The FASEB Journal • Research Communication

Mechanisms enforcing the receptor ␤ selectivity of botanical

Yan Jiang,* Ping Gong,* Zeynep Madak-Erdogan,* Teresa Martin,† Muthu Jeyakumar,† Kathryn Carlson,† Ikhlas Khan,§ Troy J. Smillie,§ Amar G. Chittiboyina,§ Sateesh C. K. Rotte,§ William G. Helferich,‡ John A. Katzenellenbogen,† and Benita S. Katzenellenbogen*,1 *Department of Molecular and Integrative Physiology, †Department of Chemistry, and ‡Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA; and §National Center for Natural Products Research, University of Mississippi, Oxford, Mississippi, USA

ABSTRACT Because little is known about the ac- Key Words: chromatin binding ⅐ gene regulation ⅐ proliferation ⅐ tions of botanical estrogens (BEs), widely consumed breast cancer cells by menopausal women, we investigated the mecha- nistic and cellular activities of some major BEs. We As the average age of the U.S. population rises, an examined the interactions of , , increasing number of women are postmenopausal for , and liquiritigenin with estrogen receptors ER␣ many years, and due to the dramatic reduction in levels of and ER␤, with key coregulators (SRC3 and RIP140) and chromatin binding sites, and the regulation of estrogens, they often experience hot flashes, night sweats, gene expression and proliferation in MCF-7 breast and mood changes, and they suffer from urogenital cancer cells containing ER␣ and/or ER␤. Unlike the atrophy and loss of bone density. The traditional treat- endogenous estrogen, (E2), BEs preferen- ment for menopausal symptoms is to restore estrogen tially bind to ER␤, but their ER␤-potency selectivity levels by hormone replacement therapy (HRT). Despite in gene stimulation (340- to 830-fold vs. E2) is the well-known advantages of HRT, studies from the enhanced at several levels (coregulator recruitment, Women’s Health Initiative revealed several adverse effects chromatin binding); nevertheless, at high (0.1 or 1 of estrogen alone or estrogen plus progestin, such as ␮M) concentrations, BEs also fully activate ER␣. increased risk of heart disease (1), stroke (2), breast Because ER␣ drives breast cancer cell proliferation cancer (3), and dementia (4, 5). Consequently, there is and ER␤ dampens this, the relative levels of these great interest from researchers, clinicians, and the public two ERs in target cells and the BE dose greatly affect in the development of new treatment strategies to avoid gene expression and proliferative response and will the adverse effects of HRT. be crucial determinants of the potential benefits vs. Botanicals containing estrogenic compounds are widely risks of BEs. Our findings reveal key and novel available and are consumed by women, in particular by mechanistic differences in the estrogenic activities of older women seeking relief from menopausal symptoms, BEs vs. E2, with BEs displaying patterns of activity dis- with the expectation that these botanical estrogens (BEs) tinctly different from those seen with E2 and provide may provide a safe, natural source of estrogens to replace valuable information to inform future studies.—Jiang, Y., the loss of endogenous estrogens in menopause. Soy- Gong, P., Madak-Erdogan, Z., Martin, T., Jeyakumar, M., based products have drawn increasing attention lately as Carlson, K., Khan, I., Smillie, T. J., Chittiboyina, A. G., alternative treatments for relief of menopausal symptoms Rotte, S. C. K., Helferich, W. G., Katzenellenbogen, J. A., because their major isoflavone components, genistein Katzenellenbogen, B. S. Mechanisms enforcing the estro- ␤ and daidzein, are known to have estrogenic effects (6). gen receptor selectivity of botanical estrogens. FASEB J. However, preclinical studies (7, 8) and studies in humans 27, 4406–4418 (2013). www.fasebj.org have given inconclusive results regarding the efficacy of soy for this purpose (9–11). Because estrogens have stimulatory effects on many tissues, including increasing Abbreviations: BE, botanical estrogen; ChIP, chromatin the growth of some breast cancers, the unregulated immunoprecipitation; ChIP-seq, chromatin immunoprecipi- tation-DNA sequencing; E2, estradiol; ER, ; FRET, fluorescence resonance energy transfer; HRT, hor- mone replacement therapy; OTUB2, otubain 2; PgR, proges- 1 Correspondence: Department of Molecular and Inte- terone receptor; RBA, relative binding affinity; RCA, relative grative Physiology, University of Illinois and College of coactivator binding affinity; RIP140, receptor interacting pro- Medicine at Urbana-Champaign, Urbana, IL 61801, USA. tein 140; SRC, receptor coactivator; TR-FRET, time- E-mail: [email protected] resolved fluorescence resonance energy transfer doi: 10.1096/fj.13-234617

4406 0892-6638/13/0027-4406 © FASEB consumption of BEs might not be contributing uniformly Technologies, Grand Island, NY, USA), supplemented with to healthy aging in women (12–16). 5% calf serum (HyClone, Logan, UT, USA) and 100 ␮g/ml Estrogens exert effects on diverse target tissues and penicillin/streptomycin (Invitrogen, Carlsbad, CA). For es- cells, and they act through two different estrogen recep- trogen-free experiments, the cells were seeded in phenol ␣ ␤ ␣ ␤ red-free DMEM (Gibco/Life Technologies) plus 5%charcoal- tors (ERs), ER and ER (17–20). ER and ER are dextran-treated calf serum at a density of 2.5 ϫ 105 cells/well encoded by different genes and have different tissue of 6-well plate (for mRNA studies) or 1 ϫ 106 cells/10-cm distributions and different ligand binding specificities. tissue culture dish (for ChIP assays) for 3 d before siRNA ER␣ is generally the more potent regulator of gene transfection and adenovirus infection. Recombinant adenovi- expression and appears responsible for mediating the ruses were constructed and prepared as described (22, 24). proliferative drive of estrogens in some target tissues, such Cells were infected with either control adenovirus (Ad) ␤ expressing ␤-galactosidase or adenovirus expressing ER␤ as the uterus and some breast cancers, whereas ER when ␤ present together with ER␣ has a generally restraining (AdER ) for 72 h. Conditions used were those described ␣ previously (24, 29) to generate MCF-7 cells expressing levels effect on ER activities (21–25). Because we and others of ER␤ equal to that of the endogenously expressed ER␣. The have shown that many estrogens isolated from botanicals siRNA experiments for knockdown of the endogenous ER␣ in (e.g., genistein, daidzein, equol, liquiritigenin) are prefer- MCF-7 cells were performed as described previously and ential ligands for binding to ER␤ (6, 17, 26–28), they resulted in knockdown of ER␣ mRNA and protein by greater differ in this respect from endogenous estrogens and than 95% (24). siER␣ sequences (Dharmacon, Lafayette, CO, most estrogen pharmaceutical agents, which are ER␣- USA) were forward, 5=-UCAUCGCAUUCCUUGCAAAdTdT- binding preferential. Thus, one might expect the ER␤- 3=, and reverse, 5=-UUUGCAAGGAAUGCGAUGAdTdT-3= ␣ ␤ preferential botanicals to have different biological activi- (24). Because ER knockdown did not affect ER levels, the level of ER␤ obtained in the ER␤-only cells (24) was similar to ties than estradiol (E2). that of ER␣ in the original MCF-7 cells. E2, genistein, and To gain mechanistic information and to examine dose- were from Sigma-Aldrich (St. Louis, MO, dependent effects of BEs that might provide a new USA), liquiritigenin from Tocris Bioscience (Bristol, UK), conceptual framework for understanding whether BEs daidzein from Indofine Chemical Co. (Hillsborough, NJ, have similar or unique activities compared to those of USA), R-equol from Cayman Chemical Co. (Ann Arbor, MI, other estrogens, such as E2, we have studied in detail 4 USA). Racemic and S-equol were prepared as described (26, BEs, genistein, daidzein, S-equol, and liquiritigenin. We 30). All compounds were checked for identity and purity by measured their binding affinities to ER␣ and ER␤, and mass spectrometry and NMR. the affinity with which their complexes with ER recruit the key steroid receptor coactivator (SRC), SRC3; we exam- Relative binding affinity assay ined the chromatin binding of these ligand-receptor complexes along with that of the coregulators SRC3 and Relative binding affinities were determined by a competitive receptor interacting protein 140 (RIP140) at estrogen- radiometric binding assay as described previously (31, 32) using regulated genes by chromatin immunoprecipitation 2nM[3H]-E2 as tracer ([2,4,6,7-3H]-estra-1,3,5(10)-triene- ␤ (ChIP) assays, and we assessed their potency and efficacy 3,17 -diol, 70–115 Ci/mmol; Perkin Elmer, Waltham, MA, ␣ ␤ in regulating gene expression and their effects on prolif- USA), and purified, full-length, human ER and ER purchased from PanVera/Invitrogen. Incubations were for 18–24 h at 0°C. eration of human breast cancer cells (MCF-7) containing ␣ ␤ ␣ ␤ Hydroxyapatite (Bio-Rad, Hercules, CA, USA) was used to only ER , only ER , or both ER and ER , thereby absorb the receptor-ligand complexes, and free ligand was mimicking the different ratios of these two ERs present in washed away. The binding affinities are expressed as relative different ER target tissues and human breast tumors. Our binding affinity (RBA) values with the RBA of E2 set to 100%. studies highlight that BEs bind, induce coactivator recruit- The values given are the average Ϯ range or sd of2to3 ␣ ment, and stimulate chromatin binding preferentially of independent determinations. E2 binds to ER with a Kd of 0.2 ␤ ER␤ to enhance expression of ER␤-regulated genes. In nM and to ER with a Kd of 0.5 nM (31). addition, our findings provide new insight that, compared ␤ ␣ to E2, the ER vs. ER potency selectivity of BEs is Coactivator titration time-resolved fluorescence resonance enhanced at distinct levels (e.g., coregulator recruitment, energy transfer (FRET) assay to determine relative chromatin binding) beyond their binding affinities for coactivator-binding affinity (RCA) the two ERs. However, at high concentrations, rather than dampen cell proliferation through ER␤, they stimulate The assay was conducted exactly as described previously and proliferation through ER␣, highlighting that BE dose and utilized SRC3-fluorescein and biotinylated-ER␣ or ER␤ la- the presence of ER␣ and/or ER␤ in cells markedly affect beled with a streptavidin-terbium conjugate (33–35). the biological effects of BEs. RNA isolation and real-time PCR

MATERIALS AND METHODS Total RNA was isolated from cells using TRIzol (Invitrogen), RNA samples were reverse transcribed by MMTV reverse Ligands, cell culture, and construction of MCF-7 breast transcriptase (New England Biolabs, Ipswich, MA, USA), and cancer cells containing ER␣,ER␣, and ER␤,orER␤ only real-time PCR was carried out on the ABI Prism 7900HT (Applied Biosystems, Foster City, CA, USA) using SYBR Green MCF-7 cells [American Type Culture Collection (ATCC), PCR Master Mix (Roche, Basel, Switzerland) as described Manassas, VA, USA] were cultured in DMEM (Gibco/Life previously (22).

BOTANICAL ESTROGEN ACTIONS, ER␣,ER␤, COREGULATORS 4407 Western blot analysis sem of Ն3 independent experiments. Values of P Ͻ 0.05 were considered significant. Whole-cell extracts were prepared using 1ϫ ChIP lysis buffer supplemented with 1ϫ complete protease inhibitor (Roche). Proteins were separated on 10% SDS-PAGE gels and trans- ferred to nitrocellulose membranes. Western blotting used RESULTS antibodies against ER␣ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ER␤ (CWK-F12 produced by our laboratory; BEs show preference in binding and coactivator ␤ ref. 36), and -actin (Sigma-Aldrich). recruitment to ER␤ vs. ER␣

ChIP assays The BEs studied include genistein, daidzein, racemic equol, R-equol, S-equol, liquiritigenin, (the ChIP for ER␣ or ER␤ was carried out as described previously monomethyl ether of genistein), and formononetin (the (37) and used the ER␣ antibody HC-20 (Santa Cruz Biotech- ␤ monomethyl ether of daidzein). These compounds are nology). ER antibodies were a combination with equal parts displayed in Fig. 1 in the orientation that best shows their of CWK-F12 produced by our laboratory (36), GTX70182 ␤ (GeneTex, SanAntonio, TX, USA), GR40 (Calbiochem, La relationship to the structure of 17 -E2. Binding affinities Jolla, CA, USA), and PA1-311 (Thermo Fisher Scientific, for human full-length ER␣ and ER␤ were determined by Rockford, IL, USA), RIP140 (H-300; Santa Cruz Biotechnol- a radiometric competitive binding assay using tritiated ogy), and SRC3 (M397; Santa Cruz Biotechnology). The ChIP 17␤-E2 as tracer and E2 as standard (31, 32). Affinities are DNA was used for quantitative real-time PCR. expressed as RBA values where the affinity of E2 for ER␣ or ER␤ was set at 100% (Table 1). E2 has an absolute ␣ Cell proliferation assays binding affinity (Kd) of 0.2 nM for ER and 0.5 nM for ER␤ (31). All of the BEs have Ն10-fold lower affinity for WST-1 assay (Roche) was used to quantify cell viability, as ER␤ and Ն50-fold lower affinity for ER␣, compared to E2, described previously (38). Absorbance was measured at 450 but all of the naturally occurring BEs are quite ER␤ nm using a Bio-Rad 680 Microplate Reader, and all assays were performed in triplicate. preferential in their binding affinities. As seen in Table 1, the highest-affinity ER␤ ligand is genistein; its RBA of 6.8% corresponds to a K of 7.4 Statistics d nM, and it also has by far the greatest ER␤ selectivity ␤ ␣ϭ Statistically significant differences of gene expression changes ( / 324). Daidzein, the most abundant isoflavone in on different ligand treatments were analyzed by 1-way soy, binds much less well to both ERs than genistein ANOVA followed by Bonferroni post hoc test in GraphPad and with a reduced ER␤ preference. The reduced (GraphPad, San Diego, CA, USA). Results are the average Ϯ affinity of daidzein compared to genistein has been

OH O OH O OH H

H H O O O HO HO H HO Estradiol (E2) Genistein Daidzein

O OH O OH O OH

H H H HO HO HO S-Equol R-Equol (+/-)-Equol

O O OH O OH

O OH H O OH O MeO MeO HO

Biochanin A Formononetin Liquiritigenin

Figure 1. Structures of the BEs studied.

4408 Vol. 27 November 2013 The FASEB Journal ⅐ www.fasebj.org JIANG ET AL. TABLE 1. RBA of BEs for ER␣ and ER␤ and comparison then transcriptional potency must, in some way, reflect with E2 both ligand-binding affinity and coactivator-binding affin- ity (33). Thus, with the BEs, the selectivity of their ␤ ␣ BE RBAER␣ (%) RBAER␤ (%) / transcriptional activity through ER␤ (see below) should E2 100 100 1 be enhanced by both ligand-binding preference and Genistein 0.021 Ϯ 0.009 6.80 Ϯ 1.1 324 coactivator-binding preference (Table 3), an aspect that is Daidzein 0.003 Ϯ 0.001 0.051 Ϯ 0.02 17 discussed further in Discussion. S-equol 0.144 Ϯ 0.02 3.50 Ϯ 0.51 24 R-equol 0.374 Ϯ 0.05 0.327 Ϯ 0.02 0.9 Relative levels of ER␣ and ER␤ in cells affect Ϯ Ϯ Ϯ ( )-Equol 0.201 0.02 1.52 0.41 8 response to different BEs Biochanin A ϳ0.001 Ϯ 0 0.024 Ϯ 0.006 24 Ϯ Ϯ Formononetin 0.003 0.001 0.003 0.001 1 The potency of the BEs, acting through either ER␣ or Liquritigenin Ͻ0.001 0.013 Ϯ 0.003 Ͼ13 ER␤, in regulating the expression of estrogen-respon- RBA is calculated from the competitive radiometric binding sive genes was evaluated in MCF-7 cells containing 3 ϫ ␤ ␣ϭ curves: (IC50 E2/IC50 BE) 100. / RBAER␤/RBAER␣. different complements of ER: cells that contain ER␣ only, cells with ER␣ϩER␤ at equal levels, or cells with ER␤ only. These cells were constructed using ER␣ attributed to the absence of the phenolic hydroxy siRNA for ER␣ knockdown and/or adenovirus gene group that in genistein forms an intramolecular hydro- delivery for expression of ER␤, as we described previ- gen bond with the ketone; this leaves this polar func- ously (24, 46, 47). The expression of ER␣ and ER␤ tional group in daidzein isolated in the ER ligand- protein in the 3 cell types is shown in Fig. 3.ER␣-only binding pocket, which lowers its affinity because the ER MCF-7 cells contain endogenous ER␣,ER␣ϩER␤ MCF-7 cannot supply a suitable polar residue to partner with cells contain both ER␣ and ER␤, and ER␤-only MCF-7 cells this ketone (39). contain ER␤ and very little ER␣. Daidzein is metabolized in the gut by some micro- flora to S-equol (40), a conversion that increases its affinity for both ERs by 50- to 70-fold while still preserv- ing substantial ER␤ preference; thus, the ER␤ affinity of S-equol rivals that of genistein. The unnatural enan- tiomer, R-equol, also binds much better than daidzein, but its ER␣ binding affinity is more markedly increased, so it is very slightly ER␣ preferential. The binding of (Ϯ)-equol is, as expected, the average of that of R- and S-equol on both ERs. Not surprisingly, the methyl ethers, formononetin, and biochanin A, also have very low binding affinities (Ͻ0.03%) for both receptors, reflecting the well-known importance of having a free phenol that can mimic the A-ring phenol of E2 for high affinity (41). Liquiritigenin also has a very low affinity for both ERs but is distinctly ER␤-binding preferential. And as opposed to the isoflavonoid structures of the other BEs, liquiritigenin has a flavonoid structure that is a poorer structural mimic of E2 (see Fig. 1). We have developed a time-resolved fluorescence energy transfer (TR-FRET) assay to quantify the inter- action of coactivators and corepressors with nuclear hormone receptors (33, 42, 43), and we used this method in a coactivator-titration mode (33) to compare the affinity with which SRC3, a major coregulator found at high levels in breast cancer (30, 44, 45), binds to ER␣ and ER␤ complexes of these BEs. The titration curves are shown in Fig. 2, and affinities are summarized in Table 2. SRC3 binds to the ER␣-E2 complex very well, with ␣ ␤ an EC50 of 0.13 nM (which for comparison with BEs is Figure 2. Coactivator binding affinity to ER or ER com- considered to be an RCA of 100). The BEs, by contrast, form plexes with the BEs. In a coactivator titration protocol, complexes with ER␣, with which SRC3 has lower affinity increasing concentrations of the coactivator SRC3 (labeled (E2 Ͼ genistein Ͼ liquiritigen Ͼ S-equol Ͼ R-equol). All of with fluorescein) are added to a fixed concentration of the ER-BE complex (ER labeled through biotin with streptavidin- the BEs, however, form ER␤ complexes with which SRC3 ␤ terbium). The SRC3-ER interaction is monitored by time- has very good affinity, similar to that of the ER -E2 resolved FRET and expressed as receptor/donor emission ,as described previously (33 ,(1000ءcomplex. If one proposes that the transcriptional activity intensities ϫ 1000 (A/D of ER requires that it bind both ligand and coactivator, 42, 43). RCA, relative coactivator affinity with E2 set at 100%.

BOTANICAL ESTROGEN ACTIONS, ER␣,ER␤, COREGULATORS 4409 TABLE 2. Binding affinity of the coregulator SRC3 to ER␣ and ER␤ complexes with E2 and BEs expressed as RCA values

ER␣ ER␤ ␤ ␣ BE EC50 (nM) RCA EC50 (nM) RCA /

E2 0.13 Ϯ 0.004 [100] 0.12 Ϯ 0.002 [100] [1.0] Genistein 1.1 Ϯ 0.1 11.30 Ϯ 0.7 0.20 Ϯ 0.01 61 Ϯ 0.9 5.4 Daidzein 14.2 Ϯ 1.1 0.88 Ϯ 0.05 0.22 Ϯ 0.02 54.4 Ϯ 2.1 61.8 S-equol 16.7 Ϯ 0.6 0.73 Ϯ 0.01 0.18 Ϯ 0.01 78.5 Ϯ 4.2 108.0 R-equol 33.9 Ϯ 1.8 0.36 Ϯ 0.008 0.33 Ϯ 0.1 43 Ϯ 2.1 119.0 (Ϯ)-Equol 25.9 Ϯ 4.2 0.47 Ϯ 0.02 0.25 Ϯ 0.11 56 Ϯ 4.2 119.0 Biochanin A 88.8 Ϯ 6.4 0.14 Ϯ 0.02 0.26 Ϯ 0.01 46.7 Ϯ 1.5 334.0 Formononetin ND ND 0.53 Ϯ 0.02 22.9 Ϯ 1.7 — Liquiritigenin 2.2 Ϯ 0.4 4.60 Ϯ 0.5 0.13 Ϯ 0.01 88.9 Ϯ 6.7 19.3

ϭ ϫ ␤ ␣ϭ RCA (EC50 E2/EC50 BE) 100. / RCAER␤/RCAER␣. ND, not detectable.

For our studies of endogenous gene expression reg- also effectively elicited in cells containing ER␣ only ulation, we selected the 4 most significant BEs: (Fig. 4). In addition, gene stimulation by BEs and E2 in genistein, daidzein, S-equol, and liquiritigenin. R-equol the 3 receptor cell backgrounds was completely sup- is not found naturally but was studied for comparison. pressed by an excess of the ICI 182,780 We investigated the effect of the 4 BEs on the expres- () implying ER mediation (data not shown). sion of the progesterone receptor (PgR), an ER␣- The 4 BEs displayed a 5- to 10-fold ER␤ potency responsive gene (48), and otubain 2 (OTUB2), an preference for gene regulation. Considering that E2 ER␤-responsive gene (24), in the ER␣-only, ER␣ϩ has a gene regulation potency preference ϳ50-fold in ER␤, and ER␤-only cells over a dose range, comparing favor of ER␣, the BEs thus have a 250- to 500-fold them with the response to E2. E2 showed a marked ER␣ greater ER␤ potency preference relative to E2 in terms potency preference in gene stimulation: In ER␣-only of their regulation of gene expression (Table 3). This ␤ cells, E2 had an EC50 of 0.02 nM for stimulating PgR likely reflects both the preference of ER for binding gene expression, whereas in ER␣ϩER␤ or ER␤-only the BEs and of coactivators for binding to the ER␤-BE cells, it had considerably higher EC50 values for stimu- complexes. The unnatural R-equol enantiomer showed lating OTUB2 gene expression (Fig. 4A). Thus, al- little if any ER␤ potency selectivity in gene regulation though E2 binds to ER␣ only 2–3-fold better than to (Fig. 4E), consonant with its nearly equal binding ER␤, its ER␣ potency preference for gene regulation affinity for ER␣ and ER␤ (Table 1). was much greater than this (ϳ50-fold; red curve is to the left of green curves), and it was especially poor in Potency and efficacy of liquiritigenin through ER␤ stimulating gene expression in ER␤-only cells. are greater than expected from its ER␤ binding Notably, by contrast, the 4 naturally occurring BEs affinity: gene stimulation and chromatin binding of showed a very different (more ER␤ preferential) pat- receptors and coregulators tern of potency in gene regulation (green curves to left of red curve). As seen in Fig. 4B, low concentrations of In terms of ligand binding, the RBA of liquiritigenin for genistein stimulated gene expression more effectively ER␣ is only ϳ0.001% and for ER␤ is only 0.021% that of when ER␤ was present in cells. Daidzein, S-equol, and E2 (Table 1). Thus, the RBA of liquiritigenin is 105-fold liquiritigenin (Fig. 4C, D, F) also showed ER␤ potency- lower than that of E2 for ER␣ and 5000-fold lower for ␤ preferential gene regulation. However, with all com- ER . However, the EC50 values for liquiritigenin stimula- pounds at high concentrations, gene stimulation was tion of OTUB2 gene expression in ER␣ϩER␤ or

TABLE 3. Potency of the BEs and their preferential activities through ER␤ vs. ER␣ in cellular (gene stimulation) and cell-free (ligand binding and coactivator binding) assays

Potency from cellular activities Potency from cell-free activities ␤/␣ potency ␤/␣ selectivity Gene stimulation, EC50 (nM) selectivity RBA ϫ RCA ␤/␣ RCA ␤/␣ RBA Ligand ER␤ only, OTUB2 ER␣ only, PgR [relative to E2] [relative to E2] selectivity selectivity

E2 1.67 0.02 0.012 [1.0] [1.0] [1.00] [1.00] Genistein 4 24 6 [500] 1750 5.4 324 S-equol 1.4 14 10 [830] 2600 108 24 Daidzein 50 200 4 [340] 1024 62 17 Liquiritigenin 60 300 5 [420] 400 19 21

OTUB2, otubain 2; PgR, progesterone receptor.

4410 Vol. 27 November 2013 The FASEB Journal ⅐ www.fasebj.org JIANG ET AL. ABment of ER␣,ER␤, and key coregulators to their ERα ERα+ERβ ERα ERβ AdERβ - + binding sites on target genes. Based on the dose- AdERβ - + ERα siRNA - + response studies for liquiritigenin shown in Fig. 4, 2 ERβ concentrations were selected. The low concentration ERβ (50 nM) elicited little stimulation of PgR gene expres- ERα ␣ ERα sion in ER MCF-7 cells but did stimulate some OTUB2 gene expression when ER␤ was present; by contrast, at β-actin β-actin the high concentration (1 ␮M), liquiritigenin was able to stimulate both PgR and OTUB2 gene expression in ␣ ␤ Figure 3. Western blots show ER␣ and ER␤ levels in cells with cells containing ER or ER , respectively (Fig. 5B, E). the 3 complements of ERs. A) MCF-7 cells were infected with These two concentrations of liquiritigenin (50 nM control adenovirus or ER␤-expressing adenovirus to generate and 1 ␮M), and 10 nM E2 for comparison, were then cells containing ER␣ only and ER␣ϩER␤, respectively. used in ChIP assays in cells with these three different B) Cells designated as ER␤ only were generated by knock- ␣ ␣ ␤ complements of ERs to examine the recruitment of down of ER using ER siRNA and then infection with ER ER␣,ER␤, SRC3, and another coregulator, RIP140, to adenovirus. binding sites on the PgR and OTUB2 genes (Fig. 5). Using ChIP-DNA sequencing (ChIP-seq; ref. 47), we ER␤-only cells are ϳ100 nM (Fig. 4F), while that of E2 is have identified binding sites of ER␣,ER␤, and the ϳ1 nM. This means that liquiritigenin only needs ϳ100- coregulators SRC3 and RIP140 on the PgR and OTUB2 fold higher concentrations than E2 to achieve the same genes after treatment of cells with E2 (Fig. 5A, D). As effects through ER␤ in ER␣ϩER␤ or ER␤-only cells. expected, 10 nM E2 recruited ER␣ to binding sites on Thus, liquiritigenin appears to have greater potency and the PgR gene in ER␣-only or ER␣ϩER␤ MCF-7 cells, efficacy through ER␤ in gene regulation than expected and it recruited ER␤ to binding sites on PgR in from its ER␤-binding affinity, which could be a reflection ER␣ϩER␤ or ER␤-only cells (Fig. 5C, solid bars). E2 of the very good SRC3 binding to the ER␤-liquiritigenin also recruited SRC3 and RIP140 to the same binding complex (Fig. 2 and Table 2). sites on the PgR gene, with highest recruitment ob- Because liquiritigenin has been a BE of considerable served in ER␣ cells, lowest in ER␤ cells, and interme- interest (27, 44), we sought to further understand the diate in ER␣ϩER␤ cells. The high concentration of mechanism for its increased potency and efficacy in liquiritigenin (Fig. 5C, shaded bars) recruited ER␣, stimulating gene expression by examining the recruit- ER␤, SRC3, and RIP140 to binding sites on PgR, in a

A Estradiol B Genistein C Daidzein OTUB2mRNA (fold change) ERα, PgR OTUB2 mRNA (fold change) OTUB2 mRNA (fold change) 10 10 25 5 25 15 ERα+ERβ, OTUB2 ERβ, OTUB2 8 8 20 4 20 10 6 6 15 3 15

4 4 10 2 10 5 2 2 5 1 5 PgR mRNA (fold change) (fold mRNA PgR PgR mRNA (fold change) mRNA PgR PgR mRNA (fold change) mRNA PgR 0 0 0 0 0 0 -12 -11 -10 -9 -8 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 Estradiol (log M) Genistein (log M) Daidzein (log M)

D S-Equol E R-Equol F Liquiritigenin OTUB2 mRNA (fold change) OTUB2 mRNA (fold change) OTUB2 mRNA (fold change) 30 10 25 8 20 6

8 20 6 15 20 4 6 15 4 10 4 10 10 2 2 5 2 5 PgR mRNA (fold change) mRNA PgR 0 0 (fold change) mRNA PgR 0 0 (fold change) mRNA PgR 0 0 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 S-Equol (log M) R-Equol (log M) Liquiritigenin (log M)

Figure 4. Effects of E2 and BEs on gene regulation in MCF-7 cells containing ER␣ only, ER␣ϩER␤,orER␤ only. Effects of E2 (A), genistein (B), daidzein (C), S-equol (D), R-equol (E), and liquiritigenin (F)onER␣-preferential progesterone receptor (PgR) gene expression (red) or ER␤-preferential OTUB2 gene expression (green) monitored after 4 h treatment.

BOTANICAL ESTROGEN ACTIONS, ER␣,ER␤, COREGULATORS 4411 PgR AB150 Veh 10 nM E2 50 nM Liq 100 1 µMLiq

50

PgR Fold induction (mRNA) 0 α β β ER ER α+ER R E C 15 6 6 6 β α

10 4 4 4

5 2 2 2 (% of Input) of (% (% Input) of (% Input) of (% of Input) of (% Recruitment of ER of Recruitment Recruitment of ER Recruitment of SRC3 of Recruitment Recruitment of RIP140 0 0 0 0 α β β α β β α β β α β β R R ER ER ER ER ER E ER ER E α+ER α+ER α+ER α+ ER ER ER ER D E OTUB2 5 Veh 4 10 nM E2 50 nM Liq 3 1 µMLiq

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Fold induction (mRNA) 0 α β β R R OTUB2 E E α+ER ER

F 1.5 5 1.5 2.0 α β 4 1.5 1.0 1.0 3 1.0 2 0.5 0.5 (% of Input) (% of (% Input) of (% of Input) (% of Input) 0.5 1 Recruitment of ER Recruitment of Recruitment of ER of Recruitment Recruitment of SRC3 of Recruitment Recruitment of RIP140 of Recruitment 0.0 0 0.0 0.0 α α α β β α β β β β β β R ER ER E ER ER ER ER ER α+ER α+ER α+ER α+ER ER ER ER ER

Figure 5. Recruitment of ER␣,ER␤, and coregulators SRC3 and RIP140 to chromatin by E2 or liquiritigenin to mediate PgR and OTUB2 gene expression. A, D) Binding sites identified by ChIP-seq for ER␣,ER␤, and cofactors SRC3 and RIP140 at the PgR (A) and OTUB2 genes (D). B, E) Effects of 10 nM E2 and 50 nM or 1 ␮M liquiritigenin on PgR (B) or OTUB2 (E) gene expression monitored at 4 h. mRNA level in control vehicle MCF-7 ER␣ cells is set at 1. C, F) Recruitment of ER␣ or ER␤, SRC3, or RIP140 to chromatin binding sites at the PgR gene (C) or OTUB2 gene (F) in MCF-7 cells containing ER␣ only, ER␣ϩER␤,orER␤ only that were treated for 45 min with 0.1% control ethanol vehicle, 10 nM E2, or 50 nM or 1 ␮M liquiritigenin prior to ChIP. manner similar to that of 10 nM E2. The low concen- liquiritigenin was able to recruit ER␤ in ER␣ϩER␤ or tration of liquiritigenin (Fig. 5C, hatched bars), how- ER␤-only cells, it was not effective in recruiting ER␣, ever, showed a different pattern. Even though 50 nM SRC3, and RIP140 to the same binding sites on PgR

4412 Vol. 27 November 2013 The FASEB Journal ⅐ www.fasebj.org JIANG ET AL. (Fig. 5C). Since PgR is an ER␣-responsive gene and (Fig. 6). E2 stimulated proliferation of MCF-7 cells needs ER␣ and cofactor recruitment to initiate gene containing ER␣ only at ligand concentrations as low Ϫ Ϫ expression, this explains the observation that the low as 10 12 M and elicited maximal stimulation at 10 11 concentration of liquritigenin did not stimulate PgR M. Expression of ER␤ along with ER␣, however, gene expression in the 3 types of MCF-7 cells (Fig. 5B). greatly reduced the magnitude of E2-stimulated cell Both low and high concentrations of liquiritigenin, as proliferation (Fig. 6A). well as 10 nM E2, up-regulated OTUB2 gene expression Consistent with the results of the dose-response ␣ϩ ␤ ␤ in ER ER or ER -only MCF-7 cells (Fig. 5E). Both gene expression studies, low concentrations (Ͻ10 ␤ concentrations of liquiritigenin recruited ER , SRC3 and nM) of genistein, daidzein, S-equol, R-equol, and RIP140 to binding sites on OTUB2, but liquiritigenin was liquiritigenin did not stimulate proliferation of ER␣- ␣ less good in recruitment of ER , showing recruitment only MCF-7 cells, but they were able to effectively ␮ only at the high 1 M concentration (Fig. 5F). Since promote proliferation of ER␣-only cells at high con- ␤ OTUB2 is an ER -responsive gene, its gene expression centrations (Fig. 6). S-equol was the most effective in was highest in ER␣ϩER␤ or ER␤-only cells, consistent ␣ ␤ stimulating proliferation through ER , with good with the strong recruitment of ER and the two coregu- stimulation being observed at 10 nM. Notably, the lators in these cells (Fig. 5F). By contrast, E2 recruited copresence of ER␤ dampened the stimulation of cell ER␣ to binding sites on the OTUB2 gene in ER␣-only or proliferation through ER␣ by the high concentra- ER␣ϩER␤ cells, and E2 recruited ER␤ to the binding tions of the BEs. The effect of ER␤ in reducing site on the OTUB2 gene in ER␣ϩER␤ or ER␤-only cells. proliferation was most marked with E2, liquiritige- E2 also recruited SRC3 and RIP140 to the same binding nin, and S-equol (Fig. 6, red arrows), and least with sites on the OTUB2 gene, with highest recruitment in ␣ϩ ␤ daidzein and genistein (Fig. 6, beige arrows). ER ER cells. These results indicate that despite its ␤ very low binding affinity for the ERs, relatively low con- In cells containing ER only, the basal, control centrations of liquiritigenin can recruit ER and coactiva- rate of cell proliferation monitored by Ki67 gene ␣ tor proteins and activate gene transcription in a markedly expression was reduced compared to that of ER -only ER␤-preferential manner. cells, and 10 nM E2 enhanced expression of Ki67 mRNA in both ER␣-only or ER␤-only cells (Fig. 7A). Consistent ␤ ␣ BEs vary in their effectiveness in promoting the with the greater efficacy of BEs through ER vs. ER , ER␤-dampening effect on the proproliferative activity the low concentration of genistein and daidzein ␤ ␣ of ER␣ elevated Ki67 in cells containing ER but not ER (Fig. 7B, C). At a high concentration (1 ␮M), all BEs We examined the effects of the BEs on the prolifer- except S-equol elicited increased Ki67 expression in ation of cells containing ER␣-only or ER␣ϩER␤ both ER␣-only and ER␤-only cells(Fig. 7B–E). S-

A 5 C E Estradiol 5 Daidzein R-Equol 5 4 ERα 4 4 3 ERα 3 ERα 3 2 ERα+ERβ 2

(fold change) 2 (fold change) Cell Proliferation (fold change) Cell Proliferation

1 Cell Proliferation 1 ERα+ERβ 1 ERα+ERβ

0 -9 -8 -7 -6 3 1 -9 -8 -7 -6 -5 1 -9 -8 -7 -6 -5 -13 -12 -11 -1 -1 -12 -1 -10 -13 -12 -1 -10 Concentration (Log M) Concentration(LogM) Concentration (Log M) B GenisteinD S-Equol F Liquiritigenin 5 5 5 ERα 4 4

4 on ti

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3 ERα 3 fera 3 li ro

2 2 P 2 (fold change) (fold change) (fold cha ell Cell Proliferation Cell Proliferation ERα+ERβ C ERα+ERβ 1 1 ERα+ERβ 1

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Figure 6. Effects of E2 (A), genistein (B), daidzein (C), S-equol (D), R-equol (E), and liquiritigenin (F) on the proliferation of ER␣-only or ER␣ϩER␤ cells. Proliferation was monitored after 5 d. Arrows indicate magnitude of suppression of proliferation by the specified ligand in cells containing ER␣ϩER␤ vs. cells with ER␣ only. Red arrows indicate greater suppression; beige arrows indicate lesser suppression.

BOTANICAL ESTROGEN ACTIONS, ER␣,ER␤, COREGULATORS 4413 A Estradiol B Genistein C Daidzein 2.5 ERα cells 2.5 ERα cells 2.5 ERα cells ERβ cells ERβ cells ERβ cells ** ** ** 2.0 2.0 ** 2.0 *

1.5 1.5 1.5 * * ** ** * * * 1.0 1.0 1.0 Ki67 mRNA (fold change) (fold mRNA Ki67 0.5 change) (fold mRNA Ki67 0.5 Ki67 mRNA (fold change) (fold mRNA Ki67 0.5

0.0 0.0 0.0

2 M M M M E µ E2 µ E2 µ E2 µ Veh Veh nM Veh nM Veh 6 nM 1 6 1 1 0 nM 1 Veh Veh nM nM nM 20 2 nM E2 10 Genistein 10 Genistein 10 Daidzein 10 nM Daidzein 10 nM E2 10

D S-Equol E Liquiritigenin 2.5 2.5 ERα cells ERα cells ERβ cells ERβ cells ** ** 2.0 * 2.0 ** Figure 7. Effects of E2 (A), genistein (B), daidzein (C), S- 1.5 1.5 equol (D), and liquiritigenin (E) on expression of the pro- * * liferation-related gene Ki67 in ER␣-only or ER␤-only cells. 1.0 1.0 * RNA was isolated from cells after 24 h, and gene expres- sion was assessed by qPCR. Ki67 mRNA (fold change) mRNA Ki67 Ki67 mRNA (fold change) (fold mRNA Ki67 0.5 0.5

0.0 0.0

M 2 M M M µ µ M µ E2 µ Veh Veh nM Veh Veh nM M E2 1 0 1 1 0 1 20 nM 2 20 n nM 2 10 n 10 nM E 10 nM E2 10 S-Equol S-Equol Liquirit Liquirit equol did not elevate Ki67 in ER␤-only cells, but did BEs becomes progressively reinforced as their activity at both the lower and higher concentration in ER␣- proceeds down the ER axis and involves key coregulator only cells, perhaps reflecting the fact that S-equol and chromatin binding aspects. Although the 4 major had the highest binding affinity of the natural BEs naturally occurring BEs we studied in detail bind to for ER␣ (Table 1). ER␤ with a 20- to 300-fold preference over ER␣, using our TR-FRET assay, we found that the BEs recruit SRC3 to ER␤ as well as does E2, whereas their potency in DISCUSSION recruiting SRC3 to ER␣ was only 1–10% that of E2, thereby reinforcing their ER␤ preference at a second ER␤ selectivity of BEs becomes progressively (coregulator) level. Further evidence for the amplifica- amplified along the ER signaling axis tion of the ER␤ potency preference of the BEs came from dose-response studies of ER␣- vs. ER␤-responsive We undertook these studies to examine whether the gene expression in breast cancer cells containing dif- estrogenic activity of BEs was similar to or different ferent complements of ER␣ and ER␤. Here we found from that of E2, and if different, through what mech- that the relative potency of BEs for activating ER␤- vs. anistic components did these differences arise. While it ER␣-regulated genes compared to E2 was as high as was well known that BEs have preferential binding 500. Consequently, at low concentrations, the BEs affinity for ER␤, our findings are novel in revealing regulated gene expression that was preferentially that, relative to E2, the ER␤-preferential potency of the through ER␤; however, this preferential regulation of

4414 Vol. 27 November 2013 The FASEB Journal ⅐ www.fasebj.org JIANG ET AL. estrogen-responsive gene expression by ER␤ was no liquiritigenin did not stimulate the proliferation of longer observed in cells treated with higher concentra- ER␣-only cells, while high concentrations stimulated tions of BEs, when ER␣ and ER␤ were equally well ER␣-only MCF-7 cell proliferation to an extent as great recruited to gene regulatory sites on chromatin and as E2. A recent report on the dynamic racemization of both functioned as effective ligand-regulated transcrip- the liquiritigenin enantiomers and the equilibration of tion factors and stimulators of proliferation through these flavonoids with the ring-opened ER␣. We previously noted this dose-dependent ER␤ vs. chalcone isomer suggests that the net biological activity ER␣ selectivity for genistein in gene stimulation (24). of this compound may actually be the combined result Table 3 contains a summary of our dataset, arranged of the activity from an equilibrating mixture of these 3 in terms of the preference of the 4 principal BEs for different chemical species, regardless of which compo- activity through ER␤ vs. ER␣ from both cell-free assays nent has been administered (44); this is most likely true and cell-based assays. When the cellular potency selec- for most, cell-free, cellular, and in vivo studies with this tivity values are referenced to the selectivity of E2, the compound. ER␤ potency preference of the 4 BEs in cells ranged Metabolism of other BEs may also be occurring in from 340 to 830. These values are greater than their MCF-7 cells, since there is evidence for phase II metab- ER␤-binding selectivities and are notably similar to that olism and its up-regulation by botanicals, e.g., UGT predicted from the product of their ER␤-binding selec- 2B15 up-regulation by E2 and genistein (50) and tivity (RBA) and their SRC3 coactivator recruitment sulfation of genistein and biochanin A (51). However, potency selectivity (RCA) (400–2600). Thus, it appears the extent of metabolism and its relationship with oral that both their ER␤ ligand-binding preference and first-pass effects in vivo are not known. their coactivator ER␤-binding preference (33) make significant contributions to the very high ER␤ potency BE proliferative effects on breast cancer cells: ligand selectivity of the BEs in a cellular context. dose and ER␣ and ER␤ cell context

S-equol and liquiritigenin as unusual BEs: mechanism It is now well established that ER␣ is the major driver of and potency the proliferative effects of estrogens in both breast cancer and normal reproductive tissues, with ER␤ Two other aspects of altered potency relate to S-equol serving largely as a brake on ER␣-driven proliferation and liquiritigenin. S-equol is an intestinal metabolite of (22, 23, 48, 52). Notably, while normal breast tissue daidzein, the most abundant isoflavone in soy, and this contains both ER␣ and ER␤,ER␤ levels decline with metabolic conversion results in a 50- and 70-fold in- the development and progression of breast cancer to a crease in binding affinity for ER␣ and ER␤, respec- more malignant state, so that ER␣ becomes the major tively. Thus, unlike its low-affinity metabolic precursor, player in hormone-responsive advanced breast cancer daidzein, S-equol had a potency similar to that of (53–55). The MCF-7 breast cancer cells we studied genistein in terms of stimulation of gene expression having ER␣ only, ER␣ϩER␤, and ER␤ only provide a and regulation of proliferation through ER␣ or ER␤. convenient model system to examine the proliferative The enteric production of S-equol from daidzein, how- and gene regulatory effects of various estrogens ever, depends on the constituent microflora, with indi- through the two ERs in breast cancer (22, 24). Previ- viduals being “low” and “high” equol producers (40). ously we and others reported that E2 markedly stimu- Thus, the exposure of individuals to S-equol depends lated cell proliferation in ER␣-only containing breast not only on their intake of the precursor, daidzein, but cancer cells, while proliferation in ER␣ϩER␤ cells was the degree to which daidzein undergoes metabolic more limited (23–25, 29, 47). conversion to S-equol. All 4 BEs had little or no effect on ER␣-only MCF-7 There is much current interest in understanding the cell proliferation at low concentrations, whereas at high effects of liquiritigenin, the most active estrogenic concentrations they stimulated proliferation of these compound from licorice root (Glycyrrhizae uralensis). cells quite well. And, as with E2, expression of ER␤ Liquiritigenin is known to be a highly selective ER␤ along with ER␣ reduced the proliferative response of agonist (27) and has been shown to significantly reduce cells to the BEs at all concentrations. Thus, stimulation tumor growth in a glioma xenograft model that con- of proliferation appears to track with the potency of a tains only ER␤ (49). Even though liquiritigenin has a BE for ER␣, with ER␤ always having a growth-suppres- very low affinity for both ER␣ and ER␤, its ER␤ sive effect on ER␣, which was observed with all BEs and preferential binding and activity were evident in our with E2. Of note, however, the copresence of ER␤ studies. In the 3 types of MCF-7 cells, liquiritigenin along with ER␣ reduced proliferation with E2, proved to be more potent in stimulating gene expres- liquiritigenin, and S-equol more so than with daid- sion than expected based on its binding affinity. Our zein and genistein. Also, we found that proliferation results suggest that this increment in potency might be was slower in ER␤-only vs. ER␣-only cells. In this due to the competence of the ER-liquiritigenin com- regard, it is of interest that patients with human plex for the recruitment of coactivators and for binding breast tumors that express ER␤ in the absence of ER␣ to regulatory chromatin sites of estrogen-responsive are reported to have a generally good prognosis and genes. As observed with the other 3 BEs, low doses of clinical outcome (56).

BOTANICAL ESTROGEN ACTIONS, ER␣,ER␤, COREGULATORS 4415 Potential health effects of BEs This project was made possible by U.S. National Institutes of Health (NIH) grant P50AT006268 (to W.G.H., I.K., B.S.K., BEs are being widely consumed by women seeking and J.A.K.) from the National Center for Complementary and relief from menopausal symptoms, and while their Alternative Medicines (NCCAM), the Office of Dietary Sup- plements (ODS), and the National Cancer Institute (NCI). Its efficacy for this therapeutic purpose is uncertain, much contents are solely the responsibility of the authors and do still remains to be understood about potential benefits not necessarily represent the official views of the NCCAM, and risks of BEs. To provide a better basis for further ODS, NCI, or NIH. Partial support to Z.M.E. and T.M. was understanding of these aspects, we investigated the from NIH T32ES007326. estrogenic character of 4 major BEs in a broader context; this involved examination of their binding to ER␣ and ER␤, interaction of the ligand-receptor com- plexes with coregulators, receptor and coregulator re- REFERENCES cruitment to chromatin binding sites of estrogen-re- 1. Manson, J. E., Hsia, J., Johnson, K. C., Rossouw, J. E., Assaf, A. R., sponsive genes, and effects on the regulation of ER- Lasser, N. L., Trevisan, M., Black, H. R., Heckbert, S. R., subtype-selective gene expression and breast cancer cell Detrano, R., Strickland, O. L., Wong, N. D., Crouse, J. R., Stein, proliferation. The BEs are clearly different from E2 in E., and Cushman, M. (2003) Estrogen plus progestin and the affinity and potency, but most notably in their selectiv- risk of coronary heart disease. N. Engl. J. Med. 349, 523–534 ␤ ␣ 2. Wassertheil-Smoller, S., Hendrix, S. 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4418 Vol. 27 November 2013 The FASEB Journal ⅐ www.fasebj.org JIANG ET AL.