(Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture

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Abstract

In the public opinion, phytochemicals (PCs) present in the human diet are often considered beneficial (e.g. by preventing breast cancer). Two possible mechanisms that could modulate tumor growth are via interaction with the estrogen receptor (ER) and inhibition of aromatase (CYP19). Multiple in vitro studies confirmed that these compounds act estrogenic, thus potentially induce tumor growth, as well as aromatase inhibitory, thus potentially reduce tumor growth. It is thought that in the in vivo situation breast epithelial (tumor) cells communicate with surrounding connective tissue by means of cytokines, prostaglandins and estradiol forming a complex feedback mechanism. Recently our laboratory developed an in vitro co-culture model of healthy mammary fibroblasts and MCF-7 cells that (at least partly) simulated this feedback mechanism (M. Heneweer et al., TAAP vol. 202(1): 50–58, 2005). In the present study biochanin A, chrysin, naringenin, apigenin, genistein and quercetin were studied for their estrogenic properties (cell proliferation, pS2 mRNA) and aromatase inhibition in MCF-7 breast tumor cells, healthy mammary fibroblasts and their co-culture. The proliferative potency of these compounds in the MCF-7 cells derived from their EC50s decreased in the following order: estadiol (4*10 3 nM) > biochanin A (9 nM) > genistein (32 nM) > testosterone (46 nM) > naringenin (287 nM) > apigenin (440 nM) > chrysin (4 µM). The potency to inhibit aromatase derived from their IC50s decreased in the following order: chrysin (1.5 μM) > naringenin (2.2 μM) > genistein (3.6 μM) > apigenin (4.1 μM) > biochanin A (25 μM) > quercetin (30 μM). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1–10 μM). Results from co-cultures did not elucidate the dominant effect of these compounds. MCF-7 cell proliferation occurs at concentrations that are not uncommon in blood of individuals using food supplements. Results also indicate that estrogenicity of these PCs is quantitatively more sensitive than aromatase inhibition. It is suggested that perhaps a more cautionary approach should be taken for these PCs before taken as food supplements.

Introduction

Breast cancer is the predominant type of cancer in industrialized countries and the second leading cause of cancer-related deaths in women (Nkondjock and Ghadirian, 2005). Breast cancer is diagnosed more frequent in postmenopausal women and the hormone sensitive tumor incidence is higher in postmenopausal women (60%) than premenopausal women (Brodie et al., 1997).

Numerous risk factors for breast cancer etiology have been identified, including gender (women more than male), family history (heritability/loss of function of susceptibility genes BRCA-1 and BRCA-2), socioeconomic status, life-style (alcohol consumption, smoking, exercise, obesity) and steroid-hormonal status (including age of first/last menarche and first pregnancy, breast feeding) (reviewed by Veronesi et al., 2005).

Estrogen production in the ovaries of postmenopausal women ceases and plasma levels drop 80–90% compared to those of premenopausal women (Lonning et al., 1997, Shields-Botella et al., 2005). In the postmenopausal period testosterone levels, produced in the adrenal cortex, increase and testosterone is converted to estrogens in adipose, muscle and connective tissue expressing CYP19 (Longcope et al., 1978). This conversion is catalyzed by aromatase, the protein product of CYP19 and the rate limiting enzyme converting androgens to estrogens. Peripheral produced estrogens are secreted from cells that express aromatase and can significantly increase the levels in surrounding tissue (e.g. in mammary tissue) to concentrations that are in the same range of those observed in premenopausal women (van Landeghem et al., 1985). These locally produced estrogens, e.g. in breast tissue, can potentially initiate, via catechol E2, and stimulate the growth of estrogen sensitive breast tumors. Supporting evidence for this mechanism is provided by the fact that estradiol levels have been reported to be higher in breast tumors than in healthy tissue (Brodie et al., 1997). This observation is also mechanistically supported by observed elevated expression of aromatase activity in breast adipose tissue of breast cancer patients (Zhou et al., 2001). Aromatase expression can be induced by cytokines e.g. prostaglandin (PGE2) and interleukin-6 (IL-6) that are among others secreted by tumor cells, but also by infiltrating macrophages and lymphocytes (Singh et al., 1995, Reed and Purohit, 1997, De Jong et al., 2001, Richards et al., 2002, Simpson, 2003). In healthy tissue the expression of aromatase is under the control of the promoter region (pr) I.4, regulated by class I cytokines such as IL-6, IL-11 and Tumor Necrosis Factor-α (TNF-α), but also glucocorticoids (GREs) are required for its regulation. Noticeable, a promoter switch from pr I.4 to pr I.3 and pr pII is commonly found in breast tumor tissue and linked to elevated CYP19 expression (Chen, 1998). In contrast these promoters I.3 and pII are under the control of a cAMP responsive element (CRE) and not by e.g. cytokines (Simpson, 2003).

Recently, this interaction between breast tumor cells and surrounding tissue has been studied in an in vitro model (Heneweer et al., 2005a). In these experiments the modulation by compounds such as dexamethasone (DEX), 17β-estradiol (E2), diethylstilbestrol (DES), fadrozole (FAD) and fulvestrant (ICI 182780) was studied by measuring pS2 gene and CYP19 expression in different cell types. In literature the expression level of pS2 has been described to be positively correlated to exposure of MCF-7 cells to estrogens (Rio and Chambon, 1990, Duda et al., 1996). Therefore in our co-culture experiments the pS2 expression level serves as a measure for exposure to estrogenic compounds. CYP19 expression, on the other hand, greatly determines aromatase activity in this co-culture model and occurs predominantly if not exclusively in the breast fibroblasts (Fig 1).

Certain dietary phytochemicals (PCs) like apigenin, biochanin A, chrysin and naringenin have been shown to act as competitive inhibitors in various in vitro systems (Kellis and Vickery, 1984, Campbell and Kurzer, 1993, Almstrup et al., 2002, Sanderson et al., 2004, Edmunds et al., 2005). The aromatase inhibitory properties of PCs have also been confirmed with computer analysis using structure activity relation for the pocket binding domain of this enzyme (Chen et al., 1997, Kao et al., 1998). Thus, these PCs act similar to pharmaceutical aromatase inhibitors such as fadrozole and could therefore have a chemo preventive role in breast cancer therapy. On the other hand, some of these PCs are also known to bind and activate the estrogen receptor (ER) (Kao et al., 1998, Kuiper et al., 1998) in breast tumor cell lines, thereby causing growth stimulation of ER positive tumor cells. As these phytochemicals are part of a regular diet, the possible beneficial as well adverse effects on human health should be considered in conjunction. The objective of this study was to investigate possible counteracting effects of various PCs using ER-dependent cell proliferation and aromatase inhibition as endpoints. In this study test compounds were investigated in two separate cells systems, human breast adenocarcinoma MCF-7 cells and primary fibroblasts isolated from healthy mammary tissue, as well as a co-culture system from both cell types as described above. The results from these experiments were used to evaluate the overall (anti)estrogenic effect of these PCs in relation to human (internal) exposure.

Section snippets

Chemicals and plastics

17β-estradiol (E2), testosterone (T), 4-androstene-3,17-dione (A-dione), biochanin A (BioA), genistein (GEN), naringenin (NAR) and dexamethasone (DEX) were purchased from Sigma (St. Louis, MO, USA). Chrysin (CHR) was obtained from Aldrich (Zwijndrecht, The Netherlands). Apigenin (APG) was purchased from Lancaster Synthesis (Muhlheim am Main, Germany). The catalytic aromatase inhibitor fadrozole (FAD) was kindly provided by Novartis Pharma AG (Basel, Switzerland). The ER antagonist fulvestrant

Cell proliferation experiments with MCF-7 cells and mammary fibroblasts

The MCF-7 cell proliferation assay was used as a measure for estrogenicity and to determine estrogenic properties of the test compounds. In this assay the individual reference compounds (E2, T, A-dione, DEX, FAD and ICI 182780) and the PCs (APG, BioA, CHR, GEN and NAR) were tested. As a control for later co-culture experiments, E2, BioA, CHR, GEN and NAR, were tested in a cell proliferation assay using fibroblasts instead of MCF-7 cells.

E2, T, APG, BioA, CHR, GEN and NAR produced a

Discussion

In this paper results are presented from experiments with primary fibroblasts from healthy human mammary tissue, human epithelial estrogen sensitive breast tumor cells (MCF-7) and co-cultures of both cell types. The effects of APG, BioA, CHR, GEN, NAR and QUE on aromatase activities in mammary fibroblasts and/or cell proliferation in MCF-7 cells were studied. These PCs are known dietary components and can induce cell proliferation in ER positive breast tumor cell lines like MCF-7, and T47D (

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