2', 3', 4'-trihydroxychalcone is an Estrogen Receptor Alpha Coagonist

Estrogens in hormone replacement therapy (HRT) decrease menopausal symptoms, but increase the risks of reproductive cancers. The beneficial effects of estrogen on peripheral tissues and the adverse proliferative effects on the uterus and mammary gland are mediated by ERalpha. Currently HRT is approved only for short-term use. Short-term HRT works for decreasing symptoms associated with menopause, however, long-term usage is needed to prevent subclinical diseases. Because estradiol-bound ERalpha is an agonist in all tissues there is a need for development of more tissue-selective estrogens that can be used for both short and long-term HRT. Chalcones display antiproliferative activity through ERbeta and may have benefits on menopause-induced hot flashes, however activity through ERalpha and their effects on both estradiol gene regulation and physiology are less known. The present study aimed at identifying a chalcone compound which could change the activity of ERalpha in the presence of estradiol as a coagonist, thereby modulating the response of ERalpha on gene regulation and increasing its tissue specificity. After screening a panel of five chalcone compounds for estrogenic activity in cells cotransfected with ERalpha and an ERE upstream of tk-luciferase, 2', 3', 4'-trihydroxychalcone was identified as a unique ERalpha coagonist. 2', 3', 4'-THC displayed no estrogenic activity on its own, but synergized the activation of the ERE in the presence of estradiol. Competitive binding assays with [3H]-estradiol demonstrated that 2', 3', 4'-THC binds to both ERalpha and ERbeta. Estradiol and SERM-induced genes, KRT-19 and NKG2E, were not regulated by 2', 3', 4'-THC alone. Both KRT-19 and NKG2E were synergized with the combination of 2', 3', 4'-THC and estradiol. Tamoxifen and raloxifene induced expression of NKG2E, but did not synergize the expression in the presence of estradiol. The data demonstrates that 2', 3', 4'-THC behaves as a novel coagonist and not a SERM on gene regulation. A unique gene expression profile was induced in U2OSalpha cells treated with a combination of estradiol and 2', 3', 4'-THC for 24 hours with doses that would allow binding of both ligands to ERalpha at the same time. Functional analysis utilizing the binding affinities of estradiol, 2', 3', 4'-THC and another ERalpha binding chalcone, 2, 2', 4'-THC, demonstrated that a heteroligand complex consisting of estradiol and 2', 3', 4'-THC in ERalpha is possible. Despite the synergistic activation of estradiol regulated genes in U2OSalpha cells, the combination of 2', 3', 4'-THC and estradiol did not induce proliferation of MCF-7 cells. In the same cells 2', 3', 4'-THC blocked estradiol-induced G1 to S phase cell cycle transition without blocking proliferative genes regulated by estradiol. In female ovariectomized mice on a soy-free chow diet treated for four weeks (n=5, per group), 2', 3', 4'-THC did not cause uterine proliferation and blocked estradiol-induced proliferation and gene expression. Although 2', 3', 4'-THC blocked estradiol gene expression in uterine tissue, it regulated and modulated estradiol-induced genes in adipose tissue. Because 2', 3', 4'-THC displays unique coagonist activity through ERalpha without causing proliferation, it may be useful for future HRT and expanding our knowledge of ERalpha regulation and ligand interaction