Environmental Signaling in Urban Ecosystems
7th Annual Symposium on the Environment and Hormones
April 13-16, 2008
Hosted by: Tulane/Xavier Center for Bioenvironmental Research
We contend that the phenomenon known as endocrine disruption is a manifestation of a larger biological issue – that is, the complex signaling networks that exist within and between organisms. These signaling networks can be maintained or perturbed by environmental factors. At e.hormone we intend to develop the tools for a new science of environmental signaling and to invite participants to further develop the Environmental Signaling Network (an NSF-sponsored activity).
Toxic activities in wood dust
Mark Wilson, Roy Rando, and Chuck Miller
Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine
Wood dust is a complex agent that causes numerous human health effects, including cancer. Epidemiological studies indicate professional woodworkers have a two- to ten-fold increased risk for developing cancers in the upper respiratory (nasal) tract. Few mechanistic and molecular toxicity studies exist for wood dust, so we are employing various cellular assays to better define the toxic nature of wood dust. Species-specific wood dust was generated using a radial sander and plywood dust was collected at a plywood manufacturing facility. Following collection, the dusts were extracted in methanol, dried, and re-suspended in dimethyl sulfoxide (DMSO). Methanol extracts from various wood types activated human aryl hydrocarbon receptor signaling in a yeast bioassay. Methanol extracts from red oak wood dust reduced growth and killed cultured Chinese hamster ovary (CHO) cells. Some surviving cells were multi-nucleated. There is evidence of toxicity associated with high doses of some dust extracts in the yeast assay. Collectively, the data suggest that wood dust, perhaps in conjunction with other agents present in woodworking operations, contains a compound or compounds that could contribute to carcinogenic processes.
DIF-1, a putative morphogen of the cellular slime mold Dictyostelium discoideum, possesses anti-cancer properties
Whitney L. Stutts*, Melyssa R. Bratton*+, John A. McLachlan*
*Tulane/Xavier Center for Bioenvironmental Research, +Tulane University Medical Center, Hematology/Oncology Division
According to the American Cancer Society, the number of new cases of breast cancer in men and women was estimated at 214,640 in 2006, while the estimated death toll due to breast cancer was 41,430. Although many treatment options are available to breast cancer patients, many of them are accompanied by harsh side effects or eventual drug resistance. Phytoestrogens have been studied for their impact on cancer cell growth; however, little research has been done with mycoestrogens. Differentiation-inducing factor-1 (DIF-1) is a putative morphogen of the cellular slime mold Dictyostelium discoideum and has been shown to possess anti-cancer properties. Previous work in our lab has shown DIF-1 acts as an inhibitor of ERα activity in ERE luciferase assays. To further understand the mechanism of DIF-1 antagonism of ERα activity, we exposed MCF-7 breast cancer cells to DIF-1 followed by western blot analysis which indicated that DIF-1 caused a significant reduction in expression of ERα. Using RT-PCR, we can show that DIF-1 is not only capable of down-regulating expression of ER-sensitive genes but also causes a decrease in expression of the receptor itself by ~85% compared to estrogen alone, explaining the western blot results. Consistent with these data, colony assays reveal that 1 μM DIF-1 reduces cell proliferation in MCF-7 cells by ~80% compared to estrogen alone. These results show that DIF-1 is able to antagonize ERα activity by disrupting the receptor’s transcription in MCF-7 breast cancer cells, thereby resulting in reduced proliferation of these cells.
Reproductive Abnormalities In The Giant Toad Bufo marinus Are Associated With Agriculture.
Krista A. McCoy 1, 2,3 Lauriel J. Bortnick 2, Chelsey Campbell 2, Heather J. Hamlin2, Colette St. Mary 1,2, Louis Guillette1,2
1 School of Natural Resources and Environment, College of Agriculture and Life Sciences University of Florida, 103 Black Hall, PO Box 116455, Gainesville Florida 32611-6455
2 Department of Zoology, College of Liberal Arts and Sciences University of Florida, 223 Bartram Hall, PO Boz 118525, Gainesville Florida 32611-8525
3 Department of Biology, College of Arts & Sciences Boston University, 5 Cummington St. Boston Massachusetts 02215
Endocrine disrupting chemicals (EDCs) are ubiquitous pollutants that do not typically induce mortality at ecologically relevant concentrations, but instead can negatively affect physiological, behavioral or developmental processes that are influenced by the endocrine system. Agricultural contaminants are known to disrupt endocrine systems of wildlife, including amphibians, and recent studies have suggested that agricultural contaminants are associated with amphibian reproductive abnormalities and population declines. We survey abnormalities in gonadal morphology in the giant toad, Bufo marinus, and quantify the association between gonadal abnormalities and agricultural habitats. We find that the number of abnormalities per individual as well as the frequency of intersex gonads is higher at agricultural sites. We then evaluate whether these abnormalities are associated with altered gondal function. Testosterone, but not 17β estradiol, concentrations were altered and secondary sexual traits were either feminized (increased mottling) or demasculinized (reduced forearm width and nuptial pad number) in intersex toads. Females did not differ across sites. However, males from agricultural areas had hormone concentrations and secondary sexual traits that were intermediate between intersex toads and non-agricultural male toads. In summary, B. marinus living in agricultural areas have altered gondal morphology and function. Steroid hormone concentrations and secondary sexual traits are known to correlate with reproductive activity and success, thus these effected toads may have reduced reproductive success. Further, other amphibian species living in the same agricultural areas could be similarly affected.
Effect of Propylthiouracil (PTU)-induced Hypothyroidism on the Developing Female American Alligator Reproductive Tract
Nicole L. Botteri, Ashley Boggs, Lori Albergoti, Dieldrich Bermudez, and Dr. Louis J. Guillette, Jr.
Department of Zoology, University of Florida, Gainesville, Fl
Thyroid hormones are essential for normal female vertebrate reproduction, and an imbalance of thyroid hormones can cause a suite of well-documented reproductive abnormalities including ovarian atrophy, amenorrhea, spontaneous abortion, and infertility. A common thyroid disorder, hypothyroidism, results in a decrease in the circulating levels of plasma thyroxine (T4) and triiodothyronine (T3), which has been subsequently linked to a decrease in follicle stimulating hormone (FSH) and luteinizing hormone (LH). Studies have been performed where hypothyroidism has been induced through administration of propylthiouracil (PTU) to observe the effects on the mammalian female reproductive system. However, comparative studies on the effects of PTU-induced hypothyroidism on the developing female reproductive system are lacking. American alligator (Alligator mississippiensis) eggs were collected from Lakes Apopka and Woodruff in Florida and treated in ovo with PTU during the developmental periods of sex determination and gonadogenesis. Treatment groups included PTU dosage at 40 pg/dose, 40µg/dose, and a vehicle (ethanol) control. Animals were raised to one month post-hatching and given a FSH injection 24 hours prior to necropsy. Paired reproductive tracts were collected and are currently being analyzed using real-time quantitative PCR to determine changes in gene expression and histologically to assess potential morphological changes. This study will provide insight into potential developmental and reproductive effects that can be acquired from areas of perchlorate, nitrate, and thiocyanate contamination, as these contaminants are associated with hypothyroidism.
Differential Gene Expression between Normal Uterine Smooth Muscle and Leiomyoma Cells is Associated with Epigenetic Differences
Tung-chin Chiang 1, Mary M. Nguyen 1, Erica Nierth-Simpson 1, Ashley Fornerette 1, Jovanny Zabaleta 2,3 L Joseph Su 3,4, John A. McLachlan 1
1 Tulane/Xavier Center for Bioenvironmental Research, Tulane University, New Orleans, LA 70112
2 Department of Genetics, Louisiana State University, New Orleans, LA 70112
3 Stanley S Scott Cancer Center Louisiana State University, New Orleans, LA 70112
4 School of Public Health, Louisiana State University, New Orleans, LA 70112
Differential gene expression between uterine leiomyoma (LM) and adjacent normal smooth muscle (SMC) has been found in clinical tissues and it is likely that this differential gene expression contributes to leiomyoma pathology. Genetic studies of leiomyomas have not found key mutated genes and chromosomal alterations are believed occur after the initiations of leiomyoma tumors during clonal expansion. Epigenetic changes that alter expression without changing DNA sequence are found in many cancers and we hypothesize that they may contribute to leiomyomas. The purpose of this study is to investigate whether there are differential epigenetic signatures, such as DNA methylation, between LM and SMC and their involvement in differential gene expression between these two cell types.
In order to alter epigenetic status to observe downstream effect on gene expression, two in vitro uterine stable cell lines, utLM-hT and utSMC-hT, were used. We first analyzed promoter DNA methylation status using a methyl bead array, which contains 1505 CpG sites of 807 genes. Our data showed a differential methylation profile between the two cell lines. There are 317 CpG sites methylated and 255 CpG sites demethylated in utLM-hT cells versus utSMC-hT cells (p<0.05). In addition, cells treated with the demethylation agent, 5-Aza-2'-deoxycytidine (DAC), resulted in altered gene expression in 50% of selected genes between the two cell lines. This is the first study investigating the possible role of epigenetic changes in uterine leiomyoma. The long-term goal of this study is to learn whether the differing epigenetic signatures contribute to LM’s estrogen sensitivity.
Nitrate as an Endocrine Disrupting Contaminant in Cultured Siberian Sturgeon
Heather J. Hamlina, b,c,*, Brandon C. Moorea, Thea M. Edwards a, Iskande L.V. Larkin a, Ashley Boggs a, William J. High a, Kevan L. Main b, Louis J. Guillette Jr. a
a Department of Zoology, University of Florida, 223 Bartram Hall, PO Box 118525, Gainesville, FL 32611-8525, USA
b Mote Marine Laboratory, Center for Aquaculture Research and Development, 1600 Ken Thompson Pkwy, Sarasota, FL 34236, USA
c Department of Fisheries and Aquatic Sciences, University of Florida, 7922 NW 71st Street, Gainesville, FL 32653, USA
The endocrine disrupting actions of various environmental contaminants have become the focus of much research. Although nitrate is a ubiquitous component of aquatic environments, and has become a global pollutant in a variety of aquatic systems, it has only recently begun to receive attention for its ability to alter endocrine function. Aquaculture environments with limited water exchange often contain nitrate levels far in excess of natural environments, yet nitrate’s impact on the reproductive health and endocrine function of commercially important species residing in these environments has been absent from this research. Understanding the endocrine disruptive effects of nitrate is especially important to sturgeon, whose economic viability relies heavily on the proper production of eggs. This study examined the effects of elevated nitrate on molecular endpoints of steroidogenesis, as well as plasma concentrations of cortisol, glucose, 17β-estradiol, testosterone and 11-ketotestosterone, on cultured Siberian sturgeon (Acipenser baeri). In the first study, two groups of fish were exposed to a low and high nitrate concentration (11.5 and 57 mg/l nitrate-N respectively) for a period of 1-month. The second study exposed two groups of fish to 1.5 and 57 mg/l nitrate-N respectively. Results revealed that elevated nitrate caused an increase in plasma sex steroids, but did not alter the gene expression of P450SCC or ERβ. Since gene expression of P450SCC or ERβ did not correlate with increases in circulating concentrations of plasma sex steroids, these results indicate another mechanism, such as altered liver clearance, could be involved.
Effect of Novel Sphingosine Kinase Inhibitors on Estrogen Receptor in Endocrine Sensitive and Endocrine Resistant Breast Cancer
Meacham WD1, Antoon JW1, Burow ME2, Smith CD3, Slaughter EM1, Rhodes LV2, Muir SE1, Guillot LM2, Bratton MR2, Beckman BS1
1 Tulane University, Dept. of Pharmacology
2 Tulane University, Dept. Of Medicine, Tulane University Health Sciences Center
3 Medical University of South Carolina, Charleston, SC
Endocrine resistance is a major cause of tamoxifen and fulvestrant treatment failure in breast cancer. Recent research suggests that irregularities in endogenous sphingolipids contribute to chemoresistance. More specifically, altered levels of ceramide and sphingosine-1-phosphate are present in resistant breast cancer cells. We found that exogenously administered Sphingosine Kinase Inhibitors (SKIs) decrease breast cancer colony formation in vitro. The clonogenic survival (IC50) of exogenous 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) was 1.735 uM in endocrine sensitive MCF-7K cells and was 1.3645 uM in tamoxifen resistant MCF-7T cells. SKI-II was also shown to decrease the amount of estrogen receptor expressed in MCF-7 cells, though the mechanism of action of this event is currently unknown. We found that SKI-II markedly reduced ERE-luciferase activity in estrogen receptor transfected HEK-293T cells by 42% when treated at the IC50 value. The SKI 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) was shown to be effective in vivo at both low and high doses. 14 days of intraperitoneal treatment at 20 mg/kg/day and 100 mg/kg/day reduced tumor volume by 63% and 67%, respectively (n=10). At day 14 of both concentrations of dosing, there was no marked weight loss or visual signs of metastasis to the heart, lungs, liver or uterus. These results show that sphingosine kinase-based therapy represents a potential chemotherapeutic treatment of endocrine sensitive and endocrine resistant breast cancer.
Effects of an Anti-androgenic Pesticide, Vinclozolin, on Song Production in the Dark-eyed Junco (Junco hyemalis).
Danielle Satre; Michael S. Reichert; Cynthia Corbitt
University of Louisville Biology
Endocrine disrupting chemicals (EDCs) produce subtle changes in physiology and behavior by acting as hormone mimics or antagonists. The fungicide vinclozolin acts as an anti-androgen and has been shown to affect reproductive and other behaviors in rodents. To investigate the possible effects of exposure to EDCs on the development of song (i.e., song crystallization), we examined the effects of vinclozolin in wild-caught adult Dark-eyed Juncos (Junco hyemalis). Songbirds sing to establish and defend territories, as well as attract mates, and this behavior is largely under hormonal control. For many temperate species, testosterone has powerful activational effects on neuroplasticity and song production in adulthood. We hypothesized that vinclozolin would disrupt song production. Starting in February, adult male juncos received daily oral gavage for 10 weeks with 100uL of either vehicle (organic canola oil) or 2mM vinclozolin dissolved in vehicle. After one week of dosing, juncos were photostimulated (16L:8D) and once singing began, were audiotaped using a directional microphone, twice for 15 minutes each. In addition, they were videotaped during song sessions. Audiotapes were digitized and analyzed using Syrinx software. Spectrograms produced by Syrinx were then used to describe each individual's song repertoire. While some song measures did not differ significantly between groups (# songs types and song duration), we found a statistically significant difference in the variation of inter-syllable distance (the time between syllables), suggesting that vinclozolin may disrupt seasonal song development by affecting the degree of song crystallization in adult male juncos. Support contributed by NSF IBN0137548 (CC).
The Application of SuperArray Focused PCR Arrays to the Study of Genes Regulated by Endocrine Disrupting Chemicals
H. Chris Segar, Elena V. Skripnikova, and Thomas E. Wiese
Xavier University College of Pharmacy
One of the central questions regarding exposure to estrogen-active environmental endocrine disruption chemicals is that of altered endogenous gene regulation. While traditional gene arrays have been used to characterize the activity of a number of xenoestrogens, the process is expensive, the very large amount of data generated can be daunting to analyze and the function of genes identified may be unknown. In this report, we describe the application of the SuperArray focused PCR Array to define genes in breast cancer cells that are regulated by environmental endocrine disrupting chemicals and plant extracts. The SuperArray focused PCR Arrays combine real-time PCR detection with array technology in a 96 well format such that the regulation of 84 known genes are evaluated between treatments. Running the PCR Array involves loading super mix on to all wells followed by 40 PCR cycles and a first derivative dissociation curve for each well. The specific array plates used in this study are focused on estrogen receptor and breast cancer signaling pathway genes. We have shown that the gene induction data obtained from the PCR Array plates is reproducible between RNA-cDNA preps as well as between cell passage. We present the application of the PCR Array system to determine the gene regulation activity of the pesticide DDT and the extracts of dietary supplements used to treat postmenopausal symptoms. This technology appears to be a simple method to identify abnormal gene regulation events induced by xenoestrogens in cell culture models at a lower cost and more specific focus than the traditional microarray systems.
Validation of new screening method for endocrine disrupting chemical using crustacean, Daphnia magna.
Norihisa Tatarazako(1), Shigeto Oda(1), Taisen Iguchi(2)
1 National Institute for Environmental Studies
2 National Institute for Basic Biology
Crustaceans are major constituents to aquatic ecosystems that provide a variety of ecological and economic services. Ecdysteroids and terpenoids are two major classes of terminal signaling molecules in these cascades. Hormones from these two classes function independently or in concert to regulate various processes. Disruption of the ecdysteroid/terpenoid signaling pathways in crustaceans has been associated with aberrations in growth, metamorphosis, reproductive maturation, sex determination, and sex differentiation. TG 211, Daphnia magna Reproduction Test is one of the Test Guidelines for Testing of Chemicals, which was revised in September 1989 from TG 202, Part II, Daphnia sp. Reproduction Test. On the basis of an understanding of the endocrinology of cladocerans in recent years, this Test Guideline was updated for the purpose of screening for endocrine disrupting chemicals in crustaceans. The previous TG 211 was covering various endpoints among which the change in offspring sex ratio could be included. In this test guideline, new endpoints such as offspring sex ratio and molt inhibition are explicitly included for detection of the effect of endocrine disrupting chemicals in crustaceans. The main difference between this enhanced version and the previous version of the Guideline is an explicit inclusion of new endpoints for the detection of endocrine disruption in Daphnia. We introduce about the validity of this testing method here.
Effects of Dietary Phytoestrogens in a Rodent Model of Aging
Monica Unselda, Mirna Bajramovica, Jared Woodsa, Anne Oliphanta, Andrew Vonberga, Michael Giurgevicha, Angy Mounira, Kesha Rhodesa, Shafeeq Sheikha, Alison Wheatleya, Andrew M. Robertsb, and Cynthia Corbitta
a Dept. of Biology, University of Louisville, Louisville KY 40292
b Dept. of Physiology and Biophysics, University of Louisville, Louisville KY 40292
Women approaching menopause may consider using phytoestrogen (plant-made estrogenic compounds) supplements to relieve menopausal symptoms because these products are viewed as being 'natural' and potentially less harmful than traditional Hormone Replacement Therapy. Among the various effects of estrogens on different tissues, studies have found them to be potentially neuroprotective against age-related deficits in cognition and lung function. Using a rodent model of aging, we tested two hypotheses; that phytoestrogens are also neuroprotective for memory and that they protect the lungs from loss of elasticity after ovariectomy. Two different cohorts (summer, fall) of rats (n=48) were ovariectomized to eliminate ovarian estrogen, then were fed a diet either with (Soy+) or without (Soy-) soy phytoestrogens for 3 weeks before being tested for anxiety-like behaviors in an open field protocol and in object, placement and context memory trials over the course of 3 weeks. Experimental groups (n=12) were Middle Age Soy-, Middle Age Soy+, Young Soy-, and Young Soy+. The memory tests took advantage of a rodent's attraction to novelty so no training or task acquisition is necessary. Results from the mazes failed to disclose any general pattern of performance based on diet or age. After the memory trials were completed, static lung compliance measures were taken on a subset of the rats (n=4/group). We found a statistically significant effect of diet, but not age, on static lung compliance, suggesting a possible phytoestrogen-induced difference in lung elasticity. Supported by NIH grant R03AG25144-2 to CC.
Conformational Searching, Flexibility, and Docking/Scoring to ER LBD of Five Mycoestrogens and their similarity to 17β-Estradiol and Diethylstilbestrol
K Y B Williamsa,c, John A. McLachlanb,c , and Thomas E. Wiesec,d
a Biomedical Sciences Program, Tulane University, New Orleans, Louisiana 70112
b Department of Pharmacology, Tulane University, New Orleans, Louisiana 70112
c Center for Bioenvironmental Research, Tulane University, New Orleans, Louisiana 70112
d Xavier University College of Pharmacy, New Orleans, Louisiana 70125
Mycoestrogens are mycotoxins produced by the Fusarium species of fungus that are known to induce estrogen activity. Mycoestrogens are metabolized to less active compounds and they are found on corn or wheat products. As contaminants, mycotoxins have been found in corn products and cereals, and upon digestion they can have an effects on the endocrine system. This study focuses on Mycoestrogens that are known to activate the estrogen receptor (ER). We believe mycotoxins have the ability to activate the estrogen receptor by mimicking the structure of its endogenous estrogen, 17β-Estradiol (E2). Therefore, we investigated how the conformation, flexibility, and general structure of the mycotoxins relate to the binding that occurs between the mycotoxin and the ER, and how this correlates to actual Estrogen Receptor binding activity. Molecular models of all isomers and enantiomers of five Mycoestrogens were generated using the computer program Stereoplex and the 3D structures were produced using the program Concord. All unique conformations of the models were generated using Confort followed by geometry optimization and a root means squared similarity criteria of 0.75Å. Comparison of the compounds to available crystal structures of the same Mycoestrogen from the Cambridge Structural Database was done to ensure the correct structures were produced, and the compounds where then aligned using FlexS with DES and 17β-Estradiol (E2) as templates. The models were docked to the alpha isoform of the Estrogen Receptor ligand binding domain from the 1ERE crystal structure using Surflex-Dock, and scored using CScore. The docking profiles show that very subtle differences exist within the compounds that allows them to mimic and bind to the estrogen receptor to allow activation of the receptor, and their alignment suggest features that are consistent to allow for mimicking 17β-Estradiol. We conclude that the conformation searching, flexibility analysis, and the docking profiles focuses on features of the compounds that allow for activation of the estrogen receptor.
AKT Cross-talk with ERβ in Breast Cancer.
Syreeta L. Tilghman 1,2, Melyssa Bratton 1,2, Tung-Chin Chiang 2, Lyndsay Vanhoy 1, Steven Elliot 1, Erica Nierth 2, Ashley Fornerette 2, John A. McLachlan 2 and Matthew E. Burow 1,2.
1 Tulane University, Department of Hematology and Medical Oncology
2 Tulane/Xavier Center for Bioenviromental Research
The cross-talk between growth factor activation of signaling proteins such as phosphatidylinositol 3-kinase (PI3K)-AKT and estrogen receptor α (ERα) has been shown to mediate tamoxifen resistance in breast cancer. Recently our lab demonstrated for the first time the cross-talk between AKT and the poorly understood steroid receptor, estrogen receptor β, (ERβ) pathways and has implicated that this may be an important aspect that may influence breast cancer responsiveness to endocrine therapy. Here, we investigate the proliferation and viability of MCF-7 breast cancer cells treated with estrogen (E2), the ERβ specific agonist diarylpropionitrile (DPN), insulin-like growth factor (IGF), antiestrogens(AE) (fulvestrant and tamoxifen) and AKT inhibitor II. Colony formation assays showed that as expected E2 and DPN to a lesser extent caused the greatest stimulation in colony formation while both AE and the AKT inhibitor decreased colony formation. Reporter gene assays in both HEK 293T and MCF-7N cells demonstrated that DPN caused a more potent induction of ERβ potentiation than E2. The DPN effect was further enhanced by overexpressing AKT or the constitutively active AKT. IGF had a similar effect as E2, while both AEs and the AKT inhibitor reduced ERβ potentiation to levels slightly lower than control. Given that estrogen induces a cascade of rapid signaling events we investigated the involvement of DPN to activate several components of the PI3K-AKT signaling cascade. Time course studies (0 min, 30 min, 1 hr, 2 hr and 24 hr) were performed with MCF-7 cells using either E2 or DPN treatment. While both treatments did not cause a statistically significant change in total AKT expression, phospho-AKT (ser 473) expression increased as well as phospho-GSK-3β. Gene expression of several estrogen responsive genes [progesterone receptor (PgR), stromal cell derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF)] was also examined using real-time PCR. Results demonstrated that both E2 and DPN treatment induced gene expression of PgR and SDF-1 3-6 fold above the control. Taken together this data begins to elucidate a potential mechanism by which AKT regulates ERβ transcription activity in breast cancer which may prove to be useful in developing treatment strategies for endocrine resistance breast cancer.
Phytochemical Glyceollin I, A Novel Anti-estrogenic Isolated From Soy
Maria C. Zimmermann*, Melanie H. Howell*, James W. Antoon*, Steven M. Boue***, Steven Elliott*, John A. McLachlan*, Thomas E. Wiese**, Matthew E. Burow*,
Barbara S. Beckman*
* Tulane University Health Sciences Center, New Orleans LA.
** Xavier University, New Orleans LA
*** US Department of Agriculture
The anti-estrogenic activities of glyceollin isomers (I, II and III) was investigated, for the first time, in a competition binding assay with estrogen receptor α and in an estrogen response element luciferase assay. We were able to identify the isomer glyceollin I as the active component of the mix. We also report the effects of glyceollin I in comparison to 4-hydroxy-tamoxifen, ICI 182,780, and 17β-estradiol in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on the expression patterns of two genes: progesterone receptor and stromal derived factor-1α. In addition, the influence of this compound on cell proliferation and viability utilizing a clonogenic assay method was assessed. Furthermore, inhibition of proliferation on MDA-MB 231 ER-negative breast cancer cells was demonstrated. The effects of glyceollin I on proliferation were determined by colony assays. Colony formation was determined after 10 days of incubation. Gene expression was determined by ERE-luciferase and real time RT-PCR assays. ER-dependent cancer cell lines were transfected with an ERE-Luc plasmid, treated with glyceollin I in the presence and absence of estrogen stimulation, and harvested for luciferase activity. The cells were also analyzed for the expression of PgR and SDF-1 genes after glyceollin treatment. A competitive binding assay with fluorescent detection was used to assess the ability of the glyceollins to bind to the ER-α. We have established the ability of glyceollin I to significantly suppress proliferation on ER-dependent and independent breast cancer cells. We further demonstrate that the effects of glyceollins in suppression of cell growth correlate with inhibition of estrogen stimulated gene expression and suppression of ERE-reporter gene activation. Taken together, our results establish a novel in vitro inhibition of cell proliferation. Although further studies will be needed, glyceollin I may represent an important component of a soy-based diet in terms of chemoprevention and treatment of estrogen-related cancer.