Effects Of Equol Research Articles

Abstract 3050: Equol as a radiosensitizer and therapeutic agent in prostate cancer

Abstract The gastrointestinal microbiome and the metabolites it can produce have been linked to response to various cancer treatments, including in prostate cancer. Equol is a microbial metabolite that is exclusively produced by the conversion of soy isoflavones in the diet by specific intestinal bacteria. Interestingly, equol has previously been shown to exhibit anti-androgen effects on prostate cells and likewise may act as a radiosensitizer. We analyzed the microbial composition of fecal samples collected from men receiving stereotactic ablative radiation (SABR) versus observation to treat oligometastatic prostate cancer as part of the ORIOLE trial and found that patients harboring equol-producing bacteria had a lower risk of disease progression. We therefore aimed to study the effect of this compound on prostate cancer cell lines in vitro. MTT assay was used to assess the effects of equol treatment on cell viability in androgen receptor (AR) positive LNCaP cells and AR null PC3 cells over time. We assessed the ability of equol to reduce AR protein levels and/or subcellular localization in LNCaP cells using immunohistochemistry. Finally, western blotting was used to assess the effects of equol treatment on prostate specific antigen (PSA) levels to assess effects on the AR signaling pathway. Cell viability was significantly decreased over time in equol treated cells, but only in prostate cancer cell lines that were AR positive. The effects of equol on LNCaP viability was abrogated in an AR knockdown LNCaP cell line. Interestingly, AR protein levels were found to be markedly decreased upon equol treatment of LNCaP cells. We likewise observed a decrease in PSA protein levels in AR positive cells after equol treatment. Our results support the notion that equol can inhibit prostate cancer growth by inhibition of the androgen receptor. We are currently investigating whether combining equol exposure with radiation treatment produces a synergistic effect on DNA damage and reduction of prostate cancer cell viability and survival in vitro. Our future studies will investigate the effects of equol and equol-producing bacteria on tumor growth in in vivo models. Citation Format: Carli B. Jones, Jonathan B. Coulter, Janielle P. Maynard, Luke A. Mummert, Ryan M. Phillips, Phuoc T. Tran, Karen S. Sfanos. Equol as a radiosensitizer and therapeutic agent in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3050.

Soy-Derived Equol Induces Antioxidant Activity in Zebrafish in an Nrf2-Independent Manner.

Antioxidant effects of soy-derived isoflavones are predicted to be mediated by the Keap1-Nrf2 pathway. Recently, we constructed an assay system to evaluate the antioxidant effects of dietary phytochemicals in zebrafish and revealed a relationship between these effects and the Keap1-Nrf2 pathway. In this study, we used this system to examine the antioxidant effects of seven isoflavones. Among those seven, equol showed strong antioxidant effects when arsenite was used as an oxidative stressor. The antioxidant effect of equol was also shown in Nrf2-mutant zebrafish nfe2l2afh318, suggesting that this effect was not mediated by the Keap1-Nrf2 pathway. To elucidate this unidentified mechanism, the gene expression profiles of equol-treated larvae were analyzed using RNA-seq and qRT-PCR, while no noticeable changes were detected in the expression of genes related to antioxidant effects, except weak induction of Nrf2 target genes. Because nfe2l2afh318 is an amino acid-substitution mutant (Arg485Lue), we considered that the antioxidant effect of equol in this mutant might be due to residual Nrf2 activity. To examine this possibility, we generated an Nrf2-knockout zebrafish nfe2l2ait321 using CRISPR-Cas9 and analyzed the antioxidant effect of equol. As a result, equol showed strong antioxidant effects even in Nrf2-knockout larvae, suggesting that equol indeed upregulates antioxidant activity in zebrafish in an Nrf2-independent manner.

Urinary equol levels are positively associated with urinary estradiol excretion in women

Isoflavones found in soy products are a promising class of nutrients that may have a positive effect on human health. In particular, the phytoestrogen metabolite equol is associated with a reduced risk of developing female hormone-related diseases. However, the effect of equol on estrogen remains unclear. Equol can modify blood and urinary estradiol (E2) levels. The aim of this cross-sectional study was to examine the associations between urinary estrogen levels, equol levels, and equol production status in Japanese women. We analyzed urine samples from 520 women by gas chromatography-mass spectrometry. Urinary E2 and 4-hydroxylated E2 levels were higher in equol producers (EQP) than in non-EQPs (P < 0.0001 and P=0.00112, respectively). After adjusting for age and tobacco use by analysis of covariance, the association remained significant (β = 0.299, P < 0.0001). Analysis of covariance demonstrated that equol levels in urine were also positively associated with urinary E2 (β = 0.597, P < 0.0001). The log equol concentration showed a significant, but moderate, negative association with the serum E2 concentration (β = − 0.0225, P = 0.0462). Our findings suggest that equol may promote urinary E2 excretion and modify blood E2 levels in women.

Association of equol with obesity in postmenopausal women.

Equol, which is an active metabolite of isoflavone, has a beneficial impact on metabolic diseases such as dyslipidemia and hyperglycemia. However, the effect of equol on obesity remains uncertain. This study was performed to determine the association between equol and obesity in postmenopausal women. We evaluated 386 women in their 50s-60s who underwent health check-ups from February 2018 to January 2019 at Watari Hospital Health Center in Fukushima, Japan. Overweight and visceral obesity were defined as a body mass index ≥25 kg/m2 and waist circumference ≥ 90 cm, respectively. Participants were asked to complete a questionnaire about their ordinary lifestyle. Participants were defined as equol producers when urinary equol level was 1.0 μM or more. The proportion of individuals with obesity (overweight and visceral obesity) and lifestyle factors were compared between equol producers and nonproducers. In addition, the association between equol and obesity was examined using logistic regression analysis with adjustment for lifestyle factors. Of the 386 participants, 106 (27.5%) women were equol producers. The proportions of women who were overweight (13.2% vs 25.7%) and had visceral obesity (6.6% vs 20.7%) were significantly lower in the equol-producing group than in the nonproducing group. Multivariable logistic regression analysis showed that equol production was significantly associated with overweight (odds ratio =0.47, 95% confidence interval: 0.25-0.88) and visceral obesity (odds ratio =0.30, 95% confidence interval: 0.13-0.68). Equol is significantly associated with obesity in postmenopausal women.

(±)-Equol does not interact with genistein on estrogen-dependent breast tumor growth

Equol (EQ) is a prominent microbial metabolite of the soy isoflavone, daidzein, with estrogen-like properties. The major soy isoflavone, genistein (GEN), stimulated growth of estrogen-dependent breast cancer (EDBC) cells in vitro and tumor growth in vivo but EQ did not. To understand possible interactions of EQ and GEN on EDBC, EQ was used with GEN in combination in vitro and in vivo. Effects of EQ, GEN and EQ + GEN were evaluated using MCF-7 and T47D EDBC. Ovariectomized athymic mice were used as a model for in vivo tumor growth. Dietary EQ had no effect on MCF-7 tumor growth and the absence of effect was confirmed using a T47D EDBC in vivo model. EQ alone or in combination with GEN increased EDBC cell proliferation in vitro. EQ alone neither stimulated EDBC tumor growth in vivo at various doses nor suppressed tumor growth induced by dietary GEN. In summary, EQ has similar estrogenic effect as GEN in vitro but does not interact with GEN on EDBC tumor growth. Based on the evidence presented here, dietary EQ is unlikely to have estrogenic effects in vivo.

Equol: A Bacterial Metabolite from The Daidzein Isoflavone and Its Presumed Beneficial Health Effects.

Epidemiological data suggest that regular intake of isoflavones from soy reduces the incidence of estrogen-dependent and aging-associated disorders, such as menopause symptoms in women, osteoporosis, cardiovascular diseases and cancer. Equol, produced from daidzein, is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. Consequently, equol has been endorsed as having many beneficial effects on human health. The conversion of daidzein into equol takes place in the intestine via the action of reductase enzymes belonging to incompletely characterized members of the gut microbiota. While all animal species analyzed so far produce equol, only between one third and one half of human subjects (depending on the community) are able to do so, ostensibly those that harbor equol-producing microbes. Conceivably, these subjects might be the only ones who can fully benefit from soy or isoflavone consumption. This review summarizes current knowledge on the microorganisms involved in, the genetic background to, and the biochemical pathways of, equol biosynthesis. It also outlines the results of recent clinical trials and meta-analyses on the effects of equol on different areas of human health and discusses briefly its presumptive mode of action.

Abstract 4390: Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation

Abstract We previously reported that dietary daidzein, a soy isoflavone, increased breast cancer progression in a nude mouse model of breast cancer metastasis. Further studies indicated that equol, a metabolite of the soy isoflavone daidzein is responsible for the cancer promoting effects of soy isoflavones in breast cancer cells in vitro. We reported that equol increases the expression of the eukaryotic protein synthesis initiation factor eIF4G1, and the transcription factor c-Myc. Increased eIF4G in response to equol resulted in the non-canonical protein synthesis of pro-cancer molecules such as Cyclin D, Bcl-XL, p120, matrix metalloproteinases, etc., as confirmed by ribosome profiling and eIF4G1 knockdown studies. This study tested the hypothesis that equol promoted breast cancer progression via eIF4G1 upregulation. Immunocompromised nude mice were used to establish mammary fatpad tumors from human metastatic breast cancer cells expressing eIF4G1 knockdowns via a Tetracycline-inducible promoter. Oral gavage of 50 mg/kg BW Doxycycline 3X a week for 4 weeks was enough to knockdown eIF4G1 expression by ~80%. Therefore, one week following tumor establishment, the mice were treated with vehicle (90% corn oil, 10% ethanol), equol, Doxycycline or, equol and Doxycycline. Data show that as expected, equol treatment increased tumor growth relative to vehicle treatment. Moreover, eIF4G1 knockdown abolished the effect of equol on tumor growth. Interestingly, the effect of eIF4G1 knockdown on mammary tumor growth was not evident until 55 days of doxycycline treatment. Therefore, we tested the cell and tumor lysates for potential expression of the eIF4G1 isoform Dap5 when eIF4G1 is knocked out. Dap5 protein was expressed in eIF4G1 knockdown cells and was increased by equol treatment, indicating that Dap5 may compensate for eIF4G1 knockdown by inducing alternative cap-mediated protein synthesis. In conclusion, the cancer promoting effect of equol is abolished in breast tumors where eIF4G1 expression was stably knocked down, thus, validating our hypothesis that equol increases breast cancer progression via upregulation of eIF4G1 dependent protein synthesis initiation. Citation Format: Ailed M. Cruz-Collazo, Columba De la Parra, Robert Schneider, Suranganie Dharmawardhane. Dietary soy isoflavone equol promotes breast cancer progression via regulation of protein synthesis initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4390.

Effect of Equol on Vasocontractions in Rat Carotid Arteries Treated with High Insulin.

Previous research has indicated that high insulin affects vascular function. Equol is an active metabolite of daidzein, an isoflavone produced from soy by intestinal microbial flora, with beneficial effects on the vascular system. This study investigated whether equol was beneficial for vascular function under high insulin conditions. Using organ culture techniques, rat carotid arteries were treated for 23 ± 1 h with a vehicle, high insulin (100 nM), or equol (100 µM) plus high insulin (100 nM). Vascular isometric forces were measured by the organ bath technique. In each endothelium-intact ring, the contractions induced by high-K+, noradrenaline, or by serotonin (5-HT) were similar for the vehicle, insulin, and equol + insulin treatments. Contractions induced by a selective 5-HT2A receptor agonist (TCB2) increased with insulin treatment (vs. vehicle), but less so with equol + insulin. Under basal conditions, a selective 5-HT2B receptor agonist (BW723C86) did not induce contraction; following precontraction by a thromboxane analog, it induced contraction but not relaxation. These responses were similar across the three treatments. Acetylcholine-induced relaxations were also similar for the three treatments. In the endothelium-denuded preparations, 5-HT-induced contraction was augmented with insulin treatment (vs. vehicle) but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by iberiotoxin, a large-conductance calcium-activated K+ channel (BKCa) inhibitor. These results suggest that equol exerts a preventive effect on the enhancement of 5-HT-induced contraction by high insulin (possibly mediated by the 5-HT2A receptor), and that these effects may be attributed to the activation of BKCa channels in vascular smooth muscle.

Abstract P2-05-06: Effects of a soy isoflavone in breast cancer treatment

Abstract Background : Isoflavones are phytoestrogens that may be effective in preventing osteoporosis, reducing cardiovascular events, and improving menopausal symptoms such as hot flash due to their estrogen-like actions. Isoflavones are currently used as a supplement for improving symptoms of menopause. Epidemiological studies have shown that ingestion of soy products may also reduce the risk of breast cancer, and antitumor effects on hormone receptor (HR)-positive breast cancer cells have been shown in vitro for equol, an isoflavone metabolite. Equol binds to estrogen receptors (ERα, β) and is thought to antagonize ERα-estradiol binding in the presence of estradiol. Since equol shows an antiestrogenic effect, similar to that of the hormonal agent tamoxifen, it is thought that equol acts as a selective estrogen receptor modulator (SERM), but the mechanism is still unclear. In this study, we evaluated the antitumor effects of equol alone and in combination with existing therapeutic agents in HR-positive breast cancer cells, and examined the mechanism of these effects. Methods : The antitumor effects of equol alone and in combination with hormone drugs (4-hydroxytamoxifen (Tam), fulvestrant (Ful)) and chemotherapeutic agents (paclitaxel (Ptx), doxorubicin (Dox)) were examined using a MTS assay. Combination indexes (CIs) were determined in HR-positive MCF-7, T-47D, and ZR-75-1 cell lines. The mechanisms of the drug effects were evaluated by Western blot for assessment of changes in chemoresistance factors at the protein level. Results : In MCF-7, T-47D and ZR-75-1 cells, there was a concentration-dependent antitumor effect of equol and of the other hormonal agents. CIs showed an antagonistic effect of equol with Tam and a synergistic effect with Ful in all cell lines. Equol also had an antagonistic effect with the two chemotherapeutic agents, with the strongest antagonism occurring at a low dose of equol. Western blot showed that ER, PgR, Cyclin D1 and Bcl-2 were upregulated via ER at a low concentration of equol, similarly to the effect of 17-β-estradiol (E2), and ER, PgR, Cyclin D1 and Bcl-2 were downregulated at a high concentration, similarly to the effect of Tam. In addition, ER, PgR and cyclin D1 were downregulated with the combination of equol and Tam, while the expression of Bcl-2, a chemoresistance factor, increased. Conclusion : These results suggest that equol has a concentration-dependent antitumor effect in HR-positive breast cancer cell lines and may antagonize the effect of existing therapeutic agents (hormone drugs and chemotherapeutic agents). In particular, it was considered that in combination of equol and Tam, the apoptosis inducing action of each drug was attenuated and antagonistic effect was shown. Citation Format: Hatono M, Ikeda H, Taira N, Suzuki Y, Kajiwara Y, Kawata K, Takahashi Y, Tsukioki T, Kochi M, Iwamoto T, Shien T, Doihara H. Effects of a soy isoflavone in breast cancer treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-05-06.

Abstract 841: Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS)

Abstract Soy isoflavones such as daidzein and its bioactive metabolite equol converted from daidzein by human intestinal bacterial flora, have been proposed to reduce the risk of breast cancer incidence in Asian women probably due to higher number of the inhabitants capable of producing equol in these countries. Although several in vitro studies shows the anti-cancer activity of equol against human breast cancer cells, there have been few reports that have directly showed the preventive role of equol in the experimental animal model of mammary carcinoma. We investigated here the effects of equol on the development of breast cancer using the EMS chemically induced rat model of hormone-dependent mammary carcinoma. Female WKA rats (n = 30) were equally divided into 2 groups, subsequently given EMS orally for 12 weeks and fed the CE2 diets with or without 50 mg equol/g diet throughout the experiments. All EMS-treated rats fed either diet developed ER- and/or PgR- positive hormone-dependent mammary carcinoma by 24 weeks. Compared with the equol-free diet, the diet supplemented with equol could not significantly delay the development of mammary carcinoma, showing any preventive activity on the development of EMS-induced hormone-dependent mammary carcinoma. The body weight, total EMS uptakes, and urine estradiol concentrations were not significantly different between these 2 groups. These data indicate that high dose administration of equol alone does not exert clear preventive effects on hormone-dependent EMS-induced mammary carcinogenesis, and may require other soy isoflavone components such as genistein that has previously been shown to exhibit a great synergy when combined with equol. The in vivo combination studies using genistein and equol are underway. Citation Format: MISAKI ONO, Takako Higuchi, Mikako Takeshima, Shuji Nakano. Effect of equol on hormone-dependent rat mammary carcinoma induced by ethyl methanesulphonate (EMS). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 841.

The impact of equol-producing status in modifying the effect of soya isoflavones on risk factors for CHD: a systematic review of randomised controlled trials.

Recent studies suggest that the ability to produce equol, a metabolite of the soya isoflavone daidzein, is beneficial to coronary health. Equol, generated by bacterial action on isoflavones in the human gut, is biologically more potent than dietary sources of isoflavones. Not all humans are equol producers. We investigated whether equol-producing status is favourably associated with risk factors for CHD following an intervention by dietary soya isoflavones. We systematically reviewed randomised controlled trials (RCT) that evaluated the effect of soya isoflavones on risk factors for CHD and that reported equol-producing status. We searched PubMed, EMBASE, Ovid Medline and the Cochrane Central Register for Controlled Trials published up to April 2015 and hand-searched bibliographies to identify the RCT. Characteristics of participants and outcomes measurements were extracted and qualitatively analysed. From a total of 1671 studies, we identified forty-two articles that satisfied our search criteria. The effects of equol on risk factors for CHD were mainly based on secondary analyses in these studies, thus with inadequate statistical power. Although fourteen out of the forty-two studies found that equol production after a soya isoflavone intervention significantly improved a range of risk factors including cholesterol and other lipids, inflammation and blood pressure variables, these results need further verification by sufficiently powered studies. The other twenty-eight studies primarily reported null results. RCT of equol, which has recently become available as a dietary supplement, on CHD and its risk factors are awaited.

Effects of equol on deoxycorticosterone acetate salt-induced hypertension and associated vascular dementia in rats.

Oxidative stress is the major cause of neuronal cell degeneration observed in neurodegenerative diseases including vascular dementia (VaD), and hypertension has been found to increase the probability of VaD. Here, we investigated the effects of equol in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats (DHRs) and the associated VaD. The systolic blood pressure of rats treated with low- (10 mg per kg body weight) and high-dose (20 mg per kg body weight) equol for 4 weeks was lower than that of the control group by 12.18 and 17.48% in a dose-dependent manner, respectively (p < 0.05), which was regulated by inhibiting angiotensin-converting enzyme (ACE) activity and increasing the nitric oxide (NO) production. Equol-treated DHRs showed a significant decrease in both the swimming distance and time required to reach the escape platform (78.20 to 82.56%, p < 0.05). In addition, the probe trial session and working memory test indicated that equol improved the long- and short-term memory of the rats. Moreover, the brain antioxidant activity was increased by elevating the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels, and the malondialdehyde (MDA) content and acetylcholinesterase (AChE) activity were decreased, indicating that equol suppressed oxidative stress. In conclusion, we demonstrated that equol exhibited comparable blood pressure (BP)-lowering and VaD-improving effects with the clinically used drug, lisinopril in DHRs while there was a positive correlation between the doses. Therefore, this bioactive compound may be useful for developing functional foods, thereby extending the application of equol-containing crops.

Effect of soy isoflavone supplementation on endothelial dysfunction and oxidative stress in equol-producing postmenopausal women.

Dietary isoflavones are considered to be cardioprotective because of their structural similarity to estrogen. Equol is a metabolite of daidzein that is more potent than daidzein itself. The effect of equol on endothelial function and oxidative stress remains unclear. The objective of this study was to determine the effect of 100 mg/day soy isoflavone supplementation on the following markers: vascular cell adhesion molecule-1 (VCAM-1) and nitric oxide (NO) as markers of vascular endothelial function, and malonyldialdehyde (MDA) as oxidative stress marker in equol-producing postmenopausal women. A stratified randomized double blind controlled trial was conducted among 190 postmenopausal Indonesian women aged 47 to 60 years. They were stratified into equol producers and equol nonproducers. The random allocation of intervention was carried out separately in each stratum. The intervention group received tablets consisting of 100 mg soy isoflavones and calcium carbonate 500 mg, and the control group received 500 mg calcium carbonate. The concentrations of VCAM-1, NO and MDA were measured at baseline and post-supplementation at 6 months. After 6 months of supplementation the MDA concentrations were significantly lower in the soy-isoflavone equol producers compared with equol nonproducers (p=0.021). Similar results were also found for VCAM-1 and NO concentrations, but these were statistically not significant (p = 0.413 and p= 0.724, respectively). This study demonstrates that isoflavone supplementation in postmenopausal women with equol-producer status had a more beneficial effect by decreasing the MDA concentration, but did not improve VCAM-1 and NO concentrations.

Equol Production and Increased Leukocyte Mitochondrial DNA in Postmenopausal Women

Background: Equol, a metabolite of diadzein, is produced by some intestinal bacteria. Equol acts as an estrogen receptor agonist and has been reported to have several beneficial health effects. Leukocytes play an important role in the pathogenesis of autoimmune, metabolic, and cardiovascular diseases. Decreased leukocyte mitochondrial DNA (mtDNA) content, as an index of mitochondrial function, is associated with metabolic syndrome, bone mineral density, and aging. The possible association between equol production and leukocyte mitochondrial function has not been studied to date. Therefore, we investigated whether equol production is associated with leukocyte mtDNA copy number in postmenopausal women. Methods: This observational cross-sectional study included 71 postmenopausal women. They completed a lifestyle questionnaire and medical history. In addition, a dietary assessment using a 24-hour recall method and food frequency questionnaire, anthropometric evaluation, and blood sampling were conducted. Serum equol concentration was measured in the fasting state. Leukocyte mtDNA copy number was measured by real-time polymerase chain reaction. Results: Among older females, 33.8% were equol producers. The leukocyte mtDNA copy number was lower in non-equol producers versus equol producers. Furthermore, the leukocyte mtDNA copy number was positively associated with the serum equol concentration (r=0.42, P<0.01). Stepwise multiple regression analysis showed that equol production (β=47.864, P<0.01) was an independent factor associated with mtDNA copy number. Conclusions: Equol production was associated with elevated mtDNA content in the peripheral blood of postmenopausal women. This finding suggests that the beneficial health effects of equol in postmenopausal women may be related to increased mitochondrial function.

Therapeutic effect of equol on local skin wounds caused by mustard gas in rabbits and related mechanism

Therapeutic effect of equol on local skin wounds caused by mustard gas in rabbits and related mechanism

Bi-Phasic Effect of Equol on Adipocyte Differentiation of MC3T3-L1 Cells

We investigated the effects of equol on adipogenesis by measuring lipid accumulation and analyzing the change in adipocyte-related gene expression in MC3T3-L1 cells. Treatment with 10 µM equol tended to increase adipocyte-related gene expression, whereas 100 µM equol reduced lipid accumulation and suppressed the expression of these genes and proteins. Our results suggest that equol regulated adipogenesis in a bi-phasic fashion.

Abstract 577: Equol enhances tamoxifen's antitumor effect by induction of caspase-mediated apoptosis in MCF-7 breast cancer cells

Abstract Soy phytoestrogens, such as daidzein and its metabolite equol, have been proposed to be responsible for the low breast cancer rate in Asian women. However, since these compounds possess both estrogenic and anti-estrogenic properties, it remains unknown whether they provide protection against tumor development and progression. Since the majority of estrogen receptor positive (ER+) breast cancer patients are treated with the selective estrogen receptor modulator tamoxifen, the basic objective of this project is to determine whether equol enhances or inhibits tamoxifen's anti-tumor effect and to identify the molecular mechanisms involved. For this purpose, we examined the individual and combined effects of equol and tamoxifen on the ER+ breast cancer cell line MCF-7 (ERα+/−Rα+). We found that equol at concentrations higher than 10 μM significantly reduced the viability of MCF-7 cells. Furthermore, the combination of equol (100 μM) and 4-hydroxytamoxifen (4-OHT; 10 μM) reduced MCF-7 cell viability and induced apoptosis more effectively than each compound alone. Subsequent treatment of MCF-7 cells with the pan-caspase inhibitor Z-VAD-FMK and the calpain/cathepsin inhibitor ALLN demonstrated that equol and 4-OHT induce apoptosis via both caspase-depended and -independed mechanisms. In order to further investigate the apoptotic pathways employed by equol, 4-OHT and their combination, we evaluated the expression of key proteins involved in apoptosis such as PARP, α-fodrin, AIF, bcl-2, bax, p21 and p53 using Western blot analysis. The combination of 4-OHT and equol induced PARP and α-fodrin cleavage which was inhibited by Z-VAD-FMK indicating that their combination induces caspase-depended apoptosis. This was accompanied by reduced bcl-2:bax ratio confirming a pro-apoptotic effect. The combination of equol and 4-OHT induced caspase-7 cleavage indicating a significant role for caspase-7 in equol- and tamoxifen-induced apoptosis. Finally, upon determination of the effect of equol and tamoxifen on estrogen receptor expression in MCF-7 cells, it was found that equol reduces the ERα:ERα ratio, while 4-OHT increases it. When cells were treated with both compounds we observed that the effect of equol prevailed, providing an additional explanation for the beneficial combined effects of equol and tamoxifen. In conclusion, the effects of equol in potentiating tamoxifen's anti-tumor effect may be explained by the induction of the caspase-mediated apoptotic pathway and the reduction of the ERα:ERα ratio. These results can find applications in clinical or pre-clinical cancer chemoprevention studies against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 577. doi:1538-7445.AM2012-577

Abstract 1988: Increased expression of eukaryotic protein synthesis initiation factor eIF4G by the daidzein metabolite equol may contribute to breast cancer malignancy

Abstract The role of dietary soy in cancer has been the subject of intense investigation and is thought to be cancer preventive. However, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein and combined soy isoflavones (genistein, daidzein, and glycitein) promoted breast cancer progression in a nude mouse model by increasing both primary mammary tumor growth and metastasis. Dietary daidzein significantly upregulated cancer promoting molecules including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E. Herein, using tumors from mice treated orally with daidzein, we show that increased eIF expression is associated with expression of mRNAs with long structured 5′ untranslated regions and internal ribosomal entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. We then tested the hypothesis that daidzein upregulates protein synthesis initiation in breast cancer, but found that daidzein treatment did not affect the levels of eIFs in the same MDA-MB-435 cell line in vitro. Therefore, we tested the effect of equol that is metabolized from daidzein by the gut bacteria in mice and humans. Results show that equol specifically upregulated eIF4G, but not eIF4E, in MDA-MB-435 and MDA-MB-231 human breast cancer cells. Equol treatment also increased gene and protein expression of c-MYC and protein expression of other cell survival and proliferation promoting molecules with IRES sites. The elevated eIF4G in response to equol was not associated with eIF4E in a cap binding co-capture assay. Therefore, upregulation of eIF4G by equol may regulate cap-independent protein synthesis initiation resulting in cancer cell survival, proliferation, and thus, tumor progression. This research was supported by grant numbers US Army/BCRP W81XWH-11-1-0199 to CD, to NIH/NIGMS SC3GM084824 to SD Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1988. doi:1538-7445.AM2012-1988

Comparative Effects of R- and S-equol and Implication of Transactivation Functions (AF-1 and AF-2) in Estrogen Receptor-Induced Transcriptional Activity

Equol, one of the main metabolites of daidzein, is a chiral compound with pleiotropic effects on cellular signaling. This property may induce activation/inhibition of the estrogen receptors (ER) a or b, and therefore, explain the beneficial/deleterious effects of equol on estrogen-dependent diseases. With its asymmetric centre at position C-3, equol can exist in two enantiomeric forms (R- and S-equol). To elucidate the yet unclear mechanisms of ER activation/inhibition by equol, we performed a comprehensive analysis of ERa and ERb transactivation by racemic equol, as well as by enantiomerically pure forms. Racemic equol was prepared by catalytic hydrogenation from daidzein and separated into enantiomers by chiral HPLC. The configuration assignment was performed by optical rotatory power measurements. The ER-induced transactivation by R- and S-equol (0.1–10 µM) and 17b-estradiol (E2, 10 nM) was studied using transient transfections of ERα and ERβ in CHO, HepG2 and HeLa cell lines. R- and S-equol induce ER transactivation in an opposite fashion according to the cellular context. R-equol and S-equol are more potent in inducing ERα in an AF-2 and AF-1 permissive cell line, respectively. Involvement of ERα transactivation functions (AF-1 and AF-2) in these effects has been examined. Both AF-1 and AF-2 are involved in racemic equol, R-equol and S-equol induced ERα transcriptional activity. These results could be of interest to find a specific ligand modulating ER transactivation and could contribute to explaining the diversity of equol actions in vivo.

Equol is more active than soy isoflavone itself to compete for binding to thromboxane A 2 receptor in human platelets

Introduction Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A 2 (TxA 2) receptor. Material and methods Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA 2 receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA 2 was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA 2 mimic) and the calcium ionophore A23187. Results The data showed that aglycone isoflavones and equol bind to TxA 2 receptor in the µmol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [ 3H]-SQ29585 binding was: equol > genistein > daidzein > glycitein ≫ genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA 2 binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA 2 analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. Conclusions The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A 2 receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.