Mammalian lignans and genistein decrease the activities of aromatase and 17β-hydroxysteroid dehydrogenase in MCF-7 cells

Abstract

Estrogen plays a major role in breast cancer development and progression. Breast tissue and cell lines contain the necessary enzymes for estrogen synthesis, including aromatase and 17β-hydroxysteroid dehydrogenase (17β-HSD). These enzymes can influence tissue exposure to estrogen and therefore have become targets for breast cancer treatment and prevention. This study determined whether the isoflavone genistein (GEN) and the mammalian lignans enterolactone (EL) and enterodiol (ED) would inhibit the activity of aromatase and 17β-HSD type 1 in MCF-7 cancer cells, thereby decreasing the amount of estradiol (E2) produced and consequently cell proliferation. Results showed that 10 μM EL, ED and GEN significantly decreased the amount of estrone (E1) produced via the aromatase pathway by 37%, 81% and 70%, respectively. Regarding 17β-HSD type 1, 50 μM EL and GEN maximally inhibited E2 production by 84% and 59%, respectively. The reduction in E1 and E2 production by EL and the reduction in E2 production by GEN were significantly related to a reduction in MCF-7 cell proliferation. 4-Hydroxyandrostene-3,17-dione (50 μM) did not inhibit aromatase but inhibited the conversion of E1 to E2 by 78%, suggesting that it is a 17β-HSD type 1 inhibitor. In conclusion, modulation of local E2 synthesis is one potential mechanism through which ED, EL and GEN may protect against breast cancer.

Introduction

Some studies suggest an association between the intake, plasma levels or urinary excretion of phytoestrogens such as the isoflavones genistein (GEN) and daidzein (DAID) and the mammalian lignans enterolactone (EL) and enterodiol (ED) (metabolites of plant lignans) and a reduced risk of breast cancer [1], [2], the etiology and development of which estrogen plays a major role. 17β-Estradiol (E2) has been implicated in both the initiation and the promotion of breast cancer [3], such that lifetime estrogen exposure is considered a major risk factor for the disease [4], [5].

Two thirds of breast cancers occur in postmenopausal women at a time when the major source of estrogen production is outside the ovary and circulating estrogens are low [6], [7]. However, plasma estrogen concentrations are significantly lower than those found in “normal” glandular tissue and cancerous tissue [3], [8], suggesting that circulating estrogen concentrations may not be indicative of tissue exposure.

Aromatase and 17β-HSD are enzymes involved in estrogen production within the breast [9], [10], [11]. Aromatase catalyzes the final step in the production of estrogens, i.e. estrone (E1) from androstenedione (AD) and E2 from testosterone [12], while 17β-HSD (types I and II) control the balance between E2 and the less biologically active E1 [13], [14]. The increased activity and expression of these enzymes are associated with breast cancer [15], [16], [17], [18], with tumors showing elevated activity, relative to normal tissue [19]. Hence, aromatase inhibitors such as anastrozole and letrozole are now used to follow tamoxifen treatment of breast cancer and, in some cases, have been found to be more effective than tamoxifen as a first line treatment [20].

We hypothesize that interference in the normal synthesis and metabolism of estrogen is one mechanism through which the protective effect of these phytoestrogens is mediated. In previous studies, EL has been found to inhibit aromatase activity in human preadipocytes [21] and placental microsomes [22], while GEN and DAID have been shown to inhibit 17β-HSD in human placental microsomes [23], genital skin fibroblasts [24], and granulosa luteal cells [25]. However, GEN is generally considered to have little or no ability to inhibit aromatase activity [23], [25], [26], [27]. In the estrogen receptor-positive human breast cancer cells (MCF-7), GEN modulates the activity and expression of 17β-HSD [28] but not aromatase [27]. Another study found GEN to inhibit 17β-HSD in T47D but not in MCF-7 cells [29]. The effect of EL and ED on aromatase and 17β-HSD has not been examined.

Studies on aromatase activity in MCF-7 cells are often conducted in those transfected with human placental aromatase. Yue et al. [16], however, suggested that these transfected cells may not be the best model for regulation studies because the regulation of aromatase expression and activity is tissue specific. Wild-type MCF-7 cells have been shown to have aromatase activity at levels high enough to elicit an estrogenic response [30]. Because the regulation of both aromatase and 17β-HSD is tissue specific [31], [32], and in the case of aromatase changes with the development of breast cancer [33], it is of interest to examine the effect of the lignans and isoflavones in wild-type MCF-7 cells.

The objective of this study was to first determine whether the MCF-7 cells have measurable aromatase and 17β-HSD type 1 activities, and then to investigate the effect of various concentrations of EL, ED and GEN on these activities. Changes in estrogen production induced by EL, ED or GEN were then related to cell proliferation, since it is well established that E2 dose can dependently increase the proliferation of MCF-7 cells. The results will suggest one potential mechanism whereby GEN, ED and EL may reduce tumor growth of estrogen receptor-positive human breast cancer cells.