Wesley G. Gray

Bisphenol a binds to the low-affinity estrogen binding site.

Environmental estrogens are suspected of being involved in the current increase in the incidence of human reproductive malfunctions, such as a decrease in male reproductive capacity and an increased incidence of breast cancer in women. The influences of these compounds have been proposed to be mediated through binding to macromolecules, such as estrogen receptor alpha or beta. In this study we examined whether the low-affinity Type II estrogen binding site (Type II EBS), originally identified in the rat uterus, is a possible mediator of environmental estrogens such as bisphenol A (BPA). Analysis of BPAs binding to an enriched fraction of Type II EBS, using a competition assay, indicated that BPA was able to compete with estradiol in binding to this site. At a concentration of 10-15 microM (comparable to that required to induce uterine proliferation), BPA inhibited the binding of estradiol to Type II EBS by greater than 50%. The binding affinity of BPA for the Type II EBS was only 8-10-fold lower than that of the synthetic estrogen diethylstilbestrol. The binding of BPA to Type II EBS appeared specific to BPA, in that endosulfan, another environmental estrogen, failed to displace estradiol from the site. A comparison of the relative binding affinities of BPA for rat uterine estrogen receptor alpha to that of the Type II EBS implies that BPA preferentially binds to the Type II EBS.

Identification and characterization of a phytoestrogen-specific gene from the MCF-7 human breast cancer cell

Phytoestrogens are a group of compounds present in human diet that display estrogenic-like properties. Several studies have demonstrated that populations who consume large quantities of phytoestrogens have a reduced risk of estrogen-dependent cancers. Although it has been shown that certain phytoestrogens modulate estrogen action, their biological role in cancer reduction remains unclear. Through the use of differential display reverse transcriptase-polymerase chain reaction and representational difference analysis of cDNA, we have identified several phytoestrogen-responsive genes from the human breast cancer cell MCF-7. Two of these genes, PE-13.1 and pRDA-D, have been characterized in greater detail in this study. These genes were not previously known to be regulated by phytoestrogen or estradiol. PE-13.1 is a novel gene that specifies the coding of a 1.10-kb mRNA transcript. Northern blot analysis confirmed that the PE-13.1 transcript is up-regulated by phytoestrogens (Genistein, sevenfold; Zearalenone, twofold) and is nonresponsive to estradiol. Conversely, the pRDA-D transcript was down-regulated by both phytoestrogens and estradiol. The antiestrogen ICI-182,780 inhibits the expression of PE-13.1 and reverses the inhibition of pRDA-D expression induced by phytoestrogens and estradiol. Analysis of the tissue distribution of PE-13.1 transcript by RNA blot reveals that this transcript is expressed in both normal and tumor tissues. This report demonstrates for the first time the presence of two phytoestrogen-responsive genes that may be used as molecular markers in understanding the role dietary estrogen plays in cancer prevention.

Molecular and Biochemical Effects of a Kola Nut Extract on Androgen Receptor-Mediated Pathways

The low incidence of prostate cancer in Asians has been attributed to chemopreventative properties of certain chemicals found in their diet. This study characterized the androgenic and chemopreventative properties of the Jamaican bush tea “Bizzy,” using androgen receptor positive and negative cell lines. Exposure of prostate cells to Biz-2 resulted in a growth inhibition (GI50) of 15 ppm in LNCaP cells and 3.6 ppm in DU145 cells. Biz-2 elicited a 2-fold increase in the mRNA of the anti-apoptotic gene Bcl2, with a 10-fold increase in that of the proapoptotic gene Bax. We observed a 2.4- to 7.5-fold change in apoptotic cells in both cell lines. Biz-2 at 10 ppm elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Biz-2 caused a 36% decrease in PSA secretion and a significant increase in PSA mRNA. The relative binding affinity (IC50) of Biz-2 for AR was 2- to 5-fold lower than that of the synthetic androgen R1881. Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner.

Phytoestrogens Regulate mRNA and Protein Levels of Guanine Nucleotide-Binding Protein, Beta-1 Subunit (GNB1) in MCF-7 Cells

Phytoestrogens (PEs) are non‐steroidal ligands, which regulate the expression of number of estrogen receptor‐dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen‐based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes that are regulated by PEs through either the classic nuclear‐based estrogen receptor or membrane‐based estrogen receptor pathways. SSH, using mRNA from genistein (GE) treated MCF‐7 cells as testers, resulted in a significant increase in GNB1 mRNA expression levels as compared with 10 nM 17β estradiol or the no treatment control. GNB1 mRNA expression was up regulated two‐ to fivefold following exposure to 100.0 nM GE. Similarly, GNB1 protein expression was up regulated 12‐ to 14‐fold. GE regulation of GNB1 was estrogen receptor‐dependent, in the presence of the anti‐estrogen ICI‐182,780, both GNB1 mRNA and protein expression were inhibited. Analysis of the GNB1 promoter using ChIP assay showed a PE‐dependent association of estrogen receptor α (ERα) and β (ERβ) to the GNB1 promoter. This association was specific for ERα since association was not observed when the cells were co‐incubated with GE and the ERα antagonist, ICI. Our data demonstrate that the levels of G‐protein, beta‐1 subunit are regulated by PEs through an estrogen receptor pathway and further suggest that PEs may control the ratio of α‐subunit to β/γ‐subunits of the G‐protein complex in cells. J. Cell. Physiol. 219: 584–594, 2009.

Abstract 197: Anti-cancer activity of extract from the Jamaican round-leaf yellow yam (RLY2) (Dioscorea cayenensis)

The Jamaican Round-Leaf yellow yams (RLY2), Dioscorea cayenensis, are edible tubers, bulbils or rhizomes that are of considerable economic value and possess several health benefits. These RLY2 are a rich source of anti-oxidants, vitamins and phytochemicals. In addition to its nutritional value, the Jamaican populace regards RLY2 as an herbal medicine useful in treatment of diabetes mellitus, hypertension and certain kind of cancers. Chemical analysis of RLY2 revealed the presence of a myriad of bioactive ingredients that are likely to be lost due to the method of preparation for consumption. Thus, we hypothesized that mild extraction of RLY2 will preserved the bioactive analytes that give RLY2 its anti-cancer property. To test this hypothesis, different ethanolic extracts of RLY2 were prepared, and their bioactivity demonstrated in two models of prostate cancer. The present study demonstrates dosage and time-dependence inhibition in both the androgen sensitive LNCaP and androgen insensitive DU-145 prostate cancer cell lines. We observed that DU-145 was three-times more sensitive to RLY2 ethanol extract than LNCaP. Growth of LNCaP for 3-days with varying concentration of RLY2 ethanol extract resulted in an IC50 of 750 ppm (95% C.I. 726-791ppm), whereas DU-145 resulted in an IC 50 of 250 ppm (95% C.I. 236-281ppm). The maximum growth inhibition by RLY2 extract occurs within twenty-four hours for both LNCaP and DU-145 with no significant changes after 5-days. To determine the mechanism of toxicity by RLY2, we induced both LNCaP and DU-145 with RLY2 for 0-24h and prepared cells for flow cytometer analysis. The effect of RLY2 on cell cycle and degree of apoptosis will be discussed. Our data indicated that the ethanol extract of RLY2 was more sensitive toward androgen-insensitive prostate cancer. Thus, this study suggests that RLY2 contain bioactive chemical that may be used in grade II prostate adenocarcinoma represented by the DU-145 phenotype. Citation Format: Sasha-Gay A. Wright, Wesley G. Gray, Helen Asemota. Anti-cancer activity of extract from the Jamaican round-leaf yellow yam (RLY2) (Dioscorea cayenensis) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 197. doi:10.1158/1538-7445.AM2017-197