Figure 5

MIBE inhibits gene expression and proliferation induced by E2 in MCF7 cells. (a) Evaluation of mRNA expression of Cyclin D1 (Cyc D1), IRS-1, Progesterone Receptor (PR) and pS2 by real-time PCR in MCF7 cells. Cells were treated for 24 h with vehicle, 10 nM E2, 1 microM OHT and 10 microM MIBE alone or in combination, as indicated. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. Each data point represents the mean ± SD of three independent experiments performed in triplicate. (b) Immunoblots of protein levels of Cyclin D1 (Cyc D1) and IRS-1 from MCF7 cells. Cells were treated for 24 h with vehicle (-), 10 nM E2, 1 microM OHT and 10 microM MIBE alone or in combination, as indicated. β-actin serves as loading control. Data shown are representative of three independent experiments. (c) Densitometric analysis of three independent experiments, protein expressions are normalized to beta-actin. (•), (◦) indicate P < 0.05 for cells receiving vehicle versus treatments. (d) MCF7 cells were treated for five days with vehicle, increasing concentrations (logarithmic scale) of E2, OHT and MIBE and counted on Day 6. (e) Cells were treated for five days with vehicle (-), 10 nM E2, 1 microM OHT and 10 microM MIBE alone or in combination, as indicated, and then the proliferation was evaluated by cell counts on Day 6. The proliferation of cells receiving vehicle was set as 100% upon which cell growth induced by treatments was calculated. Each data point is the average ± SD of three independent experiments performed in triplicate. (•) indicates P < 0.05 for cells receiving vehicle (-) versus treatments.