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An estimated 10 million post-menopausal women in the United States use estrogen-based hormone replacement therapy (HRT) to ease menopausal symptoms and prevent osteoporosis. Epidemiological studies from the Women’s Health Initiative (WHI) indicate that HRT may actually increase the risk of breast cancer (BC). However, whether a causative relationship exists between HRT and BC remains unclear. In the present study, we have investigated the effects of HRT on proliferation of various BC cells with different estrogen receptor (ER) status. Human BC 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+ and progesterone receptor positive (PR+)] and the other MXT (ER but 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 consisting of estrogen (E2) and progesterone, have induced cell proliferation in ER+ cells. For example, at 10 μg/cm2 of dose, Prempro-treated MCF-7 cell proliferation was 2.3-fold of control in α-MEM completed with 10% fetal bovine serum (FBS). The same dose in MXT+ cells induced 1.7-fold of control of cell proliferation. For the concentrations tested (1, 2, 5, and 10 μg/cm2), it increased in a dose-dependent manner. In ER cell lines of MCF-10A and MXT, Prempro™ didn’t show any effects on cell proliferation. To further distinguish the effects of E2 and progesterone on cell proliferation, MCF-7 cells were separately treated with E2 (1x10−9 M) or progesterone (5x10-9 M). We have shown that E2 is the main component in inducing cell proliferation and progesterone has no effects. Tamoxifen, an E2 antagonist targeting ER, can inhibit the proliferating effects of E2. Because HRT is mainly used in post-menopausal women and this population generally has a higher level of iron due to the cessation of menstrual blood, we have further tested whether iron and E2 together enhance greater cell proliferation than either alone. MCF-7 cells were 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 blot of E2 treated cells showed that E2 also induced transferrin receptor, a membrane protein involved in cellular iron uptake, in ER+ cells but not in ER cells. In conclusion, our results indicate that E2 is most likely the effective compound in HRT that induces proliferation of only ER+ breast cells. Whether iron is an inter-dependent growth factor in E2-induced ER+ breast cell growth awaits further investigation.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]