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
). 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
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
-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
).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
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.