Equol and its enantiomers inhibited urethane-induced lung cancer in mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice.@*METHODS@#A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison.@*RESULTS@#A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups.@*CONCLUSION@#Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.

Effects of equol on colon cancer cell proliferation / 北京大学学报(医学版)

Objective:To investigate the effect of equol on the proliferation of colom cancer cells and to explore the mechanisms.Methods: Colon cancer cells (DLD1,HCT15,COLO205,LOVO,SW480) were incubated, the cell proliferation was identified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay.Reverse transcription PCR and Western blot were used to measure the mRNA and the protein expression of estrogen receptor and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)in the colon cancer cells, respectively.Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay was used to investigate the effect of estrogen receptor(ER) inhibitor,ERα agonist, and estrogen receptor ERβagonist on the cell proliferation.Results: ERα was faintly expressed in the DLD-1 and HCT-15 cells.However, ERβ expression in DLD1, HCT15, COLO205, LOVO, and SW480 colon cancer cells.Different concentrations of equol (0, 0.5, 1, 5, 10 μmol/L) significantly inhibited the growth of HCT-15 cell with the expression of ERα and ERβ.More-over, different concentrations of equol (0, 0.5, 1, 5, 10 μmol/L) significantly inhibited the growth of LOVO, and SW480 cells with the ERβ expression in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay.mRNA expressions of ERα and ERβ in HCT-15 were stimulated significantly.Western blotting proved that the protein expressions of ERα and ERβ increased with the increasing of equol dose.Moreover we found significant difference of Nrf2 protein expression in HCT-15 cell stimulated by different concentrationss of equol.After the similation of estrogen receptor inhibitor, ERα agonist, or ERβ agonist, we found that only dif-ferent concentrations of ERβ agonist(0, 1, 10, 100, 1 000, 10 000 nmol/L) significantly inhibited the growth of HCT-15, LOVO, and SW480 in adose-dependent manner.Estrogen receptor inhibitor and ERα agonistdid not present significant effect on the cell proliferation of HCT-15, LOVO, and SW480.Conclusion: Equol inhibited the colon cancer cell proliferation by its estrogenic activities and antioxidant activities.

Equol and daidzein decrease migration, invasion and matrix metalloproteinase (MMPs) gene expression in prostate cancer cell lines, DU-145 and PC-3 / Equol y daidzeina disminuyen la migración, invasión y la expresión génica de metaloproteinasa de la matriz(MMPs) en las líneas celulares prostáticas cancerosas DU-145 y PC-3

This search is focused on the study of diet compounds that may have any potential chemopreventive effect against cancer. Some compounds that fulfill this requirement are phytoestrogens. Among them we find genistein (1), the most studied, daidzein (2) and equol (3) (figure 1). To compare the sensitivities of different prostate cancer cells to phytoestrogen treatment, sulphorhodamine B dye assay was performed to determine cell viability. DU-145 and PC-3 prostate cancer cell lines treated with various doses of phytoestrogen (0-12.5-25-50 and 100 μM) for different times (24, 48 and 72h). For cell invasion or migration assay cells were seeded in a Transwell chamber with or without coating Matrigel respectively. DU-145 and PC-3 cells were treated previously with phytoestrogen (50 μM) for 24h. The study showed that equol, daidzein and genistein inhibited migration and invasion in prostate cancer cell lines. Moreover, we analyzed the effects of phytoestrogens in MMP-2 and MMP-9 mRNA expression by RT-PCR. The results indicated that equol, daidzein and genistein diminished the expression of MMP-2 and MMP-9 in a cell-dependent manner. Our data suggested that equol, daidzein and genistein inhibited migration and invasion in prostate cancer cell lines. Moreover, the results also suggest that down-regulation of MMP-2 and MMP- 9 might be involved in the inhibition of invasion of PC-3 and DU-145 cells after genistein, daidzein and equol treatment.

Este trabajo se centra en el estudio de los compuestos de dieta que pueden tener potencial efecto quimiopreventivo contra el cáncer. Algunos de estos compuestos son los fitoestrógenos. Entre ellos encontramos la genisteína (1), el más estudiado, la daidzeína (2) y el equol (3) (figura 1). Para comparar el efecto de estos fitoestrogenos sobre las líneas celulares de cáncer de próstata, DU-145 y PC-3, se utilizó el ensayo de sulforodamina B para determinar la viabilidad celular tras los tratamientos con diferentes concentraciones de fitoestrógenos (0-12.5-25-50-100 μM) durante diferentes tiempos (24, 48, 72 h). Para analizar el efecto sobre la migración celular, las células DU-145 y PC-3 fueron tratadas previamente con una concentración de fitoestrógrno (50 μM) durante 24 horas y sembradas en una cámara Transwell sin recubrir. El estudio mostró que el equol, daidzeína y genisteína inhibió en MMP-2 y MMP-9 expresiones de genes en líneas celulares de cáncer de próstata, la PC-3 y DU-145. Los resultados indicaron que la daidzeína disminuyó la expresión de MMP- 2 y MMP-9 en DU-145 células. Nuestros datos sugieren que equol, daidzeína y genisteína inhiben la migración y la invasión de líneas celulares de cáncer de próstata.

Cancer therapy using natural ligands that target estrogen receptor beta / 中国天然药物

Estrogen receptor beta (ERβ) is one of the two key receptors (ERα, ERβ) that facilitate biological actions of 17β-estradiol (E2). ERβ is widely expressed in many tissues, and its expression is reduced or lost during progression of many tumors. ERβ facilitates estrogen signaling by both genomic (classical and non-classical) and extra-nuclear signaling. Emerging evidence suggests that ERβ functions as a tissue-specific tumor suppressor with anti-proliferative actions. Recent studies have identified a number of naturally available selective ERβ agonists. Targeting ERβ using its naturally available ligands is an attractive approach for treating and preventing cancers. This review presents the beneficial actions of ERβ signaling and clinical utility of several natural ERβ ligands as potential cancer therapy.

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 (beta=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.

The Effect of Eqoul, a Metabolite of Isoflavone, on Endothelial Cell-independent Vasodilatation of Human Uterine Artery In Vitro

BACKGROUND: The purpose of this study is to investigate 1) whether equol has the direct modulation on vascular tone of endothelium-denuded human uterine artery, and 2) if present, whether this equol-induced modulation of vascular tone is mediated by intracellular calcium modulation through Ca2+ & K+ channels on vascular smooth muscle cell membrane. METHODS: The uterine arteries were obtained at the time of hysterectomy from 15 women. The uterine smooth muscles were pretreated with phenylephrine, 10(-5) M & high KCl solution 70 mM. The equol at 6 different concentrations from 10(-11) to 10(-6) M were used for the evaluation of modulatory action of equol on precontracted vascular smooth. The cumulative concentration-response for equol were determined on phenylephrine-induced contractions and compared with the results without pretreatment. RESULTS: Equol 10(-11) to 10(-6) M in concentration showed relaxation effect on vascular smooth muscle contraction which was induced by phenylephrine 10(-5) M. This relaxation effect of equol was dose-dependent. Equol in same concentrations showed no significant effects on vascular smooth muscle contraction induced by high KCI solution. Phenylephrine-induced contraction was markedly reduced from 10(-7) to 10(-4) M in concentration by pretreatment of equol, but high KCI-induced contraction was not affected by pretreatment of equol. CONCLUSIONS: This vasodilatation effect of equol may be induced by calcium antagonistic action, which was mediated through antagonistic action for receptor-dependent Ca2+ channel, but not for voltage-dependent Ca2+ channel. As far as we know, this is the first report of phytoestrogen equol on vascular reactivity of human vessels.

Therapeutic effect of equol on local skin wounds caused by mustard gas in rabbits and related mechanism / 第二军医大学学报

Objective To study the therapeutic effect of estrogenic equol (Eq) on mustard gas-caused skin wounds and the possible mechanism. Methods A rabbit skin wound model was established by contaminating the hip skin with mustard gas (0. 5 mg/cm2). The contaminated wound was treated externally with different concentrations (10, 50, and 100 μmol/L) of Eq for a week, and the changes of the wound were observed macroscopically. Wound specimens were collected for histopathological evaluation. Cell apoptosis and DNA cycle changes were analyzed by flow cytometry. Levels of IL-6 and TNF-α in the contaminated wound specimens were detected. Results The scores of erythema, edema, erosion and necrosis of the contaminated wounds in the treatment groups were significantly lower than those in the control group 120 h after mustard gas exposure (P<0. 05). The scores for changes of histopathological inflammation and necrosis were significantly lower in the 50 μtmol/L Eq treatment group than those in the control group 168 h after mustard gas exposure (P<0. 05). The results of flow cytometry showed that the proportion of apoptotic cells in the control group was significantly higher than those in the treatment groups 168 h after mustard gas exposure (P<0. 05), and the proportion of cells in DNA (S+G2-M) phase in the treatment groups was significantly higher than that in the control group (P<0. 05). The levels of IL-6 and TNF-α in the treatment groups were significantly lower than those in the control group 168 h after mustard gas exposure (P<0. 05). Conclusion Eq can promote healing of mustard gas-caused local skin damaged by inhibiting inflammatory response and cell apoptosis and enhancing cell proliferation.

In Vitro Antifungal Activity of Equol against Candida albicans

In this study, we demonstrate that equol has fungicidal activities against Candida albicans. The minimum inhibitory and minimum fungicidal concentrations of equol against C. albicans were 516 and 1,032 microM, respectively. Two separate viability assays found that equol changed the integrity of the C. albicans cell membrane, possibly by formation of membrane lesions. Scanning electron microscopy demonstrated ultrastructural changes.

Marked Individual Variation in Isoflavone Metabolism After a Soy Challenge Can Modulate the Skeletal Effect of Isoflavones in Premenopausal Women

Soy-isoflavones may act as estrogenic agonists or antagonists depending on the endogenous hormone status. These clinical effects can be exerted variably in individuals by the metabolic ability to produce a more potent metabolite than precursors. The objective of this randomized, double-blind, placebo-controlled study was to investigate the skeletal effect of isoflavones according to their metabolic variability in premenopausal women. Volunteers were randomly assigned to receive either soy-extract isoflavones (n=32) or lactose (n=21) once a day for three menstrual cycles. After intervention, the urinary excretions of isoflavones and their metabolites were significantly higher in the soy group than in the placebo group and showed a large inter-individual variation. Women in the soy group were divided into subgroups according to their ability to excrete more potent metabolites. Serum osteocalcin and urine deoxypyridinoline showed a tendency to increase after a challenge in equol high-excretors. Serum osteocalcin concentration in the genistein high-excretors increased significantly after a challenge (P=0.04) but did not increase in either the placebo or genistein low-excretors. An estrogenic antagonistic effect of isoflavones on bone turnover was observed in premenopausal women who are able to produce more potent metabolites.

Effects of Dietary Habits on the Serum Isoflavones Levels / 대한비뇨기과학회지

PURPOSE: Recent studies have suggested that isoflavones have an inverse correlation with the risk of prostate cancer. In addition, the serum isoflavones levels are thought to be decided not only by the level of intake of isoflavones, but also by the metabolic processes or the genetic abilities required for ingestion of isoflavones. So, we conducted this study to determine the effects of dietary habits on the serum isoflavones levels. MATERIALS AND METHODS: One hundred age- and community-matched healthy men between the ages of 10 and 59 years were interviewed using a food frequency questionnaire that was developed and validated for Korean populations. The individuals' dietary habits during the previous one-year period and the foods they ingested during the previous one-week period before blood sampling were assessed. The serum concentrations of isoflavones were analyzed by the reversed-phase high performance liquid chromatography-multiple reaction ion monitoring-mass spectrometry method (HPLC-MS), (SRL Co. Tokyo, Japan). RESULTS: The genistein and daidzein levels were significantly correlated with age (p=0.026 and p=0.016, respectively), but the equol level was not (p=0.091). The foods associated with the genistein level were unmilled rice, beans, garlic and etc. The foods significantly related to the daidzein level were unmilled rice, garlic and etc. Of the foods ingested during the previous one-week period, fermented soybean soup, garlic, strawberries and mung-bean pancakes were associated with the equol level. CONCLUSIONS: The genistein and daidzein levels were associated with usual dietary habits, but the equol level was related to the short-period food consumption. Changes in dietary habit might induce significant changes in the genistein and daidzein levels.

Protective effect of the isoflavone equol against DNA damage induced by ultraviolet radiation to hairless mouse skin

Equol, an isoflavonoid metabolite produced from the dietary isoflavone daidzein by the gut microflora in mammals, has been found to protect not only against ultraviolet (UV) radiation-induced cutaneous inflammation and photoimmune suppression, but also have antiphotocarcinogenic properties in mice. Because the state of DNA damage has been correlated with suppression of the immune system and photocarcinogenesis, we have therefore examined the potential of equol to offer protection from solar-simulated UV (SSUV) radiation-induced DNA damage in hairless mice by the immunohistochemical approach using monoclonal antibody specific for cyclobutane pyrimidine dimers (CPDs; H3 antibody). Topical application of 20 micrometer equol lotion, which was applied both before and after SSUV significantly reduced the number of CPDs. This reduction was evident immediately after SSUV exposure, at 1 h after exposure, and at 24 h after exposure, revealing 54%, 50%, and 26% reduction in CPDs, respectively. When the same concentration was applied for 5 consecutive days after SSUV exposure, there was no significant difference in the reduction of CPDs immediately after SSUV irradiation or at 1 hour afterwards, but there were significant reductions of 23% and 42% at 24 and 48 h after SSUV exposure, respectively. Despite apparently reducing the number of CPDs post-SSUV, topically applied equol did not appear to increase the rate of dimer removal. To conclude, equol applied topically prior to SSUV irradiation offers protection against CPD formation in hairless mice, possibly by acting as a suncreen and thus inhibiting DNA photodamage.

Effects of Soybean Metabolites on Prostate Cancer / 대한비뇨기과학회지

PURPOSE: It has been postulated that soybean isoflavones act as inhibitory factors in several cancers. Recently, various in vitro and in vivo experimental studies have demonstrated that these isoflavones inhibit prostate cancer. Therefore, we investigated whether soybean isoflavones influenced the development of prostate cancer by comparing the levels of circulating isoflavones between prostate cancer patients and controls. MATERIALS AND METHODS: The serum levels of genistein, daidzein and equol were determined using reverse-phase, high-performance liquid chromatography-multiple reaction ion monitoring mass spectrometry(HPLC-MS) and compared in 122 experimental subjects(61 prostate cancer patients and 61 cancer-free controls) from 6 hospitals. RESULTS: The serum concentrations of genistein, daidzein and equol in the patients were 130.7+/-181.4ng/ml, 53.6+/-69.3ng/ml and 11.37+/-43.4ng/ml, with control values of 95.6+/-95.2ng/ml, 55.2+/-121.8ng/ml and 23.2+/-34.5ng/ml, respectively. There was no statistical difference between the 2 groups. Daidzein non-metabolizers who were unable to degrade daidzein into equol were compared between the patients and the controls, and were found to be significantly more common in the patient group (p=0.001, OR=3.44, 95% CI=1.6243-7.2855). However, the equol/daidzein ratio was significantly lower in the patients than in the controls(p=0.0072). No association between age, stage, Gleason score or isoflavone concentrations was found. CONCLUSIONS: These results suggest that the capability to produce equol (i.e., the mechanism for the metabolism of daidzein into equol) is closely involved in the lower incidence of prostate cancer, and that a diet based on soybean isoflavones would be useful in preventing prostate cancer.

THE INHIBITORY EFFECT OF EQUOL ON HUMAN OVARIAN CARCINOMA CELL SKOV-3 PROLIFERATION AND ITS MECHANISM / 营养学报

Objective To investigate the effect of equol on ERK mediated signal transduction pathway and to clarify its mechanism of proliferation inhibition on human ovarian carcinoma cell line SKOV-3. Method SKOV-3 cells were treated with 10-8,10-7,10-6,10-5,5?10-5,10-4 mol/L equol for 24,48 and 72h. After pretreatment with 10 ?mol/L U0126 an ERK signaling pathway inhibitor or ICI182,780,estrogen receptor inhibitors for 1h,then treatment with 50 ?mol/L equol for 2h,the cell viability was examined and the expressions of ERK and p-ERK protein were determined using Western blotting. Results Equol was demonstrated to inhibit SKOV-3,proliferation time-and dose-dependently. The expression of p-ERK protein was decreased in dose-dependent manner,while the expression of total ERK was unchanged. Both the single use of U0126,or ICI182,780,and combined with equol could decrease the expression of p-ERK protein. Conclusion Equol could inhibit proliferation in ovarian carcinoma cell lines SKOV-3. Its inhibitory effect appears to be due to down-regulation of p-ERK protein.