Identification of the Potent Phytoestrogen Glycinol in Elicited Soybean (Glycine max ) Stephen M. Boué, Syreeta L. Tilghman, Steven Elliott, M. Carla Zimmerman, K. Y. Williams, Florastina Payton-Stewart, Allen P. Miraflor, Melanie H. Howell, Betty Y. Shih, Carol H. Carter-Wientjes, Chris Segar, Barbara S. Beckman, Thomas E. Wiese, Thomas E. Cleveland, John A. McLachlan and Matthew E. Burow Endocrinology 2009 150:2446-2453 originally published online Dec 30, 2008; , doi: 10.1210/en.2008-1235 To subscribe to Endocrinology or any of the other journals published by The Endocrine Society please go to: http://endo.endojournals.org//subscriptions/ Copyright © The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online REPRODUCTION-DEVELOPMENT Identification of the Potent Phytoestrogen Glycinol in Elicited Soybean (Glycine max) Stephen M. Boue´,* Syreeta L. Tilghman,* Steven Elliott, M. Carla Zimmerman, K. Y. Williams, Florastina Payton-Stewart, Allen P. Miraflor, Melanie H. Howell, Betty Y. Shih, Carol H. Carter-Wientjes, Chris Segar, Barbara S. Beckman, Thomas E. Wiese, Thomas E. Cleveland, John A. McLachlan, and Matthew E. Burow United States Department of Agriculture (S.M.B., B.Y.S., C.H.C.-W., T.E.C.), Agricultural Research Service, Southern Regional Research Center, New Orleans, Louisiana 70179; Departments of Medicine, Section of Hematology and Medical Oncology (S.L.T., S.E., F.P.-S., M.E.B.), Pulmonary Diseases, Critical Care, and Environmental Medicine (S.L.T.), Surgery (M.E.B.), and Pharmacology (M.C.Z., A.P.M., M.H.H., B.S.B., J.A.M.), The Tulane Cancer Center (S.L.T., F.P.-S., J.A.M., M.E.B.), and The Center for Bioenvironmental Research (S.L.T., S.E., K.Y.W., B.S.B., T.E.W., J.A.M., M.E.B.), Tulane University Health Science Center, New Orleans, Louisiana 70112; and Xavier University School of Pharmacy (C.S., T.E.W.), New Orleans, Louisiana 70125 The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent es- trogen antagonists in vitro and in vivo. The discovery of the glyceollins’ ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 ␮M, which correlated with comparable colony formation of MCF-7 cells at 10 ␮M. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor- ␣ ⌱ ϭ 1). Competitive binding assays revealed a high affinity of glycinol for both ER ( C50 13.8 nM) and ␤ ⌱ ϭ ER ( C50 9.1 nM). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ER␣ and ER␤. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods. (Endocrinology 150: 2446–2453, 2009) pidemiologic studies support the view that consumption of form of isoflavones are formed from microorganism-induced Esoy prevents certain hormonally induced cancers and other fermentation and hydrolysis. diseases associated with estrogen deficiency (1–6). Asian women Isoflavones are considered phytoalexins, low-molecular- who consume a traditional low-fat soy diet have a 4- to 6-fold weight antimicrobial compounds and are synthesized de novo, lower risk of developing breast cancer when compared with accumulating in different plant tissues in response to stress, women living in the industrialized Western world (1–6). Many physical stimuli, or infectious agents (12–17). In soybean, of the health benefits of soybean have been attributed, in part, to several significant changes in isoflavone concentration occur the presence of isoflavones (3, 6–10). The primary isoflavones in in response to stress or elicitor treatment. The predominant soybean are genistein, daidzein, glycitein, and their respective phytoalexins produced in soybean are glyceollins I, II, and III, ␤-glucosides. In most soy foods, isoflavones are present primar- and these compounds demonstrate antifungal activity against ily as ␤-glucosides, esterified with malonic or acetic acid (11). several plant pathogens (13–18). Recent research in our lab- However, in fermented soy foods, higher levels of the aglycone oratory focused on the antiestrogenic activity of the glyceol- ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: CS, Charcoal stripped; DMSO, dimethylsulfoxide; E2, estradiol; ER, estrogen Printed in U.S.A. receptor; ERE, estrogen response element; FBS, fetal bovine serum; PgR, progesterone Copyright © 2009 by The Endocrine Society receptor; RBA, relative binding affinity; SDF, stromal-cell-derived factor. doi: 10.1210/en.2008-1235 Received August 20, 2008. Accepted December 19, 2008. First Published Online December 30, 2008 * S.M.B. and S.L.T. contributed equally to this work and both should be considered as first authors of this manuscript. 2446 endo.endojournals.org Endocrinology, May 2009, 150(5):2446–2453 Endocrinology, May 2009, 150(5):2446–2453 endo.endojournals.org 2447 lins in vitro (19) and in vivo (20, 21). Our results have led to glycinol has potent estrogenic activity and may prove beneficial the examination of other soybean phytoalexins for estrogenic for the use of postmenopausal women requiring hormone re- and antiestrogenic activities. placement therapy. Several other phytoalexins that are structurally similar to the glyceollins, occur at low concentrations including glyceollidin (22, 23), glyceocarpin (22–24), and glycinol (22–27) shown in Materials and Methods Fig. 1. Glycinol, a precursor in the biosynthetic pathway of the glyceollins, was first isolated by Lyne and Mulheirn (25). In a Chemicals and plasmids study by Weinstein et al. (24), glycinol inhibited the growth of all ICI 182,780 (Fulvestrant) was purchased from Tocris Bioscience six bacteria examined and the three fungi Phytophthora me- (Ellisville, MO). 17␤-Estradiol was purchased from Sigma (St. Louis, gasperma f. sp glycinea (race 1), Saccharomyces cerevisae, and MO). The solvents acetonitrile (HPLC grade), methanol, and ethanol were purchased from Aldrich Chemical Co. (St. Louis, MO). H O Cladosporium cucumerinium. These results suggested that gly- 2 treated with a Millipore system (Bedford, MA) was used during sample cinol possessed antimicrobial and antifungal activity; however, preparation procedures and HPLC analyses. no other biological activity was examined. Because of the struc- ER␤ cDNA was generously provided by Jan-Åke Gustafsson (Karolinska tural similarity to daidzein and coumestrol in addition to various Institute, Stockholm, Sweden) in pBluescript. ER␣ and ER␤ expression ␣ ␤ other environmental estrogens studied (28, 29), it was hypoth- vectors were constructed by inserting the ER and ER cDNA respec- tively into pcDNA 3.1 vector (Invitrogen, Carlsbad, CA). ER␣ cDNA esized that glycinol would have estrogenic activity. (2090 bp) was cleaved from plasmid (pBluescript) with BamHI/EcoRI In this study, we examined the estrogenic activity of the in- and then ligated into the pcDNA3.1. ER␤ cDNA (1460 bp) was duced soybean isoflavone glycinol using several in vitro assays. cleaved from Plasmid (pBluescript) with HindIII/BamHI and then Glycinol’s effect on estrogen receptor (ER) activity was analyzed ligated into the pcDNA3.1. Each construct was verified by detailed using an ER-positive MCF-7 human breast carcinoma cell line restriction mapping. HEK 293 cells were cultured in 5% charcoal-stripped (CS) DMEM transfected with an estrogen response element (ERE)-luciferase and seeded into a 24-well plate at a density of 50,000 cells/well and reporter. We also examined glycinol’s effect on expression levels allowed to attach overnight. Cells were transfected with 0.2 ␮g ER(2)-luc of two estrogen-responsive genes by semiquantitative real time plasmid (Panomics, Fremont, CA), 0.1 ␮g pcDNA, 0.1 ␮g pcDNA3.1B- RT-PCR: stromal-cell-derived factor (SDF)-1 and progesterone ER␤,or0.1␮g pcDNA3.1B-ER␣ plasmids the next day using Effectene receptor (PgR). Furthermore, viability and proliferative proper- transfection reagent (QIAGEN, Valencia, CA) according to the manu- facturer’s protocol. After a 6-h transfection, cells were treated with com- ties exerted by MCF-7 cells was explored using a colony forma- pounds [dimethylsulfoxide (DMSO), estradiol (E ), glycinol, or fulves- ␣ ␤ 2 tion assay. ER and ER binding assays were conducted to de- trant] overnight. On the following day, the cells were lysed with 150 ␮l termine the binding affinities of glycinol to both ER subtypes. In of the M-Per mammalian extraction reagent (Pierce, Rockford, IL). After addition, ligand-receptor docking studies using computer mod- 18 h cell lysates were measured for luciferase activity. One hundred eling were performed to analyze the interaction of glycinol with microliters of the cell extract were assayed using the Bright-glo luciferase assay substrate (Promega, Madison, WI) and determined in a AutoLumat the ER␣ and ER␤ ligand binding domains. These in vitro studies Plus lunimometer (Berthold, Bad Wildbad, Germany). support our hypothesis and demonstrate for the first time that Isolation of glycinol 2 Glycinol was isolated using a procedure developed by Qi et al. (27). Seeds from the soybean [Glycine max (L.)] cultivar Asgrow 5902 were OH O obtained from Helena Chemical Co. (Thibodaux, LA). Seeds were sur- OH face sterilized for 3 min in 70% EtOH followed by a quick deionized H2O 1.6 O rinse and two 2-min rinses in deionized H2O. Seeds were presoaked in OH sterile deionized H O for 4–5 h before placement into treatment cham- Glycinol 2 Daidzin bers (10 g/chamber). Each chamber consisted of a petri dish (100 ϫ 15 mm, four compartments), each compartment lined with two autoclaved 1.2 Glycinol filter papers (Whatman, Middlesex, UK) moistened with 0.5 ml distilled H2O.