DAMD 17 - 03 - 1 - 0717 TITLE : Hormone Replacement Therapy , Iron , and Breast Cancer

For many years it has been known that estrogen as well as hormone replacement therapy (HRT) increase the risk of breast cancer in post-menopausal women. Due to the cessation of menstrual bleeding, this population generally has a high level of iron. In the present study, we have investigated whether estrogen and iron synergistically enhance proliferation of various cells with different status of estrogen receptor (ER) and progesterone receptor (PR). Human breast cancer cell line, MCF-7 (ER) and non-cancerous breast epithelial cell line MCF-10A (ER), as well as two murine mammary cancer cell lines, one MXT (ER/PR) and the other MXT (ER /PR) were used. Crystal violet assay was employed to determine cell proliferation. Our results have shown that Prempro (Wyeth-Ayerst Pharmaceuticals), the most prescribed HRT drug, have induced cell proliferation only in ER cells. For example, at 10 μg/cm of dose, Premprotreated MCF-7 cell proliferation was 2.3-fold of control in α-MEM completed with 10% fetal bovine serum (FBS). To further distinguish the effects of estradiol (E2) and progesterone (Pg) on cell proliferation, MCF-7 cells were separately treated with E2 (1x10 M) or Pg (4x10 M). Results showed that E2 is the main component in inducing cell proliferation. MCF-7 cells were then treated with E2 and/or iron in α-MEM media containing 0.1% FBS (to minimize transferrin iron in serum, a confounding factor). Our results have shown that E2 and iron have induced higher cell proliferation (15.3-fold of control) than E2 (6.1-fold) or iron (7.9-fold) alone. Western blotting of E2-treated cells showed that E2 also induced transferrin receptor (TfR), a membrane protein involved in cellular iron uptake. In conclusion, our results suggest that iron and estrogen may be concomitantly involved in the cell proliferation of ER breast cells and together may contribute to the increased risk of breast cancer in post-menopausal women.