Diarylheptanoid Phytoestrogens Isolated from the Medicinal Plant Curcuma comosa: Biologic Actions in Vitro and in Vivo Indicate Estrogen Receptor-Dependent Mechanisms

BACKGROUND: Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phytoestrogens. However, the mechanism underlying their actions has not yet been identified. OBJECTIVES: We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo. METHODS: We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D I), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diplienyl-(4E,6,E)-4,6-heptadien-3-o1 (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice. RESULTS: All diarylheptanoids Lip-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) beta or different ER alpha functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ER alpha-ERE-driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ER beta. An ER alpha mutant lacking the functional AF2 (activation function 2) region was not responsive to 17 beta-estradiol (E-2) or to any of the diarylheptanoids, whereas ER alpha lacking the AF1 domain exhibited wild-type-like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E-2, up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation. CONCLUSIONS:, Me results suggest that the D3 diaryllieptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ER alpha selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.