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.

Equol protects PC12 neuronal cells against hypoxia/reoxygenation injury in vitro by reducing reactive oxygen species production / 南方医科大学学报

<p><b>OBJECTIVE</b>Both of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate-reduced oxidases) and Src (a non-receptor protein tyrosine kinase) play a prominent role in mediating hypoxia/reoxygenation injury of neurons. The present study was designed to investigate the neuroprotective effect of equol, a predominant active metabolite of daidzein, against hypoxia/reoxygenation injury in rat pheochromocytoma cell line (PC12) and explore the underlying mechanisms.</p><p><b>METHODS</b>PC12 cells exposed to hypoxia/reoxygenation injury were examined for reactive oxygen species (ROS) using dihydroethidium and 2', 7'-dichlorofluorescein diacetate and analyzed for changes in lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. The expression levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) were measured using Western blotting.</p><p><b>RESULTS</b>Equol dose-dependently restored the cell viability and decreased LDH activity and MDA content in culture medium of PC12 cells exposed to hypoxia/reoxygenation. Pretreatment of the cells with 10(-5) and 10(-6) mol/L equol inhibited hypoxia/reoxygenation-induced increase of ROS. PC12 cells treated with equol prior to hypoxia/reoxygenation injury showed significant enhancement of the protein levels of gp91(phox) and p-Src.</p><p><b>CONCLUSION</b>Equol confers neuroprotection against hypoxia/reoxygenation injury in PC12 cells by inhibiting the generation of ROS very likely as a result of down-regulation of gp91(phox) and inhibition of Src phosphorylation.</p>

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.

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.

Dose-response assessment of the anti-cancer efficacy of soy isoflavones in dimethylhydrazine-treated rats fed 6% fructooligosaccharide

We investigated the combinatorial effects of different doses of dietary soy isoflavones (SI) and fructooligosaccharide (FOS) in a rat model of colon cancer. We hypothesized that increased bioavailability of SI metabolites due to dietary FOS may increase production of bioactive equol and affect colon carcinogenesis in a dose-dependent manner. Sprague-Dawley male rats were injected with 12-dimethylhydrazine (DMH) and were provided experimental diets that contained 0, 10, 50, 150, or 500 mg SI per kg of diet and 6% FOS for 12 weeks. The number of aberrant crypt foci (ACF) and the expression of cyclooxygenase-2 (COX-2) in colonic tissues were significantly decreased in the 6% FOS-fed groups compared to the control group. Gut transit time and fecal pH were significantly lower, and fecal concentrations of bifidobacteria were increased with 6% FOS. However, dietary SI supplementation in combination with 6% dietary FOS did not affect ACF formation or COX-2 expression. Plasma equol concentrations were dose-dependently increased by supplementation of SI up to 500 mg/kg of diet. In conclusion, SI supplementation up to 500 mg/kg of diet appeared to have no additive beneficial effects in rats with chemically-induced colon cancer that were fed 6% FOS, although plasma equol was dose-dependently increased.

Synergistic Effect of Isoflavones on the BMP-4 MediatedOsteoblastic Differentiation of C2C12 Myoblast / 대한정형외과학회잡지

PURPOSE: Isoflavones are rich in soybean and are known to affect bone formation. This study examined the effects and modes of action of isoflavones on the differentiation of C2C12 myoblasts in the presence of the bone morphogenetic protein (BMP)-4. MATERIALS AND METHODS: The isoflavones, daidzein, genistein or equol, and/or BMP-4 were added alone or in combination to C2C12 myoblasts. After 72 hours culture, the cells were stained for the early osteoblastic differentiation marker, alkaline phosphatase (ALP). The ALP activity was determined by comparing the color of the stained images as well as by spectrophotometry. The expression profiles of the extracellular matrix (ECM) genes responsible for the extensive remodeling at the cell surface were analyzed using agene expression microarray after treating thesamples with daidzein. RESULTS: ALP staining of BMP-4 or the isoflavones-treated cells showed that BMP-4 increased the activity of ALP in a dose dependent manner, whereas the isoflavones alone did not induced any remarkable increase. However, the ALP activity increased when the cells were treated with BMP-4 and any of the three isoflavones. The macrogen mouse MAC array data showed that the ECM genes, Mmp13 and Mmp3, were up-regulated by daidzein, whereas Col4a2, Col5a1 and Mmp9 were down-regulated. CONCLUSION: Isoflavones induce osteoblastic differentiation when combined with BMP-4, which is possibly achieved by modulating the expressional levels of various ECM genes.

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.

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.