Estrogenic Activity of Mycoestrogen (3β,5α,22E)-Ergost-22-en-3-ol via Estrogen Receptor α-Dependent Signaling Pathways in MCF-7 Cells

Armillariella tabescens (Scop.) Sing., a mushroom of the family Tricholomataceae, has been used in traditional oriental medicine to treat cholecystitis, improve bile secretion, and regulate bile-duct pressure. The present study evaluated the estrogen-like effects of A. tabescens using a cell-proliferation assay in an estrogen-receptor-positive breast cancer cell line (MCF-7). We found that the methanol extract of A. tabescens fruiting bodies promoted cell proliferation in MCF-7 cells. Using bioassay-guided fractionation of the methanol extract and chemical investigation, we isolated and identified four steroids and four fatty acids from the active fraction. All eight compounds were evaluated by E-screen assay for their estrogen-like effects in MCF-7 cells. Among the tested isolates, only (3β,5α,22E)-ergost-22-en-3-ol promoted cell proliferation in MCF-7 cells; this effect was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of (3β,5α,22E)-ergost-22-en-3-ol was evaluated using Western blot analysis to detect the expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and estrogen receptor α (ERα). We found that (3β,5α,22E)-ergost-22-en-3-ol induced an increase in phosphorylation of ERK, PI3K, Akt, and ERα. Together, these experimental results suggest that (3β,5α,22E)-ergost-22-en-3-ol is responsible for the estrogen-like effects of A. tabescens and may potentially aid control of estrogenic activity in menopause.


Introduction
Menopause, the complete end of menstrual periods, typically occurs worldwide for women 45 to 55 years of age. With increasing life expectancy, the global number of menopausal women aged 50 years and over is estimated to reach 1.2 billion by 2030, with 47 million new entrants each year [1,2]. In South Korea, the postmenopausal female population aged over 50 years has increased since 2000. After 2030, over half of the female population will be postmenopausal ( Figure 1), according to the Korean Statistical Information Service database [3]. Menopause results from declining ovarian function, and the production of steroid hormones such as estrogen dramatically drops. As a result, menopause results in such vasomotor symptoms as hot flushes, night sweats, sleep disturbance, vaginal dryness, and even osteoporosis [4]. Women experiencing menopausal The total female population will peak in 2030 then decrease through to 2060. The postmenopausal female population will continuously increase. After 2030, over half of South Korean females will be postmenopausal [3].
Estrogen replacement therapy has traditionally been considered beneficial for the relief and prevention of postmenopausal symptoms and related diseases [6,7]. However, patients receiving estrogen replacement therapy over the long term are often reluctant to continue because of side effects including breast cancer, heart disease, and stroke [8,9]. Thus, there is growing interest in the use of phytoestrogens with estrogen-like activity. Phytoestrogens are natural compounds found in plants and plant-based foods and are structurally, and sometimes functionally, similar to mammalian estrogens and their active metabolites [10]. Phytoestrogens such as stilbenes, isoflavonoids, lignans, and flavonoids are reported to be abundant in red clover plants, flax seeds, and soy plants [11]. When phytoestrogens bind to the estrogen receptor (ER), they act as agonists or antagonists [12]. They are structurally similar to estrogen; in theory, therefore, they can increase the risk of breast cancer development [7]. However, some studies on the effects of phytoestrogens on breast cancer have suggested that phytoestrogens exhibit no effect on cancer or even exhibit anti-cancer effects [13].
Mycoestrogens, natural fungus-derived products with estrogen-like activity, can be produced by various Fusarium species [14]. Mycoestrogens have features similar to those of phytoestrogens and are reported to act as estrogen-receptor agonists [15]. Mushrooms-the fleshy, spore-bearing fruiting bodies of fungi-have been used as functional foods and dietary supplements because of various bioactive secondary metabolites that exhibit interesting biological actions [16]. As an example of the estrogen-like activity of a mushroom, the ethanol extract of the Pleurotus eryngii fruiting body is well documented to exhibit proliferative effects in ER-positive MCF-7 human breast epithelial cell lines and to promote ovariectomy-induced bone loss in old female rats [17]. However, the chemical contributors to the estrogen-like effects of P. eryngii have not yet been identified. The total female population will peak in 2030 then decrease through to 2060. The postmenopausal female population will continuously increase. After 2030, over half of South Korean females will be postmenopausal [3].
Estrogen replacement therapy has traditionally been considered beneficial for the relief and prevention of postmenopausal symptoms and related diseases [6,7]. However, patients receiving estrogen replacement therapy over the long term are often reluctant to continue because of side effects including breast cancer, heart disease, and stroke [8,9]. Thus, there is growing interest in the use of phytoestrogens with estrogen-like activity. Phytoestrogens are natural compounds found in plants and plant-based foods and are structurally, and sometimes functionally, similar to mammalian estrogens and their active metabolites [10]. Phytoestrogens such as stilbenes, isoflavonoids, lignans, and flavonoids are reported to be abundant in red clover plants, flax seeds, and soy plants [11]. When phytoestrogens bind to the estrogen receptor (ER), they act as agonists or antagonists [12]. They are structurally similar to estrogen; in theory, therefore, they can increase the risk of breast cancer development [7]. However, some studies on the effects of phytoestrogens on breast cancer have suggested that phytoestrogens exhibit no effect on cancer or even exhibit anti-cancer effects [13].
Mycoestrogens, natural fungus-derived products with estrogen-like activity, can be produced by various Fusarium species [14]. Mycoestrogens have features similar to those of phytoestrogens and are reported to act as estrogen-receptor agonists [15]. Mushrooms-the fleshy, spore-bearing fruiting bodies of fungi-have been used as functional foods and dietary supplements because of various bioactive secondary metabolites that exhibit interesting biological actions [16]. As an example of the estrogen-like activity of a mushroom, the ethanol extract of the Pleurotus eryngii fruiting body is well documented to exhibit proliferative effects in ER-positive MCF-7 human breast epithelial cell lines and to promote ovariectomy-induced bone loss in old female rats [17]. However, the chemical contributors to the estrogen-like effects of P. eryngii have not yet been identified.
Our group has been conducting extended natural-product research to discover bioactive compounds from Korean wild mushrooms [18][19][20][21][22][23][24][25]. In this context, we investigated potential bioactive compounds from a methanol (MeOH) extract of the fruiting bodies of Armillariella tabescens (Scop.) Sing. to show anti-gastritic activity against ethanol-induced gastric damage in rats. The previous study found that (Z,Z)-9,12-octadecadienoic acid, isolated from A. tabescens as a fatty acid, exhibited anti-inflammatory activity involved in anti-gastritic activity [23]. A. tabescens, belonging to the Tricholomataceae family, is known as the "Luminous Fungus" in China. This mushroom has been used in traditional medicine to treat cholecystitis, to regulate bile-duct pressure, and to improve liver function [26,27]. Previous studies of A. tabescens extracts have reported that the extracts exhibit antitumor and immunomodulatory activities [23,28,29]. However, few studies of A. tabescens have investigated its chemical constituents, despite the potential pharmacological applications. Previous chemical investigations of A. tabescens have reported armillarisins A and B, which exhibited biological activities including antifungal effects and anti-infection properties against gastritis and hepatitis [26,27]. In our ongoing research on A. tabescens, we found that the MeOH extract of the fruiting bodies of A. tabescens showed estrogen-like effects in the estrogen-receptor-positive MCF-7 breast cancer cell line. The estrogen-like effect of A. tabescens has not previously been reported. Thus, the present study was conducted to further investigate the active MeOH extracts to identify potential mycoestrogens. Herein, we describe the isolation and structural characterization of eight compounds and evaluate their estrogen-like effects in MCF-7 cells; we also characterize the bioactivity of the active compound as a mycoestrogen.

Bioactivity-Guided Fractionation of the MeOH Extract of A. tabescens
We examined MCF-7 cell proliferation after treatment with the MeOH extract of A. tabescens using Ez-Cytox reagents. Cell proliferation increased to 152.61 ± 4.73% after treatment, with 100 µg/mL of the MeOH extract compared with the untreated cells, and this effect was mitigated by ICI 182,780, an estrogen receptor (ER) antagonist ( Figure 2A). Based on this result, the MeOH extract was successively solvent partitioned with hexane, CH 2 Cl 2 , EtOAc, and n-BuOH to give four main fractions: hexane-soluble, CH 2 Cl 2 -soluble, EtOAcsoluble, and n-BuOH-soluble. LC/MS and TLC analyses indicated that the hexane-soluble and CH 2 Cl 2 -soluble fractions had similar chemical profiles, which allowed us to consolidate the hexane-and CH 2 Cl 2 -soluble fractions, yielding the HCF for further experiments. For the bioactivity-guided fractionation of the MeOH extract, the estrogen-like effects of the three main fractions (HCF, EtOAc-soluble, and n-BuOH-soluble) were evaluated using a cellproliferation assay to identify the active fraction. Of the fractions tested, cell proliferation increased to 169.01 ± 5.91% after treatment with the HCF fraction compared with the untreated cells, and this effect was mitigated by the ICI 182,780 ( Figure 2B).

Isolation and Identification of Compounds from the Active Fraction
Based on the results of the bioactivity-guided fractionation for estrogen-like effects, chemical investigation of the active HCF fraction was conducted to identify the chemical contributors to the estrogenic activity of the MeOH extract of A. tabescens. Chemical investigation of the EA fraction, using column chromatography and preparative and semi-pre-

Effect of (3β,5α,22E)-Ergost-22-en-3-ol on the Protein Expression of Phospho-PI3K, PI3K, Phospho-Akt, Akt, Phospho-ERα, and ERα
To support the proliferation-promoting effects of (3β,5α,22E)-ergost-22-en-3-ol (4), the activation of ERα and related pathways were evaluated using Western blot analysis. Compared with untreated cells, 25 µM, 50 µM, and 100 µM of compound 4 induced a concentration-dependent increase in the protein expression of p-ERK, p-PI3K, p-Akt, and p-ERα ( Figure 6). Furthermore, this effect was mitigated by treatment with 100 nM ICI. When ICI was present, the expression of p-ERK, p-PI3K, p-Akt, and ERα failed to increase after treatment with 100 µM of compound 4 (Figure 7). These results proved that the responses of ERK, PI3K, and Akt to compound 4 depend on the functioning of ER ( Figure  8). To support the proliferation-promoting effects of (3β,5α,22E)-ergost-22-en-3-ol (4), the activation of ERα and related pathways were evaluated using Western blot analysis. Compared with untreated cells, 25 µM, 50 µM, and 100 µM of compound 4 induced a concentration-dependent increase in the protein expression of p-ERK, p-PI3K, p-Akt, and p-ERα ( Figure 6). Furthermore, this effect was mitigated by treatment with 100 nM ICI. When ICI was present, the expression of p-ERK, p-PI3K, p-Akt, and ERα failed to increase after treatment with 100 µM of compound 4 (Figure 7). These results proved that the responses of ERK, PI3K, and Akt to compound 4 depend on the functioning of ER (Figure 8).
concentration-dependent increase in the protein expression of p-ERK, p-PI3K, p-Akt, and p-ERα ( Figure 6). Furthermore, this effect was mitigated by treatment with 100 nM ICI. When ICI was present, the expression of p-ERK, p-PI3K, p-Akt, and ERα failed to increase after treatment with 100 µM of compound 4 (Figure 7). These results proved that the responses of ERK, PI3K, and Akt to compound 4 depend on the functioning of ER ( Figure  8).   bands. * Significant difference between cells treated with compound 4 and the untreated cells. # Significant reduction in co-treatment with ICI compared to treatment with compound 4 alone (n = 3 independent experiments, p < 0.05, Kruskal-Wallis nonparametric test). Data are represented as mean ± SEM.

Discussion
Few previous studies have investigated the compounds isolated from A. tabescens. Our previous chemical studies on A. tabescens have shown the presence of steroids, alkaloids, nucleic acids, and fatty acids. (Z,Z)-9,12-Octadecadienoic acid, a fatty acid, is known to possess anti-inflammatory activity [23]. Extending from our previous study, the present study found that the MeOH extract of A. tabescens had estrogen-like effects on MCF-7 cells. Eight compounds were isolated from the active fraction, the hexane-and CH2Cl2-soluble fraction showing estrogen-like effects. Of the eight compounds tested, only (3β,5α,22E)ergost-22-en-3-ol (compound 4) promoted cell proliferation in MCF-7 cells. Co-treatment with ICI 182,780, an ER antagonist, inhibited the proliferation-stimulatory effect. These results indicated that compound 4 exhibited a proliferation-stimulatory effect via the ER in MCF-7 cells. Its proliferation-stimulatory effect was confirmed by the expression of proteins related to the ER signaling pathway. The binding of estrogen to the G-protein-coupled estrogen receptor (GPER) activates the ERK and PI3K/Akt pathways [37,38]. ERK is a family of mitogen-activated protein kinases (MAPKs) stimulated by peptide hormones, cellular stress, and cytokines. It regulates the proliferation of ER-positive breast cancer cells [39]. The activated PI3K/Akt pathway regulates cellular growth, survival, and proliferation in normal estrogen-responsive tissues [40,41]. Ginsenoside Rg1, a chemical

Discussion
Few previous studies have investigated the compounds isolated from A. tabescens. Our previous chemical studies on A. tabescens have shown the presence of steroids, alkaloids, nucleic acids, and fatty acids. (Z,Z)-9,12-Octadecadienoic acid, a fatty acid, is known to possess anti-inflammatory activity [23]. Extending from our previous study, the present study found that the MeOH extract of A. tabescens had estrogen-like effects on MCF-7 cells. Eight compounds were isolated from the active fraction, the hexane-and CH 2 Cl 2 -soluble fraction showing estrogen-like effects. Of the eight compounds tested, only (3β,5α,22E)ergost-22-en-3-ol (compound 4) promoted cell proliferation in MCF-7 cells. Co-treatment with ICI 182,780, an ER antagonist, inhibited the proliferation-stimulatory effect. These results indicated that compound 4 exhibited a proliferation-stimulatory effect via the ER in MCF-7 cells. Its proliferation-stimulatory effect was confirmed by the expression of proteins related to the ER signaling pathway. The binding of estrogen to the G-proteincoupled estrogen receptor (GPER) activates the ERK and PI3K/Akt pathways [37,38]. ERK is a family of mitogen-activated protein kinases (MAPKs) stimulated by peptide hormones, cellular stress, and cytokines. It regulates the proliferation of ER-positive breast cancer cells [39]. The activated PI3K/Akt pathway regulates cellular growth, survival, and proliferation in normal estrogen-responsive tissues [40,41]. Ginsenoside Rg1, a chemical component of ginseng, has been reported to possess estrogen-like effects and promote ER signaling via the ERK and PI3K/Akt pathways [42]. Various studies have reported the estrogen-like effect of acacetin, a flavonoid, and its possible mechanism has also been evaluated as the ERK and PI3K/Akt pathway [43][44][45]. In our study, (3β,5α,22E)-ergost-22-en-3-ol also induced a concentration-dependent increase in the protein expression of p-ERK, p-PI3K, p-Akt, and p-ERα in MCF-7 cells, similar to that of other reported phytoestrogens. Therefore, it was concluded that the estrogen-like effect of (3β,5α,22E)-ergost-22-en-3-ol was mainly mediated via ERα. These cell-based results demonstrated for the first time that the extract of A. tabescens exhibited potent estrogen-like effects. Among the isolates, (3β,5α,22E)-ergost-22-en-3-ol was the main contributor to the estrogen-like effect of A. tabescens; it may be a potential candidate for further verification in animal experiments, towards finding estrogen-like drugs to eventually help postmenopausal women.

Fungus Material
Fresh fruiting bodies of A. tabescens were collected from Hwasung, Gyeonggi-do, Korea, in September 2014. Samples of fungal material were identified by one of the authors (K.H.K.). A voucher specimen (SKKU 2015-09-BN) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Statistical Analysis
All experiments were performed in triplicate. All analyses were performed using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA). Non-parametric comparisons of samples were conducted using the Kruskal-Wallis test to analyze the results. Differences were considered statistically significant at p < 0.05.