Diarylheptanoid phytoestrogens isolated from the medicinal plant curcuma comosa: Biologic actions in vitro and in vivo indicate estrogen receptor-dependent mechanisms

Abstract

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 occuring diaryl-heptanoids 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 (D1), 1,7-diphenyl-(6E)-6hepten-3=one (D2), and (3R), 1,7--diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reacion assay, a mammalian transfection model, and a uterptropic assay in mice. Results: All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells tranfected with estrogen receptor (ER) β or different ERα functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced with ICI 182,780 (ER antagonist), whereas only D2 was active with ERβ. An ERα mutant lacking the functional AF2 (activation function 2) region was not responsive to 17β-estradiol (E2) or to any of the diarylheptanoids, whereas ERα 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 E2, 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 (Cdlen1a) ER-mediated gene regulation. Conclusions: The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERα selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.