|dc.description.abstract||Hormone dependent breast cancer is a common disease in humans as well as in dogs. In both species it concerns mostly middle aged individuals. Women have a chance of 1 in 8 to develop breast cancer and unspayed bitches even have a chance of 1 in 4.
In women it is known that mammary fibroblasts produce estrone, which can stimulate estrogen receptor positive (ER+) epithelial cells when it is converted to estradiol. Estrone production is catalysed by the enzyme aromatase and this enzyme is a target in breast cancer therapy by the use of aromatase inhibitors like fadrazole. In dogs, the only used mammary cancer therapy is surgery.
It is known that a lot of people, including a large group of cancer patients, take dietary supplements. Many of these supplements contain fytoestrogens like the isoflavone genistein. Different studies showed inhibiting as well as stimulating effects of genistein on breast cells.
We investigated the effect of genistein on breast tissue by using a co-culture model in which primary mammary fibroblasts were co-cultured with a mammary ER+ epithelial cancer cell line (MCF-7) and a mammary epithelial cell line that represents healthy breast tissue (MCF-10A). Of special interest was to find out if genistein interferes with fadrazole therapy in breast cancer patients.
We used healthy tissue derived fibroblasts as well as tumour tissue derived fibroblasts from dogs. By using healthy and tumour fibroblasts we also wanted to learn more about the influence of fibroblasts on epithelial cells and their importance in breast cancer etiology and use in a breast cancer model.
Another interest was to find out if canine breast cancer shows the same characteristerics as human breast cancer and if canine primary fibroblasts can be a good alternative in a human breast cancer co-culture model. Subsequently, we wanted to learn if aromatase inhibiters might be useful in veterinary medecin as well.
Furthermore we were interested in the relation of Cox-2 and Cyp19 (aromatase enzyme gene) expression. A positive correlation has been described before in human breast cancer and we wanted to know if this correlation can also be seen in canine mammary cancer.
We performed aromatase assays and real time PCRs on primary canine fibroblasts and we could measure aromatase activity and its gene expression (Cyp19). Both were higher in tumour derived fibroblasts than in healthy tissue derived fibroblasts. Aromatase activity can not be increased with some known inducers of human aromatase, which suggests that Cyp19 expression is under control of different promotors in dogs. Fadrazole inhibited aromatase activity in canine fibroblasts very efficient (p<0.01), like it does in human cells and it might be useful in veterinary medicine as well.
Genistein at a concentration of 30 μM did increase Cyp19 expression significantly in healthy fibroblasts, but the increase in tumour fibroblasts was not significant.
In co-cultures of healthy fibroblasts with MCF-10A and MCF-7 cells, genistein exposure caused a non significant increase of Cyp19 expression. When tumour fibroblasts were co-cultured with MCF-10A and MCF-7 cells, genistein exposure had no effect or even an inhibiting effect on Cyp19 expression, which was significant in 1 experiment (P<0.05).
We used pS2 expression as an indicator of the estrogen respons in MCF-7 cells. Genistein at a concentration of 30 μM did not alter expression of this gene when MCF-7 cells were mono-cultured, but it did increase pS2 expression when MCF-7 cells were co-cultured with both tumour and healthy fibroblasts, which was significant in a co-culture with tumour fibroblasts (p<0.05).
When we exposed co-cultures of MCF-7 cells with healthy fibroblasts to genistein and fadrazole (10 μM) together, pS2 decreased (not significantly), probably because of aromatase inhibition by fadrazole and as a consequence less estrogen stimulation on epithelial cells.
When co-cultures of MCF-7 cells with tumour fibroblasts were exposed to both compounds, pS2 expression showed the same significant (p<0.05) elevated levels as when only genistein was added. Fadrazole did not show any inhibiting effect in this model. This suggests that genistein might negate the aromatase inhibiting effect of fadrazole therapy completely. Genistein should be considered as a potential risk for breast cancer patients who are using aromatase inhibitors as a therapy.
These results also indicate that tumour derived fibroblasts act different than healthy mammary tissue derived fibroblasts and that tumour fibroblasts interact differently with epithelial cells than healthy fibroblasts. Therefore, it seems necessary to use tumour derived fibroblasts from human tissue to opitimize the breast cancer co-culture model, which can be very useful in further research. Canine fibroblasts are probably not very useful, because we have seen in our aromatase assays that Cyp19 expression is regulated differently in dogs.
With real time PCR, we also found a strong positive correlation between Cox-2 and Cyp19 in canine mammary cells. These results suggest also a (partially) similar mammary tumour etiology with women and a possible role for cox-2 inhibiters, next to aromatase inhibiters in dogs.||