Estrogenic Activity of Isoflavonoids from Onobrychis ebenoides

• Abstract
• Abbreviations
• Introduction
• Material and Methods
• General methods
• Plant material
• Plant material, extraction and isolation
• Physical and spectroscopic data
• Cell culture and assessment of cell proliferation
• Isoflavone binding to isolated ERα and ERβ
• Induction of luciferase and alkaline phosphatase
• Statistics
• Results
• Discussion
• Acknowledgements
• References

Abstract

Fractionation of the neutral extract of Onobrychis ebenoides (Leguminosae) yielded a new isoflavone, named ebenosin (1), in addition to the known ones, afrormosin (2), formononetin (3) and daidzein (4). Although the relative binding affinities of 1 - 4 for estrogen receptor α (ERα) were nearly comparable and matched those of 1 - 3 for ERβ, that of 4 for the latter receptor was significantly higher than any of the other. Compounds 1 - 4 induced cell proliferation and gene expression in breast and endometrial cancer cells in an ER-dependent manner. Nonetheless, the rank order of induction potencies (4 > 3 ≥ 2 ≥ 1) matched better that of affinities for ERβ (4 > 3 ≥ 2 ≥ 1) rather than ERα (4 ≥ 3 ≥ 2 ≥ 1). While the antiestrogen ICI 182,780 could inhibit the induction of proliferation of ER-positive breast cancer cells by 1 - 4, it could not prevent 1 from exhibiting significant ER-independent cytotoxicity at 10 μM. By contrast, 1 was much less cytotoxic and only weakly estrogenic for ER-positive endometrial adenocarcinoma cells. In conclusion, our data suggest that the C-8 isoprenyl substituent of 1 renders it cytotoxic and/or estrogenic in a cell-dependent manner.

Abbreviations

AlkP:Alkaline Phosphatase

DCC-FBS:Dextran Coated Charcoal-treated Fetal Bovine Serum

EGF:Epidermal Growth Factor

ER:Estrogen Receptor

ERE:Estrogen Responsive Element

HRT:Hormone Replacement Therapy

RBA:Relative Binding Affinity

Introduction

Many different plant-derived compounds, invariably referred to as phytoestrogens, have been reported to inhibit steroidogenic enzymes as well as to bind to the estrogen receptor (ER) and exhibit tissue-specific estrogenic and/or antiestrogenic activities [1], [2], [3]. A rapidly growing class of relatively potent phytoestrogens is the isoflavonoids, of which many have been shown to exhibit estrogenic and antiestrogenic activity in vivo as well as in vitro [3].

Interest in using phytoestrogens as alternatives to conventional hormone replacement therapy (HRT) to prevent or even treat menopausal disorders is growing rapidly [3], [4], especially in the light of reports that isoflavone phytoestrogens appear to have a beneficial impact on the overall well being of the individual in the absence of any noticeable side effects [5]. In line with this trend we have previously reported on the isolation, structural characterization and estrogenic activities of three novel arylobenzofurans from Onobrychis ebenoides (Leguminosae) [6], [7]. It is known, however, that isoflavones are frequently accounted in the Leguminosae family and that Onobrychis viciifolia reportedly contains several flavonoids [8]. Here we report on the isolation, structural characterization and estrogenic activities of four isoflavones from Onobrychis ebenoides (Fig. [1]), one new, named ebenosin (1), and three already known, namely, afrormosin (2), formononetin (3) and daidzein (4).

Material and Methods

General methods

Analytical TLC was carried out using Merck pre-coated silica gel 60 F₂₅₄ plates. Column chromatography was carried out using silica gel [Merck, 0.04 - 0.06 mm (flash) and 0.015 - 0.04 mm], with an applied pressure of 300 mbar. UV spectra were obtained using spectroscopic grade EtOH/MeOH on a Shimadzu-160A spectrophotometer. A Bruker AC200 spectrometer and a Bruker AC400 spectrometer were used for obtaining the NMR spectra. Chemical shifts are given in δ (ppm) values with TMS as internal standard. The 2D experiments (COSY, HMBC and HMQC) were performed using standard Bruker microprograms. EI-MS were run on an HP-6890 spectrometer and HR-MS were run on an AEI MS-902 spectrometer.

Plant material

Whole plant material of Onobrychis ebenoides (Boiss & Spruner) was collected in May 1998, from Mount Hymettos, Attica (Greece). A voucher specimen (no. NEK 006) was deposited in the herbarium of the Laboratory of Pharmacognosy of the Department of Pharmacy of the University of Athens.

Plant material, extraction and isolation

The whole plant, dried and pulverized (1.8 kg), was extracted at room temperature three times with 2 L of CH₂Cl₂ (for 2 days each time) and five times with 2 L of MeOH (for 2 days each time). The MeOH extract and some fractions thereof were found to stimulate the growth of MCF-7 cells and were chosen for further analysis. The MeOH extract was concentrated to give a residue (50 g), which was chromatographed on silica gel (25 × 15 cm) and eluted by a CH₂Cl₂/MeOH gradient to yield 11 fractions: 1 [1 L, CH₂Cl₂], 2 [1 L, CH₂Cl₂/MeOH (99 : 1)], 3 [2 L, CH₂Cl₂/MeOH (98 : 2)], 4, 5, 6 [2 L each, CH₂Cl₂/MeOH (95 : 5)], 7, 8 [2 L each, CH₂Cl₂/MeOH (90 : 10)], 9 [2 L, CH₂Cl₂/MeOH (80 : 20)], 10 [2 L, CH₂Cl₂/MeOH (70 : 30)] and 11 [3 L, CH₂Cl₂/MeOH (50 : 50)]. Chromatography of fraction 1 (1.42 g) on a silica gel (39 g) column (i. d. 2.7 cm) using CH₂Cl₂ /MeOH (99 : 1, 280 mL) led to the isolation of 3 (12.8mg) and 4 (15.2mg). Fractions 4 (1.15 g) and 5 (1.78 g) were combined and chromatographed on silica gel (71 g) column (i. d. 4.0 cm) using a stepwise gradient solvent system consisting of CH₂Cl₂/MeOH. Seven fractions (A - G) were collected: A [1 L, CH₂Cl₂], B [1,5 L, CH₂Cl₂/MeOH (99 : 1)], C [1,2 L, CH₂Cl₂/MeOH (98 : 2)], D [1,2 L, CH₂Cl₂/MeOH (97 : 3)], E [1 L, CH₂Cl₂/MeOH (96 : 4)], F [1 L, CH₂Cl₂/MeOH (95 : 5)] and G [1 L, CH₂Cl₂/MeOH (90 : 10)]. Fraction B (720 mg) was re-chromatographed on a silica gel (21 g) column (i. d. 2.2 cm) using CH₂Cl₂ to obtain 15 fractions of 300 mL each. Compound 1 (27.6 mg) was obtained by further purification of fraction B3 using preparative TLC (SiO₂; CH₂Cl₂-hexane, 6 : 4). Chromatography of fraction B5 (150 mg) on a silica gel (3.7 g) column (i. d. 1.5 cm) using CH₂Cl₂-MeOH (99 : 1) yielded compound 2 (11.7mg).

Physical and spectroscopic data

8-(1,1-Dimethylallyl)-formononetin (ebenosin) (1): yellow solid; UV (MeOH): λ_max (log ε) = 210 (4.27), 252 (4.38), 306 nm (sh, 3.91); ¹H-NMR (400 MHz, MeOD): δ = 8.22 (1H, s, H-2), 7.90 (1H, d, J = 8.8 Hz, H-5), 6.92 (1H, d, J = 8.8 Hz, H-6), 7.46 (2H, d, J = 8.8 Hz, H-2′, H-6′), 6.96 (2H, d, J = 8.8 Hz, H-3′, H-5′), 3.83 (3H, s, 4′-OCH ₃), 3.56 (2H, d, J = 7.0 Hz, H-1′′), 5.25 (1H, t, J = 7.0 Hz, H-2′′), 1.85 (3H, s, H-4′′), 1.69 (3H, s, H-5′′); ¹³C-NMR (50 MHz, MeOD): δ = 155.3 (C-2), 125.8 (C-3), 178.9 (C-4), 117.4 (C-4a), 125.9 (C-5), 115.4 (C-6), 161.6 (C-7), 115.9 (C-8), 157.9 (C-8a), 125.8 (C-1′), 131.0 (C-2′), 116.0 (C-3′), 161.6 (C-4′), 56.2 (4′-OCH₃), 116.0 (C-5′), 131.0 (C-6′), 23.4 (C-1′′), 123.2 (C-2′′), 133.5 (C-3′′), 18.5 (C-4′′), 26.4 (C-5′′); HR-EI-MS: m/z = 336.39008, calcd. for C₂₁H₂₀O₄ : 336.39115.

Structural assignment of the other compounds was carried out as described for 1. The spectroscopic data of 2, 3 and 4 closely resemble those already reported for afrormosin, formononetin and daidzein, respectively [9], [10].

Cell culture and assessment of cell proliferation

The effects of the isoflavone on cell proliferation were assessed using MCF-7 (express ERβ to a much lower level than ERα) and MDA-MB-231 (express some ERβ but no ERα) human mammary adenocarcinoma cells, as already described [11]. MCF-12A immortalized normal human mammary cells (express neither ER) were used as stated in the text. Briefly, cells that have been cultured and subcultured as recommended by the supplier (ATCC), were plated in 96-flat-bottomed well microplates at a density of 10,000 cells/well in Dulbecco’s MEM devoid of phenol-red and supplemented with 10 % dextran-coated charcoal (DCC)-treated fetal bovine serum (FBS). Serial dilutions of the test compounds were added to the cells 24 hours after plating and, after incubation for 6 days with both medium and test compounds being renewed every 48 hours, the number of viable cells was determined using the conventional conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) to coloured formazan. Cells that received 0.1 nM 17β-estradiol (Sigma) (hereafter referred to as estradiol) and/or 5 ng/mL of epidermal growth factor (EGF, Sigma) served as stimulated proliferation controls, whereas those that received vehicle (DMSO to a final concentration ≤ 0.2 %) only, served as basal proliferation controls. ICI 182,780 (Tocris) was used to inhibit the estrogenic response as stated in the text.

Isoflavone binding to isolated ERα and ERβ

The relative binding affinity (RBA) values of 1 - 4 were assessed as previously described [11]. Briefly, the concentrations of 1 - 4, genistein (Sigma) and estradiol that inhibited ES2 (a fluorescein-labelled estrogen from Invitrogen) binding of isolated human ERα or ERβ (Invitrogen) by 50 % (IC₅₀), as assessed using a Beacon 2000 Fluorescence Polarization Reader (Invitrogen), were used to derive the RBA values listed in Table [1], as described in the legend to the Table.

Induction of luciferase and alkaline phosphatase

Assessment of the induction by the isoflavone of estrogen-responsive element (ERE)-dependent luciferase gene expression and of alkaline phosphatase (AlkP) activity was carried out using the geneticin-resistant clone D5L of MCF-7 cells and Ishikawa endometrial adenocarcinoma cells, respectively, as previously described [11]. Ishikawa cells express ERβ as well as ERα [12].

Statistics

Data were analysed using the SPSS 10.0 statistical package for Windows and compared using one-way ANOVA with a Tukey post-hoc test for multiple comparisons. Use of the t-test with a confidence interval of 95 % was as stated in the text. Differences were considered statistically significant for values of P < 0.05.

Results

Fig. [1] shows the structures of compounds 1 - 4. Compound 1 was obtained as an amorphous yellow solid. The UV spectrum of this compound in MeOH (λ_max = 210 and 252 nm) suggested the presence of the isoflavone skeleton [13]. This was supported by a ¹H-NMR signal at δ = 8.20, which is characteristic for the H-2 of isoflavones [14]. In addition, the ¹H-NMR spectrum revealed the presence of a 1,1-dimethylallyl (δ = 1.69, 3H, s; 1.85, 3H, s; 3.56, 2H, d, J = 6.7 Hz; 5.25, 1H, t, J = 6.7 Hz) and a methoxy group (δ = 3.83, 3H, s). That ring B was monosubstituted at C-4 was indicated by the presence of an AB system at δ = 6.98 (2H, d) and 7.47 (2H, d) with J = 8.5 Hz. The HMBC correlations of Fig. [2] indicated the location of the methoxy and 1,1-dimethylallyl groups (at the C-4′ and C-8 positions, respectively).

A series of increasing concentrations of 1 - 4, genistein and estradiol were allowed to compete with the fluorescent estrogen ES2 for binding to the isolated ER. The concentrations of the competitors that displaced 50 % of ES2 binding to ER (IC₅₀) were used to deduce the RBA values for ERα (RBAα) and ERβ (RBAβ) listed in Table [1]. The RBAα and RBAβ of 1 - 4 were found to be at least ca. 20- and ca.120-fold lower, respectively, than those of genistein. Notably, the RBAβ of 4 was significantly higher (p < 0.001; ANOVA) than the RBAβs of 1 - 3 as well as the RBAα of 4.

MCF-7 cells are known to express ERα to a much higher level than ERβ [15], and estrogen stimulation of their proliferation to depend on ERα [16]. Fig. [3] A shows that increasing the concentration of 1 - 4 up to 10 μM promoted the proliferation of MCF-7 cells in a manner (Table [2], column 2) that grossly reflected the RBAα values in Table [1]. In addition, Fig. [3] B shows that 1 - 4 failed to stimulate the proliferation of MDA-MB-231 cells, which are ERα-negative [17]. That 1 - 4 stimulated the proliferation of MCF-7 cells primarily via ERα is also substantiated by the fact that MCF-12A cells, which are ERα-negative and are known to proliferate in the presence of EGF rather than estrogen [18], failed to proliferate in the presence of 1 - 4, and their proliferation in the presence of EGF was not affected by these isoflavones (data not shown). Although 1 - 4 stimulated the proliferation of MCF-7 cells significantly (p < 0.001; t-test) at 10 μM, 2 and 3 appeared to impact proliferation marginally at 100 μM and 1 compromised cell viability drastically at this concentration. Similarly, 100 μM of 1 - 3 significantly (p < 0.001; t-test) inhibited the proliferation and viability of MDA-MB-231 cells as well (Fig. [3] B), indicating that the 100 μM effects are ER-independent, as already reported for other phytoestrogens [11]. Notably, Fig. [3] C shows that, while the potent antiestrogen ICI 182,780 (1 μM) could inhibit the ER-mediated stimulation of proliferation of MCF-7 cells by 1 - 4, genistein or estradiol, it could not prevent 1 from being cytotoxic at 10 μM. Since the estrogenic effect of 1 - 4 was fully antagonized by ICI 182, 780, a non-specific oxidation of the MTT by the isoflavones may be excluded. Thus, it appears that the lower (p < 0.01; ANOVA) proliferation stimulating efficacy of 1 compared to 2 - 4 at 10 μM (Table [2] column 4) is partly due to its cytotoxicity opposing its estrogenic activity, as is probably also the case with 2 - 4 at 100 μM. Interestingly, Table [2] also shows that the proliferation stimulation potencies (column 3) of 1 - 3, as deduced using the concentrations (EC₂₅) that caused a proliferation response equal to 25 % that of estradiol (column 2), were much lower than that of 4. In fact, the rank order of proliferation stimulating potencies (4 > 3 ≥ 2 ≥ 1) matched better that of RBAβ (4 > 3 ≥ 2 ≥ 1) rather than RBAα (4 ≥ 3 ≥ 2 ≥ 1), implying that stimulation of cell proliferation by 4, while primarily mediated via ERα, is positively modulated by ERβ, as already reported for ERβ-biased deoxybenzoins [11].

The activity of 1 - 4 in inducing the expression of estrogen target genes was compared to that of estradiol and genistein using MCF-7:D5L cells (Fig. [4] A). These are stably transfected with an artificial luciferase gene which allows one to report on the ERE-dependent regulation of transcription from the β-globin gene promoter, as mediated by the endogenous ER of MCF-7 cells [11]. Table [2] shows that the efficacies (column 7) of luciferase induction by 1 - 4 at 10 μM were comparable (2 - 4) to, or ca. 1.7-fold lower (1) than those of estradiol and genistein (assessed at 0.1 nM and 1 μM, respectively). ICI 182,780 (1 μM) could fully inhibit the luciferase response to 1 - 4, genistein or estradiol (data not shown). The level of luciferase activity in the presence of both 1 and ICI 182,780 matched that in their absence (i. e., of the control), suggesting that the time required to harness the luciferase response (16 h) was not adequate to unravel the ER-independent growth inhibitory effect of the isoflavone on MCF-7:D5L cells. The concentrations (EC₅₀) that effected an induction of luciferase expression equal to 50 % that of 0.1 nM estradiol (column 5) varied significantly (p < 0.01; t-test) between 1 - 3 and 4, and provided for induction potencies at least ca. 7-fold lower than that of genistein (column 6). Again, the rank order of luciferase induction potencies (4 > 3 ≥ 2 ≥ 1) matched better that of RBAβ (4 > 3 ≥ 2 ≥ 1) rather than RBAα (4 ≥ 3 ≥ 2 ≥ 1), implying that ERα-mediated induction of luciferase expression is positively modulated by the ERβ-binding selectivity of 4, as already reported for genistein [19] and ERβ-biased deoxybenzoins [11].

Fig. [4] B shows the concentration-dependence of induction of AlkP activity in estrogen-responsive Ishikawa cells by 1 - 4 as compared to estradiol and genistein. Table [2] shows that the efficacies (column 10) of AlkP induction by 1 - 4 at 10 μM were comparable to (2 - 4) or 4.5-fold lower (1) than those of genistein and estradiol. Notably, 1 - 4 (10 μM) failed to exhibit a growth inhibitory effect (as well as to mount an AlkP response) in the presence of 1 μM ICI 182,780 (not shown). However, some cytotoxicity was evident at higher concentrations, causing the AlkP response to drop considerably (Fig. [4] B). Thus, 1 (at 10 μM) is non-cytotoxic and only weakly estrogenic for endometrial cells. The EC₅₀ for AlkP induction by 2 - 4 (column 8) varied significantly (p < 0.05; t-test) between 4 and 2 or 3, and provided for induction potencies (column 9) at least 7-fold lower than that of genistein. The rank order of AlkP induction potencies (4 > 3 ≥ 2) matched better that of RBAβ rather than RBAα, indicating that ERβ binding of 4 may positively modulate the induction of AlkP activity in Ishikawa cells.

Discussion

The ERβ-binding selectivity of 4, albeit lower than that of genistein, contrasted the lack of selectivity exhibited by 1 - 3 (Table [1]). We have previously predicted that daidzein can fit in the ERβ ligand-binding cavity with an orientation similar to that of genistein [11]. The 4′- and 7-OH of genistein form hydrogen bonds with Glu305/Arg346 and His475, respectively, of ERβ [20]. Docking calculations for 1 - 3 suggest, however, that Glu305 and Arg346 interact with the 7-OH of these isoflavones (see Supporting Information). In the light of this orientation, compounds 1 - 3 appear to bind to ERα or ERβ with an affinity (Table [1]) inversely depending on the bulkiness of the substituents at positions 6 (for 2) and 8 (for 1). The steric hindrance imposed by these substituents presumably weakens the interaction of 7-OH with Glu305 and Arg346 and the ER-ligand interaction as a whole.

The estrogenic activity of 2 has not been studied before. Ruh et al. [21] reported that 3 at 10 μM stimulated the proliferation of MCF-7 cells and induced their expression of reporter genes to a level comparable to that of estradiol. Reports comparing the estrogenic activities of 3 and 4 are scarce. A few studies compared 3 and 4 using recombinant yeast, albeit this reportedly tends to underestimate the transactivation potency of the latter compound as compared to the former, especially with respect to ERα-mediated transcription [4]. Kuiper et al. [2] determined the RBAα and RBAβ of 3 and 4 and compared their ERα and ERβ-dependent induction of an ERE-dependent reporter using transiently transfected human embryonic kidney (HEK293) cells. They reported that only 4 exhibited significant ERβ-binding selectivity; that ERα- and ERβ-mediated inductions of reporter expression by 3 at 1 μM were comparable; and that the ERα-mediated inductions of reporter expression by 3 and 4 at 1 μM were weak and strong, respectively, in good agreement with the reporter gene expression data reported here.

Table [2] shows that the proliferation, luciferase and AlkP induction potencies of 4 are significantly higher than those of 1 - 3, while their RBAαs are comparable (Table [1]). This may reflect that the estrogenic activity of daidzein, while primarily mediated via ERα, is positively modulated by its selectivity for ERβ, as already reported for ERβ-biased deoxybenzoins [11] and genistein [19]. ERβ-mediated recruitment of co-activators [22] to the ERα-ERβ heterodimer may account for the positive character of this modulation. Alternatively, the lower induction potencies of 1 - 3 as compared to 4 may be the result of their opposite-to-genistein orientation imposing a conformational barrier to the ERα-mediated recruitment of co-activators by disrupting a hydrogen bond network that is reportedly necessary for ERα-mediated gene expression [23] (see also Supporting Information).

Concerning induction efficacies, 2 - 4 exhibited full agonism at 10 μM (i. e., >70 % of the efficacy of estradiol) in all the three assays of Table [2]. Genistein, on the other hand, was fully estrogenic at ca. 0.1 μM, resembling certain 8-isoprenylated flavanones (e. g., naringenin) in this respect [24], [25]. In contrast, 1 clearly exhibited partial (30 - 70 %) and weak (< 30 %) agonism in breast and endometrial cancer cells, respectively, probably reflecting cell-specific differences in the recruitment of co-activators capable of interacting with ER in its presence. Thus it appears that, although 1 - 4 can bind ERα with comparable affinities, the C-8 isoprenyl substituent of 1 prevents the receptor from fulfilling the conformational requirements for high estrogenic activity in Ishikawa cells but not in MCF-7 cells.

Acknowledgements

The technical assistance of A. Nastou is gratefully acknowledged. Dr. Barry R. Steele is acknowledged for reviewing the manuscript. This work was supported by grants EKBAN66 from the G.S.R.T-Greece, EUREKA E! 3060 and by a ‘Irakleitos’ Research fellowship from the University of Athens.

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Dr. Michael N. Alexis

Institute of Biological Research and Biotechnology

National Hellenic Research Foundation

11635 Athens

Greece

Phone: +30-210-7273741

Fax: +30-210-7273677

Email: mnalexis@eie.gr

References

• 1 Basly J P, Lavier M C. Dietary phytoestrogens: potential selective estrogen enzyme modulators?.  Planta Med. 2005;  71 287-94
  Thieme ConnectPubMedSearch in Google Scholar
• 2 Kuiper G G, Lemmen J G, Carlsson B, Corton J C, Safe S H, van der Saag P T. et al . Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.  Endocrinology. 1998;  139 4252-63
  CrossrefPubMedSearch in Google Scholar
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Dr. Michael N. Alexis

Institute of Biological Research and Biotechnology

National Hellenic Research Foundation

11635 Athens

Greece

Phone: +30-210-7273741

Fax: +30-210-7273677

Email: mnalexis@eie.gr

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