Effects Of ERK1/2 Signaling On The Estrogenic Activities Of Genistein And Equol

The mitogen-activated protein kinases (MAPKs) are crucial components of signaling cascades that regulate numerous physiological processes during development and pathogenesis. At least four subfamilies have been identified: extracellular-regulated protein kinases1 and 2 (ERK1/2), ERK5, c-Jun NH2-terminal protein kinases (JNKs), and p38 MAPKs. Of the four major MAPKs pathways in human tissues, ERK1/2 is most relevant to breast cancer. Estrogen signaling is involved in the proliferation and differentiation of estrogen-responsive cells. The proliferative effect of estrogens on breast cancer cells is mediated mainly through estrogen receptors (ER).The two major isoflavones in soybean are genistein and daidzein, and equol is the most biologically active metabolites of daidzein. Phytoestrogens have attracted attention because of their putative value as natural alternatives to hormone replacement therapy for relieving post-menopausal symptoms. The classical effects of estrogens and phytoestrogens are mediated via transcriptional activation of estrogen-responsive genes. The receptor-hormone complex binds to a specific estrogen response element (ERE) in the promoter region of target genes, leading to transcriptional activation. Also, activation of target genes by estrogens may also be mediated by other transcription factors, independent of the ERE. Recent evidence implicates cell surface receptors in the rapid responses to estrogen and phytoestrogens. Membrane-associated ERs interact with cell signaling pathways, such as the mitogen-activated protein extracellular kinase/extracellular signal regulated kinase (MEK/ERK) and phosphatidylinositol-3-kinase (PI3K) pathways and thus initiate non-genomic effects. Signal transduction pathways may connect the nongenomic actions of estrogens to genomic responses. This type of nongenomic-to-genomic signaling is a distinct mechanism by which ERs regulate transcription at alternative response elements. ERαis a major growth regulator for many breast cancers and provides an exploitable target for therapy. A greater understanding of ER-mediated events, as well as the interactions of ER with ERK1/2 signaling pathways, should lead to more targeted strategies for the treatment of hormone-dependent breast cancer.Objectives:To study the role of ERK1/2 signaling pathway in estrogenic activity of genistein and equol, and to investigate effects of ERK1/2 signaling pathway on ERα-mediated transcription activation.Methods:Effects of genistein/equol alone or in combination with signaling inhibitors on cell proliferation of estrogen-dependent MCF-7 cells were observed with the MTT assay. The cell cycle distributions were detected by flow cytometry. The mRNA expression of ERαtarget genes were measured by Real-time PCR. The protein expressions were detected by using western blotting. The MDA-MB-231 cells were co-transfected with three plasmids including rat ERαexpression plasmid rERα/pCI, reporter plasmid pERE-aug-Luc containing the reporter gene Luc regulated by the estrogen responsive element (ERE) and the control plasmid phRL-tk to develop the rERαmediated reporter gene assays.Results: Physiological concentrations of genistein and equol stimulate cell growth and S-phase entry in MCF-7 breast cancer cells by MTT and flow cytometry respectively; these actions are blocked by the ERαantagonists, OHT and ICI, and by the MEK1/2 inhibitor, U0126, suggesting that an ER-dependent mechanism and the MEK/ERK signaling pathway are involved in these stimulations. ERαtransactivation and expression of mRNA for the estrogen-responsive gene pS2,GREB1 and EEIG1 induced by genistein and equol were measured by dual-luciferase reporter assay and real-time PCR respectively; these responses are completely blocked by U0126, indicating that MEK/ERK signaling pathway is necessary for ER-mediated transcription. Genistein and equol induce delayed and prolonged phosphorylation of ERK1/2 by Western blotting; moreover, the anti-estrogen ICI has no effect on the genistein- and equol-induced delayed phosphorylation of ERK1/2; this suggests that activation of ERK1/2 lies upstream of ER-mediated signaling.Conclusions:Low concentrations of genistein and equol (<10μM) stimulate cell growth and cell cycle progression via ER-mediated transcription and activation of ERK1/2. And delayed activation of ERK signaling is necessary for transactivation of ERαinduced by genistein and equol.