 2 hrs. The cellular material was then washed 4 times with serum free DMEM media and the cells were recovered by centrifugation and transferred to 1.6 ml of low Calcium media in a Swine Skin Gelatin coated T-25 flask to select for epithelial cell growth. The next day the supernatant (enriched for tumor cells) was transferred to a new flask. Once the cells have adhered, 3 ml of additional media were added.
MMAC-1 cells in logarithmic growth phase were washed once with phosphate buffered saline (PBS), detached with trypsin and suspended to a final concentration of 5 × 104 cells/ml in fresh low calcium medium. Cells (1 × 104) were placed in a96-well flat bottomed tissue culture plate. After 24 hours supernatant was discarded and new media containing drug was added. After 48 hrs of incubation at 37°C, viable cell growth was determined using a MTT assay (Promega G3580, Madison WI). Celecoxib was purchased from Toronto Research Chemical Inc. (Ontario, CA). N-(9-fluorenyl-methyloxycarbonyl)-L-leucine, (F-L-Leu) was purchased from Sigma-Aldrich (St. Louis, USA).
Female C3 (1)-SV40 Tag-transgenic mice were weaned at 21–30 days then put on the experimental diet as indicated. Virgin animals were used because of the acceleration of the development of mammary tumors due to pregnancy. Animals were monitored weekly for the appearance of tumor by visual inspection and palpation.
Animals were fed AIN-76A Rodent Diet as their control chow (Bio-Serv, Frenchtown NJ). Drug was added to this chow during the manufacturing process at the indicated concentration (Bio-Serve custom diets S5282, F5152, F5153, S5345, S5346, S5347, S5348).The chow was sterilized by gamma radiation.
MMAC-1 cells were collected, washed with PBS, and resuspended in lysis buffer (Cell Signaling Technologies, Danvers MA). Samples were then incubated at 4°C for 20 min followed by centrifugation at 13,000 r. p.m. for 10 min at 4°C. The supernatant was then assessed for protein concentration using the Bio-Rad protein assay solution and a Bio-Rad smartspec3000 machine (BioRad, Hercules CA). Thirty µg of protein was loaded onto a NuPage10%Tris-Acetate gel (Invitrogen, Carlsbad CA) and after electrophoresis was transferred to PVDF membrane. Membranes were blocked with 5% skimmed milk–PBS-Tween for 1 h and then probed with the primary antibodies, either anti-Vimentin (1: 1000 # V5255 Sigma-Aldrich, St. Louis MI) or anti-Keratin (1: 100 # ab8068 Abcam, Quebec Canada). After washing in PBS-Tween, the membranes were incubated with a donkey anti-rabbit HRP-linked secondary antibody (1: 1000 Amersham Biosciences NA934V, Piscataway NJ) for 1 h. The signal was visualized by chemiluminescence (Supersignal West Dura extended duration substrate; Pierce Biotechnology, Rockford IL).
MMAC-1 cells were grown to 75% confluency in 6 wells plates, either with no treatment or treatment with Celecoxib (50µM) or Ciglitazone (80µM). Cells were harvested and RNA was extracted using the Qiagen total RNA isolation kit (Qiagen, Valencia CA). RT-PCR was performed using the SuperScript one step RT-PCR Kit (Invitrogen, Carlsbad CA)according to manufacturer’s instructions. The following primers were used: COX-2; F-5′(CTT TGC CCA GCA CTT CAC CCA TC) 3′; R-5′ (TCC AAA GGT GCT CGG CTT CCA G)3′ and PPAR-γ; F-5′(TCT CCG TGA TGG AAG ACC ACT CG)3′; R-5′(TTC TGG AGC ACC TTG GCG AAC AGC)3′.
Total RNA was extracted using the PicoPure RNA Kit(Molecular Devices) according to manufacturer’s instructions. First strand cDNA was generated from total RNA from each sample using the High Capacity cDNA Kit (Applied Biosystems, Foster City CA) according to manufacturer’s instructions. COX-2, PPARγ, 18S ribosomal RNA (18S) and beta actin was measured using TaqMan® technology on a ABI Prism® 7900 Sequence Detection System. Reactions were prepared in triplicate for each gene using TaqMan® Gene Expression Master Mix and the following TaqMan® Gene Expression Assays(Applied Biosystems): COX-2 (Mm00478374_m1), PPARγ (Mm00440945_m1), beta actin (Mm00607939_s1), 18S (Hs99999901_s1). Plates were loaded and reactions cycled using TaqMan® universal cycling conditions according to manufacturer’s instructions. During thermal cycling, the threshold cycle (Ct) is defined as the cycle number when amplification of a specific PCR product can be detected. An average COX-2 or PPARγ Ct value and an average 18S Ct or beta actin Ct value were calculated from the three replicate reactions. The average Ct value for the housekeeping gene (beta actin) or (18S) were subtracted from the average Ct value for each target gene (Cox-2 and PPARγ) in order to normalize the amount of sample RNA added to the reaction. Relative quantification (RQ) describes the fold-change in expression of a gene of interest in a test sample relative to a calibrator sample. With the comparative Ct (ΔΔCt) method, the following formula was used to determine the level of the target gene mRNA in the drug-treated samples relative to the level found in the untreated sample (Applied Biosystems User Bulletin #2, October 2001): RQ= 2−ΔΔCt where ΔΔCt = avg Δ Ct (drug treated) − avg Δ Ct (untreated).
Mammary tumor material was fixed in buffered formalin and embedded in paraffin. The paraffin blocks were cut into 5-µm-thick sections that were placed on positively charged slides. The sections were dewaxed in xylene and hydrated through graded ethanol. Heat-induced antigen retrieval was then performed in 10 mmol/L sodium citrate (pH 6.0) in a microwave for 10 minutes. The endogenous peroxidase activity was blocked by immersing the slides in 3% H2O2 in PBS for 30 minutes. After 30 minutes of incubation with goat blocking serum, slides were incubated with either a 1:500 dilution of Cox-2 rabbit polyclonal antibodies (Cayman Chemical, Ann Arbor MI), or a 1:50 dilution PPARγ mouse monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz CA) at 4°C overnight followed by incubating with goat 2° anti-rabbit biotinylated anti-body for Cox2, or goat 2° anti-mouse biotinylated anti-body, for 30 minutes followed by streptavidin peroxidase for 30 minutes at room temperature. DAB (Sigma-Aldrich, St. Louis MI) substrate chromogen is applied for 4 minutes till the brown color develops. The slides were counterstained with hematoxylin and mounted/Permount. The COX-2 blocking peptide (Cayman Chemical, Ann Arbor MI) was used in combination with COX-2 Polyclonal antibody 2:1 (v/v) ratio respectively in a final concentration of 1:500 in PBS solutions then each slide was treated with 100 µl of the cocktail solution, then developed as indicated with the antibody alone. PPARγ blocking peptide (SantaCruz Biotechnology, Santa Cruz CA) was used in conjunction with PPARγ mouse monoclonal antibodies 5:1 (v/v) ratio in a final concentration of 1:50 in PBS solution then each slide was treated with 100 µl of the cocktail solution and developed as indicated with the antibody alone.
Animals were monitored for tumor formation by visual inspection and palpation. When the tumor reached 10% of the animal’s body mass, the animal was euthanized and recorded as having died of tumor formation. Animals that died for other reasons were censored. Survival analysis was performed using Graphpad Prism 5.0. Statistical testing was performed using a log rank test.
We obtained a mating pair of C3 (1)-SV40 Tag mice from Dr. Denise Connolly at Fox Chase who had recently imported them from Dr. Green’s lab for another study. As expected, upon aging we found that nearly 100% of the female C3 (1)-SV40 Tag mice developed histologically confirmed mammary carcinomas between 131 and 261 days of age with a median age of onset of 232 days (Fig. 1a). It should be noted that this was somewhat longer than the 140 days initially described in Maralokou et al. (14), and may reflect differences in the feeding and housing of the mice or perhaps a reduction in the number of integrated transgenes. The tumors that developed were quite aggressive and usually within three weeks after initial detection, the animals had to be sacrificed because tumor volume had exceeded the maximum size allowed by the Fox Chase Cancer Center Laboratory Animal Facility (LAF) (Fig. 1b). Consistent with the findings of Dr. Green and colleagues, we were able to distinguish between early lesions confined to the inner lining of the breast epithelium and more advanced lesions resembling invasive adenocarcinomas (Fig. 1c). We also examined the tumors from C3 (1)-SV40 Tag females for expression of COX-2 and PPARγ using immunohistochemistry (Figure 2) and found high-levels of both COX-2 and PPARγ protein. Based on these findings, we concluded that the C3 (1)-SV40 Tag mouse model is suitable to test the chemopreventive effects of Celecoxib and F-L-Leu.
We created a mouse breast adenocarcinoma cell line by culturing a tumor derived from a C3 (1)-SV40 Tag mouse (Fig. 3a). To confirm that the cells grown out from the tumor were actually tumor cells and not contaminating stromal fibroblasts, we examined the abundance of keratin and vimentin by western blot (Fig. 3b). Epithelial cells generally express keratin and do not express vimentin. Consistent with this expectation, we found that MMAC-1 cells exhibited abundant keratin expression and undetectable levels of vimentin. This expression pattern was identical to that observed for a human breast cancer cell line (MCF-7) and was opposite of that observed for a mouse fibroblast line and for tissue from the breast region of a healthy female mouse. We also examined COX2 and PPARγ mRNA expression in the absence and presence of both celecoxib and the PPARγ agonist F-L-Leu (Fig. 3c). From these results we conclude that the MMAC-1 cell line is epithelial in origin and expresses both COX2 and PPARγ.
The antiproliferative effect of celecoxib and F-L-Leu were evaluated on MMAC-1 cells in vitro (Fig. 4a). Significant dose dependent growth inhibition was observed with both drugs. However, Celecoxib was significantly more potent than F-L-Leu at inhibiting cell growth. The IC50 for Celecoxib was 79 µM compared to 242 µM for F-L-Leu.
We next examined how cells responded to a combination of Celecoxib and F-L Leu (Fig. 4b). In this experiment, we exposed cells to 50 µM celecoxib alone which resulted in growth inhibition of 9.9%. We also examined F-L-Leu alone at concentrations of 80 µM, 160 µM and 400 µM and found that MMAC-1 cells are inhibited 16.1%, 36.3% and 78.6%, respectively. Treating the cells with combinations of 50 µM celecoxib and either 80 µM, 160 µM, and 400 µM F-L-Leu resulted in 68%, 56.5% and 90% growth inhibition, respectively. The level of growth inhibition observed, especially at lower concentrations of F-L-Leu, was greater than what would be expected if the drugs were simply working additively. This indicates that celecoxiband F-L-Leu exhibit cooperativity with respect to growth inhibition of MMAC-1 cells.
We next examined the effects of F-L-Leu and celecoxib on breast tumor formation in C3 (1)-SV40 Tag mice. Mice at the time of weaning were given either a control diet or a diet containing celecoxib at a concentration of 1500, 750 or 375 mg/kg of chow or F-L-Leu at a concentration of 250, 125, or 62.5 mg/kg of chow. Animals were then monitored for up to 400 days for tumors.
We found that all three concentrations of celecoxib tested significantly delayed tumor onset (Fig. 5a, Table 1). For animals on 1500 mg/kg celecoxib the median age of tumor appearance was 292 days (vs. control P<0.0006). Animals on 750 mg/kg celecoxib had a median age of tumor appearance of 307 days (vs. control P<0.0007). Animals on 375 mg/kg celecoxib had a median age of tumor appearance of 293 days (vs. control P<0.0002). There was no significant difference in the age of tumor appearance for the different celecoxib concentrations used.
With regards to the PPARγ agonist F-L-Leu, we observed high levels of toxicity with 250 mg/kg doses and this arm was discontinued. We did not observe any statistical difference in the median age of tumor formation with either 125 mg/kg or 62.5 mg/kg F-L-Leu compared to controls (Fig. 5b, Table 1). These results suggest that F-L-Leu alone does not inhibit breast tumor formation in C3 (1)-SV40 Tag mice. However, addition of F-L-Leu to celecoxib did significantly increase tumor latency (Fig. 5c, Table 1). A combination of 750 mg/kg celecoxiband 62.5 mg/kg F-L-Leu resulted in an increase in median tumor latency from 307 days (750 mg/kg celecoxib alone) to 393 days (P<0.007). These results show that a combination of celecoxib and F-L-Leu are more effective than celecoxib alone at inhibiting breast adenocarcinoma development in C3 (1)-SV40 Tag mice.
In conducting these experiments we noticed that although tumor formation was delayedin celecoxib treated animals, once tumors became detectable they appeared to grow as rapidly as in the untreated animals. One possible explanation for this phenomenon would be that the tumors in the celecoxib treated animals might over express COX-2, thus making them resistant to the celecoxib. Therefore, we examined the relative amount of COX-2 mRNA present in the breast tumor of an untreated, celecoxib treated, and F-L-Leu treated mouse. In addition we examined PPARγ mRNA levels as well. As shown in Fig. 6, we found that COX-2 mRNA levels were 22-fold elevated in the tumor from a celecoxib treated mouse compared to the tumor from an untreated mouse. Interestingly, PPARγ levels were 4-fold elevated in the celecoxib treated animal. We also observed significant elevations in both the COX-2 and PPARγ mRNA in the F-L-Leu treated animal. These findings are consistent with the notion that resistance to celecoxib may be related to over-expression of the COX-2 protein. Also, these results suggest that there may be cross-talk between the COX-2 and PPARγ pathways.
In this study we have examined the effects of the selective COX-2 inhibitor celecoxib and the PPARγ agonist F-L-Leu both singly and in combination for their effects on tumor formation in a mouse model of breast cancer. We found that celecoxib, but not F-L-Leu, significantly delayed tumor development. However, the strongest inhibition occurred when both agents were combined. Our results support the idea that targeting COX-2 and PPARγ in combination may be a potential chemopreventive strategy for human breast cancer.
Our results are consistent with other published studies that show that the use of selective COX-2 inhibitors can slow breast tumor formation in mice. Lanza-Jacoby et al. showed that celecoxib in mouse diet (900 mg/kg) could inhibit breast tumor formation in HER2/neu mice, increasing the mean age of incidence from 266 to 291 days (6). We observed a somewhat larger effect in our C3 (1)-SV40 Tag mouse, with the mean age of incidence increasing from 232 days to 307 days in mice fed a diet with 750 mg/kg celecoxib. We did not see a statistically significant difference in the survival curves for the different doses of celecoxib, suggesting that lower doses may be equally effective.
We did not observe inhibition of tumor formation in mice given F-L Leu alone. We found that high doses of F-L-Leu (250 mg/kg) were associated with significant toxicity as indicated by premature death. Lower doses of F-L-Leu (125 mg/kg and 62.5 mg/Kg) were well tolerated, but failed to result in increased tumor latency. To the best of our knowledge, this is the first study to examine the effect of a PPARγ agonist on a mouse model of breast cancer. However, a recent study did find that the PPARγ agonist ciglitizone given intraperitoneally prolonged survival of nude mice injected with human OVCAR-3 ovarian carcinoma cells (21).
The most effective tumor inhibition was observed in mice fed a diet with both F-L-Leuand celecoxib. Mice given the combination diet lived on average 22% longer than mice on celecoxib alone. Our in vivo findings are consistent with our in vitro results in which we found that F-L-Leu and celecoxib acted coopertively to inhibit tumor cell growth. A possible mechanism by which PPARγ agonists effect breast tumor growth may involve PTEN. PTEN is a tumor suppressor gene that is inactivated in a variety of tumors including breast tumors. Overexpression of PTEN leads to both cell-cycle arrest and increased apoptosis (22). It has previously been demonstrated that the PTEN promoter contains a PPARγ binding site and that exposure of MCF-7 cells to the PPARγ agonists lovastatin and rosiglitazone can induce expression of the tumor suppressor gene PTEN (22). It is interesting to note that we did not observe tumor growth effects of F-L-Leu alone in vivo. This may be because it is not possible to achieve high enough levels of F-L-Leu in the blood. Alternatively, it may be that cells grown in culture are more sensitive to PTEN activation than cells inside a tissue.
Clinically, several prospective epidemiologic studies have shown that use of NSAIDS, inhibitors of COX-1 and COX-2, is associated with a lower risk of breast cancer, but other studies have not (8). With regards to selective COX-2 inhibitors, a recent case-control study indicated that women taking these compounds daily for two-years or more had a three-fold reduced risk of breast cancer (7). Less is known about the clinical effects of PPARγ agonists in humans; although, a pilot study of rosiglitazone therapy in women with breast cancer showed that it had no effect on Ki67 expression in tumor tissue (23). However, the same study did find that the drug was well tolerated without serious adverse events. The work presented here supports the idea that a combination of COX-2 inhibitors and PPARγ agonists together in subclinical doses may be useful for chemoprevention in women at high risk of developing breast cancer in either primary or secondary settings. Future clinical studies will determine if this concept is feasible.
We thank Denise Connolly and Jeffrey Green for the C3 (1)-SV40 Tag mice used in the study. We thank the following facilities at Fox Chase Cancer Center: Laboratory Animal Facility, Experimental Histopathology Facility, Tissue Culture Facility, and the Genomics Facility. We also thank Drs. Margie Clapper and Mary Daly for critical reading of this manuscript.
Grant Support: NIH R01 CA100422, CA06927, and an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center
Statement of Clinical Relevance Breast cancer is the second most frequent cause of cancer deaths in women in the United States. Chemoprevention with the use of anti-estrogens for women at high risk of developing breast cancer has been shown to be effective. However, since one-third of all breast cancers are estrogen-receptor negative, alternative chemopreventive strategies are needed. Here we show that a combination of a COX-2 inhibitor and PPARγ agonist can delay breast tumor formation in a genetically predisposed mouse model. Our findings suggest that these agents may be useful in preventing or delaying breast cancer in high risk individuals.