Equol an isoflavonoid: potential for improved prostate health, in vitro and in vivoevidence
© Lund et al; licensee BioMed Central Ltd. 2011
Received: 3 December 2010
Accepted: 13 January 2011
Published: 13 January 2011
To determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones.
First, in vitro equol binding affinity for 5alpha-DHT was determined using 14C5alpha-DHT combined with cold 5alpha-DHT (3.0 nM in all samples). These steroids were incubated with increasing concentrations of equol (0-2,000 nM) and analyzed by Sephadex LH-20 column chromatography. 14C5alpha-DHT peak/profiles were determined by scintillation counting of column fractions. Using the 14C5alpha-DHT peak (0 nM equol) as a reference standard, a binding curve was generated by quantifying shifts in the 14C5alpha-DHT peaks as equol concentrations increased. Second, equol's in vitro effects on LNCap cells were determined by culturing cells (48 hours) in the presence of increasing concentrations of dimethyl sulfoxide (DMSO) (vehicle-control), 5alpha-DHT, equol or 5alpha-DHT+equol. Following culture, prostate specific antigen (PSA) levels were quantified via ELISA. Finally, the in vivo effects of equol were tested in sixteen male Long-Evans rats fed a low isoflavone diet. From 190-215 days, animals received 0.1cc s.c. injections of either DMSO-control vehicle (n = 8) or 1.0 mg/kg (body weight) of equol (in DMSO) (n = 8). At 215 days, body and prostate weights were recorded, trunk blood was collected and serum assayed for luteinizing hormone (LH), 5alpha-DHT, testosterone and 17beta-estradiol levels.
Maximum and half maximal equol binding to 5alpha-DHT occurred at approximately 100 nM and 4.8 nM respectively. LNCap cells cultured in the presence of 5alpha-DHT significantly increased PSA levels. However, in the presence of 5alpha-DHT+equol, equol blocked the significant increases in PSA levels from LNCap cells. In vivo equol treatment significantly decreased rat prostate weights and serum 5alpha-DHT levels but did not alter LH, testosterone, and estradiol levels.
Equol administration appears to have potential beneficial effects for prostate health and other 5alpha-DHT mediated disorders. Equol administration: reduces PSA levels from LNCap cells under 5alpha-DHT stimulation, decreases rat prostate size, decreases serum 5alpha-DHT levels and androgen hormone action, while not altering other circulating sex steroids or LH levels.
Polyphenols are a group of chemical substances found in plants that include berries, grapes, walnuts, peanuts, pomegranates, and other fruits and vegetables . Many of these polyphenol preparations are available as dietary supplements . The largest and best studied polyphenols are the dietary estrogen-like molecules or phytoestrogens . Of the three main classifications of phytoestrogens: 1) isoflavones (derived principally from soybeans), 2) lignans (found in flaxseed in large quantities), and 3) coumestans (derived from sprouting plants like alfalfa), human consumption of isoflavones has the largest impact due to its availability and variety in food products. Isoflavones have been implicated as potential treatments for many disorders including cardiovascular disease, osteoporosis, age-related diseases, and hormone-dependent cancers [3, 4].
However, these isoflavone molecules do not exist at high levels in their biologically active form in natural food products, but rather are at high abundance in a precursor form [3–5]. For example, daidzin, the precursor of daidzein, is the glycosidic form that contains a carbohydrate portion of the molecule. Daidzin is metabolized in the gastrointestinal tract by intestinal bacteria, which hydrolyze the carbohydrate moiety, to the biologically active isoflavone, daidzein [4, 5]. Daidzein is then further metabolized in the intestine to equol at relatively low or high levels dependent upon several biological, dietary and presumably environmental factors [4, 5]. Although, recent evidence suggests that equol is found naturally in white cabbage .
For example, it is known that prostate cancer cells are supported in their growth by androgen stimulation and the androgen-regulated expression of the prostate specific antigen (PSA) is a biological marker of such stimulation . Logically, any treatment that could decrease PSA levels in prostate cancer cells, or antagonize specific androgen hormone action, would have great potential in addressing prostate disorders, such as benign prostatic hyperplasia (BPH) or prostate cancer. With this in mind, we sought, in the following experiments, to determine equol's effect on 5α-DHT levels in cultured human prostate cancer (LNCap) cells. We also examined equol's effect on both prostate weight and circulating hormone levels in vivo using Long-Evans rats. In brief, the present results demonstrate that equol: a) (R- and/or S-isomeric mixtures) has high binding affinity for 5α-DHT making it a potent selective androgen modulator (SAM), b) blocks the stimulatory androgen action of 5α-DHT in increasing prostate specific antigen (PSA) levels in human cancer (LNCap) cell cultures and, c) significantly decreases serum 5α-DHT and subsequently prostate weight without altering, testosterone, 17β-estradiol or LH levels. Applications for equol to improve prostate disorders and other androgen-mediated conditions is also discussed.
Experiment 1. In vitrobinding of Equol (Isomer Ratio) to 5α-DHT
In a 20 ml amber glass vial approximately 4,000 to 5,000 dpm of 14C5α- Dihydrotestosterone (DHT; Dupont/NEN, Boston, MA, USA) was added. Additionally, 3.0 nM (or approximately 865 pg/ml) of cold 5α-DHT (Sigma/Aldrich Chemical Co., St. Louis, MO, USA) was added (in all experiments) and where appropriate varying concentrations of equol (70% R-equol and 30% S-equol mixture; from 0 to 2,000 nM; obtained from LC Labs., Woburn, MA,USA or Robert Handa's Laboratory, Colorado State University, USA, R-equol or S-equol isomers only) from ethanol stock solutions. This mixture was vortexed for 30 seconds at room temperature and then dried down. Subsequently 1 ml of TEGMD (10 mM Tris CL, 1.5 mM EDTA, 10% glycerol, 25 mM molybdate and 1 mM dithrothreitol) buffer was added, capped, and mixed by inverting the vial 3-times. This mixture was incubated at room temperature for 20-24 hours before being placed on a Sephadex column .
Sephadex LH-20 (25-100 μm particle diameter) from Sigma/Aldrich Chemical Co. (St. Louis, MO, USA), was used to prepare 1.3 cm × 25 cm columns, as described by Lund et al . Sephadex (0.25 grams) was mixed with 1.5 ml of TEGMD buffer. The serological pipette was plugged with a glass bead and 1 ml of Sephadex was loaded onto the column with TEGMD buffer and equilibrated. Flow rates under gravity were approximately 0.5 ml/min and 0.5 ml fractions were collected in plastic scintillation vials.
Steroid compounds tested in Equol Binding Assays
5α-PREGNAN-3β, 21β, 21-TRIOL-20-ONE
Experiment 2. In vitroblockade of 5α-DHT stimulated Prostate Specific Antigen (PSA) levels in LNCap cells by equol: Human Prostate Cancer Cell Cultures
Human prostate cancer cell line was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA; ATCC # CRL-1740, LNCap-FGC) and cultured in a 37° C, humidified incubator with 5% CO2, in RPMI Medium (Sigma/Aldrich Cat. # R-8758) with 5% fetal bovine serum (Hyclone/Thermo Sci., Waltham, MA, USA, Cat. # SH30088.03, Lot number APC20780) and 5 mM Hepes (Sigma/Aldrich Cat. H-0887), 1× antibiotic/antimycotic (Sigma/Aldrich Cat. # A5955). Cells were expanded in T-150 flasks for three passages until cryopreservation and storage in liquid nitrogen in RPMI medium with 10% FBS and 10% DMSO as cryopreservative. A cryovial was then thawed in a 37° C water bath, expanded again one or two passages, and then plated at 10,000 cells per 96 well in 0.2 ml medium in 96 well plates in RPMI 5% FBS medium. After approximately 48 hours, the medium was changed to phenol red-free DMEM/F12 (Gibco/Invitrogen, Carlsbad, CA, USA, Cat. #21041-025) with 2% FBS and 1× antibiotic/antimycotic and test materials (5α-DHT, equol or 5α-DHT plus equol; n = 6 per treatment) and DMSO/vehicle controls were added at the appropriate concentration (either, 0.1 nM, 1.0 nM or 10 nM of 5α-DHT or 100 nM, 10 nM or 1.0 nM of equol) from 10× stocks for specific experiments. [The change to phenol red-free medium was to eliminate any potential estrogenic influence]. Cells were cultured for approximately 48 hours in the presence of the test materials and controls prior to removal of medium supernatants for prostate specific antigen (PSA) ELISA quantification.
Cytotoxicity was determined by spectrophotometric detection of reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma/Aldrich Cat # 5655) at 550 nm using a Molecular Devices Vmax 96 well plate reader and SoftMax software (n = 6 per treatments or controls). Metabolic activity can be used as a measure of cytotoxicity, in that the intensity of the reduced form of MTT by live cells is directly proportional to cellular viability, and inversely proportional to cytotoxicity.
Cellular DNA Assay
Cellular DNA was determined using a CyQuant Cell Proliferation Kit (Molecular Probes/Invitrogen, Carlsbad, CA, USA, Cat # C7026) according to the manufacture's instructions to quantify cellular proliferation.
Tissue culture supernatants were diluted 10-fold in PBS and stored at -20°C, then thawed at room temperature prior to assay. A commercial ELISA kit for free PSA (Bio-Quant, San Diego, CA, USA, Cat. # BQ 067T) was utilized according to the manufacturer's instructions, and data was determined using a Molecular Devices Vmax 96 well plate reader and SoftMax software.
Experiment 3. In vivoblockade of the stimulatory effects of 5α-DHT by equol
This is an example of equol preventing the stimulatory effects of 5α-DHT in vivo. Rats were injected with 1 mg of non-racemic equol (52% S-isomer, 48% R-isomer) for 25 consecutive days, and serum 5α-DHT levels and prostate weights were measured. Adult (50 day-old) males (n = 16), purchased from Charles River Laboratories (CRL; Wilmington, MA, USA), were caged individually and housed in the Brigham Young University Vivarium and maintained on an 11-dark, 13-hour light schedule (lights on 0600-1900). Before purchase, the male animals were fed a diet containing approximately 200 ppm of isoflavones at the supplier (CRL). At 50 days of age, upon arrival, the male rats were placed on a diet containing approximately 10 ppm of isoflavones; referred to hereafter as the low isoflavone diet (Zeigler Bros., Gardnes, PA, USA; Phytoestrogen Reduced Rodent Diet II). All animals remained on the low isoflavone diet until 216 days of age to exclude the influence of dietary isoflavones on the measured parameters. At 150 days of age the rats were divided into two groups (control or equol treatments) that were matched by age and body weight. Starting at 190 days of age the male rats received a daily subcutaneous 0.1cc injection at the nape of the neck of vehicle (n = 8) (dimethyl sulfoxide; DMSO) or equol (n = 8) at a dose of approximately 1.0 mg/kg for 25 consecutive days.
The body weights for each group were recorded weekly starting at 150 days of age before the treatments were initiated, with weights obtained immediately before and after the treatments were administered (there were no significant differences in body weights between the control and equol groups at the start of this experiment). At 216 days of age the animals were weighed [grams (g) ± 0.1 g], then anesthestized with Ketamine/acepromazine and blood was collected from the heart. Next the ventral prostate organ was dissected and weighed [milligrams (mg) ± 0.001 mg]. The collected blood samples were centrifuged and serum was stored at -20˚ C until assayed. All collection procedures were performed blind to the treatments. This animal protocol was approved by the Institutional Animal Care and Use Committee at Brigham Young University.
Serum testosterone, 5α-DHT, and 17β-estradiol were quantified by radioimmunoassay (RIA) kits purchased from Diagnostic System Laboratories (Webster, TX, USA). Luteininzing hormone (LH) was quantified by an assay utilizing standards from the National Institutes of Health (NIH) USA pituitary hormone program. The samples were run in duplicate for each RIA, with internal control samples. In all RIAs, the control values were within normal ranges. The intra-assay coefficients of variance for the assays were: testosterone = 6.0%; for 5α-dihydrotestosterone = 8%, 17β-estradiol = 5% and LH = 9%.
Reagents and supplies
All reagents and supplies not specifically mentioned (e.g., EDTA, DMSO, glycerol, dithrothreitol, pipettes, 1.3 × 25 cm columns, etc.) were purchased from Sigma/Aldrich Chem. Co., St. Louis, MO, USA.
Where appropriate, data were analyzed by analysis of variance statistics (ANOVA) followed by Newman-Keuls post hoc tests. Significance was p < 0.05. Curve fitting, scientific graphing, and analysis were completed using GraphPad Software (GraphPad Prism 3.0, San Diego, CA, USA).
In preliminary studies, Table 1 shows 33 different steroid compounds that were tested in binding assays (outlined above) to determine equol's affinity for binding to each. While equol has modest affinity for some 5α-reduced steroids, equol displayed the highest affinity for 5α-dihydrotestosterone (5α-DHT) and had no affinity for 5β-dihydrotestosterone (5β-DHT) or some of the most common natural sex steroids, such as: estradiol, estrone, estriol, progesterone or testosterone. [Notably: as tested in these assays, the isoflavones: genistein and daidzein do not bind 5α-DHT].
Notably, equol at a 70% R-isomer and 30% S-isomer ratio was tested in this experiment. However, racemic equol or equol at a 30% R-isomer and 70% S-isomer ratio when tested in these assays yielded similar results to that displayed in Figure 2. These data suggest that equol, regardless of the isomer ratio mixture can binding selectively 5α-DHT in vitro.
As shown in Figure 3: In the 1, 10 or 100 nM Vehicle (DMSO) treatments PSA levels did not differ from the No Treatment baseline group and controls were not significantly different from each other. Treatment with 0.1, 1 or 10 nM 5α-DHT stimulated PSA secretion to maximal levels (ranging from 228.3 to 260 ng/ml). PSA levels from cells treated with 1, 10 or 100 nM equol were well below DHT-treated values. In fact, the 100 nM equol treatment alone yielded a PSA level at 60 ng/ml that was significantly decreased below all vehicle-control values (at approximately 87 ng/ml). The equol treatment at 10 nM slightly but significantly increased PSA levels (to 116.7 ng/ml) compared to all the DMSO-vehicle-control values around 87 ng/ml, but not to the no-treatment group at 100 ng/ml). [This may be due to the number of replicates (6) in this experiment where standard error of the mean (sem) values were relatively small]. However, combinations of 10 nM 5α-DHT plus 100 nM equol, as well as 1 nM 5α-DHT plus 10 nM equol, and 0.1 nM 5α-DHT plus 1 nM equol treatments abrogated the increase in PSA secretion, compared to the 5α-DHT treatments alone, at the respective androgen concentrations (see Figure 3). Taken together with the in vitro binding demonstrated in Figure 2, these data from experiment 2, suggest that equol binds the 5α-DHT molecule and biologically inactivates it in human cancer prostate cells.
Serum 5α-DHT levels, prostate weights and reproductive parameters in Equol-treated male rats
Prostate Weight (PW), mg
535 ± 23
429 ± 30**
PW/100 g Body Weight
76 ± 4
61 ± 5**
Luteinizing Hormone (LH), ng/ml
1.6 ± 0.2
1.3 ± 0.1
Serum Testosterone, ng/ml
2.1 ± 0.4
2.3 ± 0.5
Serum 5α-DHT, pg/ml
100 ± 18
52 ± 5*
Serum 17β-Estradiol, pg/ml
3.4 ± 0.6
4.8 ± 0.7
When LH and testosterone were quantified between the treatment groups there were no significant differences in these hormone levels (Table 2). Since LH is the gonadotrophin regulating testosterone synthesis from Leydig cells in the testes, this is not a surprising result. Finally, when 17β-estradiol levels were determined there were no significant differences between the treatment groups. All hormone levels were within normal ranges of that expected for this strain, age and sex of rat. Notably, Long-Evans rats do not develop spontaneously prostate cancer but enlargement of prostatic tissue is observed with aging.
This in vivo study demonstrates that equol can contact and biologically inactive the 5α-DHT molecule as shown by the significant decrease in 5α-DHT levels in blood and significantly reduced prostate weights of equol-treated male rats. Finally, the in vitro and in vivo studies reported above demonstrates that equol may be effective in treating prostate disorders or other androgen-mediated diseases that are regulated by the hormone 5α-DHT.
The binding assays in the present study confirm and extend previous findings from our laboratories . However, this protein and cell-free assay represents a system that is absent of steroid-binding proteins and other biologicals that may influence the in vivo interaction between equol and 5α-DHT. Moreover, after testing over 30 steroid hormones with various androgenic, estrogenic or progestin chemical configurations, equol (racemic or non-racemic mixtures) specifically binds 5α-DHT with apparent equal affinity since the bind curves obtained were very similar using different equol isomer ratios. This suggests several important issues. First, all humans have plasma levels of S-equol circulating in their bloodstream that may represent a natural modulation of the potent androgen, 5α-DHT, regardless of whether or not they are "equol producers" [4, 5]. Second, this circulating equol is derived from consumption of soy- or high content isoflavone-containing food products or low abundant isoflavone foods such as corn, wheat, cow's milk, etc., as a primary consumer. Finally, because grazing or other animals that produce high levels of S-equol naturally that are then consumed, such as meat products, by humans represents a source of equol as a secondary consumer. Evidence to support this last notion is shown in humans that consume high meat and fat diets display higher blood isoflavone/equol levels .
This denotes an interesting 'natural' modulation of 5α-DHT in humans, since all individuals consume food products that contain the precursor daidzein molecule or equol itself (i.e., soy, corn, wheat, cow's milk or animal meat products, etc.) [4, 6, 11–13]. In fact, whether or not swine were fed a soy-containing or protein-supplemented diet the serum equol levels between the dietary treatment groups were essential the same . Since the apparent half-maximal binding of equol to 5α-DHT occurred at 4.8 nM (or approximately 1.2 ng/ml) the very low level of circulating equol may alter androgen hormone action in a manner previous unknown in normal physiological conditions. Also, the apparent half-life of R- or S-equol in humans is 5 to 6 hours , suggesting a viable time interval and mechanism of action. While the present data were derived from in vitro binding studies that does not represent the complex physiological environment this viewpoint will be covered below in reference to prostate health.
Further evidence presented in this study for equol binding 5α-DHT is presented in the experiment where equol blocked the stimulatory androgen action of 5α-DHT in increasing PSA levels in human cancer cell cultures. Although, this may be the major mechanism of action, it is known that equol (particularly S-equol) has an affinity for ERβ . Previous laboratories, including ours, have shown that isoflavones binding to ERβ in the prostate can down regulate the androgen receptor (AR) and thus decrease androgen hormone action . Since PSA is an androgen-regulated gene this idea may have merit [16, 17]. In addition to equol directly binding 5α-DHT, it may alter AR expression and hence significantly decreased PSA levels in the present study. Support for this view comes from the significant decrease in PSA levels with equol treatment alone (@ 100 nM) in experiment 2 that displayed values even below baseline (vehicle) PSA concentrations. However, it could be argued that the fetal bovine serum provided a background androgenic stimulation and this may also account for this observation. In any event, the results of this experiment demonstrate the positive influence equol has on PSA levels and implicates potential applications of equol for prostate disorders. Finally, phase II clinical trial data examining dietary intervention with isoflavone supplementation in men with recurrent prostate cancer display a decline in PSA levels that support our present in vitro data .
As mentioned earlier, equol has the ability to bind ERβ. This may explain how isoflavones accumulate in prostate tissue and prostatic fluid after oral supplementation [19–21] and it has been shown that binding ERβ in the prostate decreases inflammation and carcinogenesis . Furthermore, this not only applies to prostate health but to other tissue-specific sites in the body where ERβ is present. For example, there is an abundance of ERβ in the brain, specifically in the frontal cortex and raphe nuclei as well as other brain regions that may be involved in mechanisms of anxiety and depression [23–25]. Preliminary data from our laboratory suggest that equol treatment in aged-rats after natural ovarian failure is effective as an anti-depressive agent at low concentrations that significantly increase serotonin levels. Additionally, the skin represents the largest organ of the body where ERβ is abundant in the epidermal and dermal regions, especially the keratinocytes and fibroblasts, respectively which may be tissue sites for beneficial alterations in androgen hormone action . For instance, it is known the androgens decrease, whereas, estrogenic compounds enhance wound healing  that may have effective applications for topical treatments.
The results of the third experiment demonstrated that equol can significantly decrease serum 5α-DHT along with a significant decline in prostate weight without altering, testosterone, 17β-estradiol or LH levels. There is evidence that Asian cultures have lower prostate cancer rates compared to Western cultures [1, 3, 28]. Furthermore, when data is stratified examining individuals that produce equol naturally after soy consumption suggest that lower cancer rates are due to the beneficial influence of this isoflavonoid molecule .
Moreover, while equol has been shown to have positive benefits in human prostate health, there is evidence that R-, rather than the S - enantiomer is responsible for the in vivo chemoprotective properties of equol .
Additionally, from pilot data, serum 5α-DHT levels significantly decreased by 10 to 15% in men (50 to 60 years of age) and 20 to 26% in postmenopausal women (60 to 65 years of age) when an equol oral dose of 3 mg per day was administered (without side effects), suggesting that equol effectively binds and/or alters 5α-DHT levels. This pilot data is supported by a recent study showing isoflavone supplementation that increased the production of serum equol in equol-producers resulted in a decline in serum 5α-DHT levels in men by approximately 18% versus before supplementation values .
In summary, our in vitro studies reveal equol binds specifically 5α-DHT and prevents increases in PSA from LNCap cells, while the in vivo studies demonstrate that equol decreases rat prostate size, decreases serum 5α-DHT levels and androgen hormone action, while not altering other circulating sex steroids or LH levels. Taken together these data suggest a potential use of equol as a therapeutic agent for men's prostate health. For example, benign prostate hyperplasia (BPH or prostate enlargement) that is manifested by nocturia (need to urinate frequently during the sleep-cycle), frequency of urination during the day or night, decreased voided urine volume, sensory urgency to urinate, inability to start and stop a urine stream, etc., where equol may mediate androgen hormone action of 5α-DHT and/or androgen steroid receptor inactivation is influenced by binding ERβ in a positive manner.
Trent D. Lund, PhD, is president of Stoelting Co., Wood Dale, IL 60191, USA. Crystal Blake, PhD, is a research associate in Physiology and Developmental Biology at Brigham Young University (BYU), Provo, Utah 84602, USA. Lihong Bu, PhD, is MRDDRC Imaging Core manager and instructor, Department of Neurobiology, Children's Hospital Boston and Harvard Medical School, Boston, MA 02115, USA. Amy N Hamaker, is an undergraduate student majoring in Neuroscience at Brigham Young University, Provo, Utah 84602, USA. Edwin D. Lephart, is professor of Physiology and Developmental Biology and The Neuroscience Center at BYU, Provo, Utah, 84602, USA.
List of abbreviations used
benign prostate hyperplasia
prostate specific antigen
lymph node metastatic lesion of human prostatic adenocarcinoma
selective androgen modulator
estrogen receptor beta
enzyme-linked immunosorbent assay.
This work was supported, in part, by a grant from the United States Department of Agriculture (USDA 2004-01811 to EDL). We thank Tim Aucoin for his participation in the binding experiments and Frank Zeigler for assistance with the LNCap cell culture experiment.
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