Estrogen Receptor Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Two Major Phytoestrogens on Human Breast Cancer Cells

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the downregulation of ER mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER and ER fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER and ER confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER -dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load. Breast cancer is the most frequent malignancy of women in North America, where every year about 200,000 new cases are diagnosed and 50,000 women die from the disease (LopezOtin and Diamandis, 1998). The age-adjusted death rates from breast tumors are 2to 8-fold lower in Asian countries compared with the United States and Western Europe (Parker et al., 1996), which, together with migrant studies, suggests a primary role of dietary factors in reducing cancer risk in Asian women (Adlercreutz, 1995). Epidemiological and case-control studies have reported a negative correlation between breast cancer and the intake of soy products and the urinary excretion of phytoestrogens (Ingram et al., 1997; Kurzer and Xu, 1997). However, interpretations and conclusions have been contradictory (Messina et al., 1997; de Souza, 1998; Heaton and Lewis, 1998; Humfrey 1998; Mangtani and Silva, 1998; Tesarik and Mendoza, 1998). The phytoestrogens genistein and quercetin (Fig. 1), abundantly present in soybeans, vegetables, and fruit (Price and Fenwick, 1985), have attracted research interest and have been considered natural chemopreventive agents (Larocca et al., 1990; Peterson and Barnes, 1991; Adlercreutz et al., 1995; Kuo, 1996). Many different mechanisms have been proposed to explain the antiproliferative effects exerted by these chemicals, including direct inhibition of tyrosine kinase activity (Akiyama et al., 1987, and references therein), DNAtopoisomerase II (Markovits et al., 1989), angiogenesis (Fotsis et al., 1993), and synthesis of heat-shock proteins (Hansen et al., 1997). Flavonoids such as genistein and quercetin may prevent DNA damage as free radical scavengers (Wei et al., 1993); most importantly, however, they may act as partial estrogen agonists or antagonists (Kuiper et al., 1997; Barkhem et al., 1998). This work was supported by grants from Regione Calabria and U.E. (P.O.P.), MURST-CNR (Biotechnology Program L. 95/95) and from the Swiss National Science Foundation, the Krebsforschung Schweiz and the Canton de Genève. ABBREVIATIONS: ER, estrogen receptor; OHT, hydroxytamoxifen; OHF, hydroxyflutamide; ZK, ZK98299; HBD, hormone binding domain; PCR, polymerase chain reaction; wt, wild-type; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; CS, charcoal-stripped; RT, reverse transcription; AF, activation function. 0026-895X/01/6003-595–602$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 926/928173 Mol Pharmacol 60:595–602, 2001 Printed in U.S.A. 595 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from Significant amounts of soy are “hidden” in normal processed food. In a recent study (Hargreaves et al., 1999), none of the subjects reported having knowingly consumed soy products despite displaying significant serum levels of phytoestrogens. Nanomolar amounts of phytoestrogens were measured in soy-supplemented premenopausal women and seemed to induce an estrogenic response in the breast. For postmenopausal women, it has also been reported that flavonoids may have some estrogenic activities, inducing vaginal cell maturation (Wilcox et al., 1990), reduction of hot flushes, and hepatic cholesterol synthesis (Anderson et al., 1995; Murkies et al., 1995). The effects of 17 -estradiol (E2) and related compounds are mediated by two members of the nuclear receptor superfamily, the estrogen receptors (ER) and . Upon ligand binding, they undergo a conformational change allowing chromatin interaction and the regulation of transcription of target genes (Jensen, 1995). Estrogens stimulate the proliferation of many breast tumor cells, which has led to the use of such antiestrogens as hydroxytamoxifen for endocrine therapy. The presence of ER in breast tumor biopsies has been recognized as a positive prognostic marker that correlates with higher survival rates and lower risk of relapse (Lopez-Otin and Diamandis, 1998). Estrogenic compounds in the food, for example the natural flavonoids genistein and quercetin, might influence breast cancer progression in a dose-dependent fashion. To provide evidence for this hypothesis we have used the estrogen-dependent human breast cancer cell line MCF7, its hormoneindependent variant MCF7SH (Kalkhoven et al., 1996), and the ER-negative HeLa cell line as model systems. We examined the ability of genistein and quercetin (1) to induce the transactivation function of ER and ER , (2) to modulate ER mRNA and protein levels, (3) to bind ER and ER , and (4) to exert either growth stimulatory or antiproliferative effects at concentrations that are physiologically achievable through dietary uptake. Materials and Methods Reagents. E2, genistein, quercetin, and hydroxytamoxifen (OHT) were purchased from Sigma (St. Louis, MO). Hydroxyflutamide (OHF) and ZK98299 (ZK) were a gift from Schering (Berlin, Germa-