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

Abstract

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.

Introduction

Estrogens are involved in the growth, development, and maintenance of a number of tissues. The physiological effects of estrogens are mediated by a ligand-inducible nuclear transcription factor, the estrogen receptor (ER).¹ Estrogen binds to the ligand-binding domain (LBD) of the ER and initiates a series of molecular events culminating in the transcriptional activation or repression of target genes Beekman et al 1993, Gorski et al 1993, Cavailles et al 1994, Aranda and Pascual 2001. Transcriptional regulation of genes by the ER arises from the direct interaction of the ER with ligands such as estradiol (E2) and components of the general transcription machinery Colgan et al 1993, Robyr et al 2000. Modulation of ER-responsive genes by E2 is well known; however, the transcriptional activation of the ER genes by phytoestrogens and other nonsteroidal estrogen is poorly understood.

Phytoestrogens (PEs) are compounds present in the diet that elicit estrogenic activity. These compounds are suspected of antagonizing the estrogenic effects of the endogenous estrogen, E2. The biological effects of PEs are well documented in humans and animals Adlercreutz et al 1993, Messina et al 1994, Knight and Eden 1996, Humfrey 1998, Arai et al 2000, Kim et al 2002. Several studies have suggested that PEs may function like estrogen in lowering the risk of cardiovascular disease and osteoporosis in postmenopausal women Horowitz 1993, Rodan and Martin 2000, Mei et al 2001. Investigators using urine PE excretion as an index of PE exposure have shown a strong correlation between PE in the diet and a lower incidence of breast and prostate cancers Messina et al 1994, Barnes et al 1995, Barnes and Peterson 1995, Musey et al 1995, Brawley and Barnes 2001, Mentor-Marcel et al 2001. Furthermore, epidemiological studies demonstrate that populations with a high dietary content of soya (soya being the major source of PEs) have a low incidence of breast and prostate cancers Messina et al 1994, Barnes et al 1995. PEs can bind to ER in vitro and exert estrogenic effects in vivo (Santell et al., 1997). Although the biochemical mechanism responsible for PE action is unknown, it is generally agreed that beneficial effects of PEs are mediated through interaction with the ER.

One speculated mode of action for PEs suggests that these compounds function as antiestrogens, antagonizing the effects of the endogenous hormone, E2. The end result would be a decreased incidence or low risk of estrogen-dependent cancer. This hypothesis is supported by the observation that certain PEs such as genistein (GE) and coumestrol (CO) are able to modulate the growth of MCF-7, estrogen-responsive human breast cancer cells Komori et al 1993, Pagliacci et al 1994, Hoffman 1995. Furthermore, treatment of immature female rats with GE or CO resulted in several classical estrogen responses such as the induction of the progesterone receptor (PgR) gene and increased uterine growth Makela et al 1995, Medlock et al 1995, Santell et al 1997, zsnBoettger-Tong et al 1998. PEs also have the ability to bind with high affinity to recombinant ER-α and ER-β (ERα, ERβ) or the ER present in MCF-7 cells Miksicek 1994, Kuiper et al 1997. In addition, transient transfection assays have confirmed that the effects of PE could be mediated through transcriptional activation of the ER Barnes and Peterson 1995, Casanova et al 1999. Although PEs are able to elicit an estrogen response, no study to date has been able to demonstrate that PEs are able to inhibit E2-stimulated cell growth, which could lead to a decrease in estrogen-dependent tumors.

The estrogenic properties of PEs have been measured using a variety of assays and animal models. Admittedly, the endpoints of these assays are good indicators of PE estrogenicity. However, these assays generally measure multiple-step processes and do not provide information concerning the initial effects of PEs. Furthermore, no methodology is available to measure the direct effects of PEs independent of their estrogenic activity. Thus, the objective of this study was to identify a set of biological markers that could be used to assess the functional and mechanistic action of PEs without the influence of their estrogenic activity. To accomplish this, differential display reverse transcriptase–polymerase chain reaction (ddRT–PCR) and representational difference analysis of cDNA (cDNA-RDA) were used to isolate unique genes that are specifically regulated by PE and not by E2. In this study, we describe the identification and characterization of two PE-responsive genes that are differentially expressed in MCF-7 cells.