ERß1 Sensitizes and ERß2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERß1 and ERß2 (isoforms of ERß), the second ER isotype. At present, the impact of ERß isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERß1 or ERß2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERß1 and MCF7-ERß2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERß1 cells and cancer-promoting effects in MCF7-ERß2 cells. Our data are favorable to ERß1 being a marker of responsiveness and ERß2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones.

The purpose of this study was to evaluate the response of estrogen target cells to a series of isoflavone glucosides and aglycones from Genista halacsyi Heldr. The methanolic extract of aerial parts of this plant was processed using fast centrifugal partition chromatography, resulting in isolation of four archetypal isoflavones (genistein, daidzein, isoprunetin, 8-C-ß-D-glucopyranosyl-genistein) and ten derivatives thereof. 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein were among the most abundant constituents of the isolate. All fourteen, except genistein, displayed low binding affinity for estrogen receptors (ER). Models of binding to ERα could account for the low binding affinity of monoglucosides. Genistein and its glucosides displayed full efficacy in inducing alkaline phosphatase (AlkP) in Ishikawa cells, proliferation of MCF-7 cells and ER-dependent gene expression in reporter cells at low concentrations (around 0.3 µM). ICI182,780 fully antagonized these effects. The AlkP-inducing efficacy of the fourteen isoflavonoids was more strongly correlated with their transcriptional efficacy through ERα. O-monoglucosides displayed higher area under the dose-response curve (AUC) of AlkP response relative to the AUC of ERα-transcriptional response compared to the respective aglycones. In addition, 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein displayed estradiol-like efficacy in promoting differentiation of MC3T3-E1 cells to osteoblasts, while genistein was not convincingly effective in this respect. Moreover, 7,4΄-di-O-ß-D-glucopyranosyl-genistein suppressed lipopolysaccharide-induced tumor necrosis factor mRNA expression in RAW 264.7 cells, while 7-O-ß-D-glucopyranosyl-genistein was not convincingly effective and genistein was ineffective. However, genistein and its O-glucosides were ineffective in inhibiting differentiation of RAW 264.7 cells to osteoclasts and in protecting glutamate-challenged HT22 hippocampal neurons from oxidative stress-induced cell death. These findings suggest that 7-O-ß-D-glucopyranosyl-genistein and 7,4΄-di-O-ß-D-glucopyranosyl-genistein display higher estrogen-like and/or anti-inflammatory activity compared to the aglycone. The possibility of using preparations rich in O-ß-D-glucopyranosides of genistein to substitute for low-dose estrogen in formulations for menopausal symptoms is discussed.

Estrogenic activity of isoflavonoids from the stem bark of the tropical tree Amphimas pterocarpoides, a source of traditional medicines.

Various preparations of the African tree Amphimas pterocarpoides Harms are traditionally used to treat endocrine- related adverse health conditions. In the ovariectomized rat, the enriched in phenolics fraction of the methanol extract of stem bark of A. pterocarpoides acted as vaginotrophic agent of considerably weaker uterotrophic activity compared to estradiol. Evaluation of the fraction and 11 isoflavonoids isolated therefrom using Ishikawa cells and estrogen receptor (ER) isotype-specific reporter cells suggested that the estrogenic activity of the fraction could be attributed primarily to daidzein and dihydroglycitein and secondarily to glycitein. The potency-based selectivity of daidzein, dihydroglycitein and glycitein for gene expression through ERß versus ERα, expressed relative to estradiol, was 37, 27 and 20, respectively. However, the rank order of relative-to-estradiol potencies of induction of alkaline phosphatase in Ishikawa cells, a reliable marker of estrogenic activity, was daidzein>dihydroglycitein>>glycitein. The considerably higher estrogenic activity of dihydroglycitein compared to glycitein could be attributed to the partial agonist/antagonist activity of dihydroglycitein through ERß. Calculation of theoretical free energies of binding predicted the partial agonism/antagonism of dihydroglycitein through ERß. The fraction and the isolated isoflavonoids promoted lactogenic differentiation of HC11 mammary epithelial cells at least as effectively as premenopausal levels of estradiol. This data suggests that the estrogenic activity of the fraction likely depends on the metabolism of glycitein to dihydroglycitein; that the fraction could exert vaginotrophic activity likely without challenging endocrine cancer risk more than estrogen-alone supplementation; and that the fraction's safety for the reproductive track warrants a more detailed evaluation.

A new class of phytoestrogens; evaluation of the estrogenic activity of deoxybenzoins.

Although deoxybenzoins are intermediates in the synthesis of isoflavones, their estrogenic activity has not been investigated. Eleven deoxybenzoins were synthesized and their estrogenicity was evaluated. While their affinities for estrogen receptors (ER) ERalpha and ERbeta were found grossly comparable to those of daidzein, some exhibited considerable selectivity and transcriptional bias toward ERbeta, which appeared to allow for enhancement of ER-mediated transcription via deoxybenzoin binding of ERbeta. Their activity to stimulate the proliferation of ER-positive breast cancer cells and regulate the expression of endogenous and stably transfected reporter genes differed considerably, with some inhibiting cell proliferation while effectively inducing gene expression at the same time. Molecular modeling confirmed that deoxybenzoins fit well in the ligand binding pocket of ERbeta, albeit with different orientations. Our data support the view that deoxybenzoins constitute a promising new class of ERbeta-biased phytoestrogens.