Soy Isoflavone Genistein Research Articles

Abstract 4278: Soy isoflavones and their metabolites modulate IL-12-induced NK cell IFN-γ production

Abstract Soybeans and foods derived from them are a rich source of phytochemcials that have gained attention due to their ability to affect obesity, metabolism, cancer, and inflammation. Soy isoflavones (genistein and daidzein) have also been shown to be differentially metabolized between individuals. This is presumably due to differences in gut microbiota, and can lead to production of terminal isoflavone metabolites such as O-desmethylangolensin (ODMA) and equol. In a prior phase-I clinical study conducted by our group, patients with prostate cancer received a novel soy-enriched bread product. Analysis of blood and urine following soy intervention revealed that men clustered into distinct groups based on their ability to metabolize daidizein into either ODMA or equol. Reduced pro-inflammatory cytokines and percentages of Tregs and MDSC were also observed in the blood of patients after 8 weeks of soy bread intervention as compared to baseline. Given these differences, we hypothesized that individual isoflavones and metabolites would have unique immunomodulatory effects upon human natural killer (NK) cell function. Although a limited number of prior studies have evaluated how soy isoflavones or their metabolites modulate inflammation and different immune subsets (monocytes, macrophages, DCs), their role in NK cell biology remains poorly defined. Understanding the role of soy isoflavones and their metabolites in regulating NK cell activity is critical due to NK cells’ importance in the anti-tumor response and immunosurveillance. We found that the soy isoflavones and their metabolites did not affect the viability of healthy donor PBMC (n = 3) at concentrations as high as 25μM. However, pre-treatment of healthy donor PBMC (n = 3) with genistein (mean 7.1 ng/ml ±1.3) or equol (mean 6.7 ng/ml ±1.8) for 4 hours and then stimulated with IL-12/18 for 72 hours decreased IFN-γ production compared to unstimulated controls (mean 26.7 ng/ml ±16.1). In contrast, daidzein and ODMA had no effect. We next utilized intracellular flow cytometry to determine the cellular subset(s) that were modulated by soy isoflavones or their metabolites. These data revealed that only genistein and equol decreased the percentage of IL-12/18 induced IFN-γ producing NK cells (CD56+CD3-) by 30% and 53%, respectively. There was no observed difference in other IFN-γ producing immune subsets (T and NKT cells) when cultured with the soy isoflavones or metabolites. Furthermore, we found soy isoflavones and metabolites altered in vitro cytotoxicity of human IL-12 stimulated NK cells. Ongoing studies in our laboratory are elucidating the precise signaling pathways downstream of IL-12 in human NK cells that are modulated in response to soy isoflavones and their bioactive metabolites. This study provides critical insight regarding the impact of dietary soy on NK cell mediated responses potentially informing the use of soy during chemopreventive or immune based therapies. Citation Format: Thomas A. Mace, Samantha King, Matthew Farren, Elizabeth McMichael, Steven Scoville, William E. Carson, Gregory Young, Jennifer Thomas-Ahner, Kenneth M. Riedl, Steven Schwartz, Steven K. Clinton, Gregory Lesinski. Soy isoflavones and their metabolites modulate IL-12-induced NK cell IFN-γ production. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2015-4278

Role of flavonoids in future anticancer therapy by eliminating the cancer stem cells.

Despite the numerous recent advances made in conventional anticancer therapies, metastasis and recurrence still remain the major problems in cancer management. The current treatment modalities kill the bulk of the tumor, leaving cancer stem cells behind and therefore, the agents specifically targeting this cancer initiating cell population may have important clinical implications. In this review article, the data about the inhibitory action of flavonoids, both natural as well as their synthetic derivatives, on the self-renewal capacity and survival of cancer stem cells of different origins are compiled and analyzed. These data indicate that several plant secondary metabolites, including soy isoflavone genistein, green tea catechins and a widely distributed flavonol quercetin, have the potential to suppress the stemness markers and properties, traits of the epithelial-to-mesenchymal transition and migratory characteristics, being also able to sensitize these cells to the standard chemotherapeutic drugs. These polyphenolic compounds act through multiple signal transduction pathways, providing thus the maximal therapeutic response and offering some promise to be included in the future cancer treatment schemes in combination with the conventional therapies. Such approach may give an important contribution to the shift of cancer management from palliative to curative mode, likely leading to the disease-free survival. Thus, flavonoids can serve as attractive candidates for novel anticancer agents by eliminating the roots of cancer.

Genistein Reduces Glyburide Efflux by the Human Placental BCRP Transporter: Transcriptional Regulation and Direct Inhibition

The hypoglycemic drug glyburide is routinely used in the treatment of gestational diabetes. Fetal exposure to glyburide is typically low because the BCRP/ABCG2 transporter on placental syncytiotrophoblasts prevents its maternal‐to‐fetal transfer. The purpose of this study was to determine the effect of the dietary soy isoflavone genistein on the transcriptional regulation and direct inhibition of BCRP. For the regulation studies, human choriocarcinoma BeWo placental cells were incubated with genistein (0‐10 µM). After 48 h exposure to genistein, placental BeWo cells exhibited a 40% reduction in BCRP mRNA and protein. Down‐regulation of BCRP led to a 50% increase in the accumulation of the BCRP substrate, Hoechst 33342 in BeWo cells treated with genistein. Further, down‐regulation of BCRP by genistein was prevented by removal of serum from the media. In addition to modulating the expression of BCRP, genistein also competitively inhibited its transport in HEK cells overexpressing the wild‐type (WT) or functionally‐reduced BCRP variant (C421A). As expected, C421A‐BCRP cells had 10% greater accumulation of 3H‐glyburide than WT‐BCRP cells. Genistein increased the accumulation of 3H‐glyburide in both WT‐ and C421A‐BCRP cells by 50%. Taken together, pregnant women expressing the common loss‐of‐function C421A‐BCRP variant and/or consume genistein through a soy diet may be at an elevated risk for fetal exposure to glyburide and subsequent neonatal hypoglycemia. Support: ES020522, ES005022, ES007148, AFPE and PhRMA Predoctoral Fellowships.

Downregulation of miR‐155 Contributes to The Anti‐proliferative Effects of The Soy Isoflavone Genistein in Estrogen Receptor Negative Breast Cancer

Consumption of the soy isoflavone genistein has been associated with reduced breast cancer risk. We previously reported that dietary genistein inhibits mammary tumor growth and metastasis of estrogen receptor (ER) (‐) cancer cells in mice. The objective of this study was to investigate a role for the oncomir miR‐155 in the anticancer effects of genistein. At low physiologically relevant concentrations, genistein inhibited cell viability, induced apoptosis, and downregulated miR‐155 in the ER (‐) MDA‐MB‐435 and Hs578t breast cancer cells, without significantly affecting the viability or miR‐155 levels of ER (+) MCF‐7 breast cancer cells. In parallel, the pro‐apoptotic and anti‐cell proliferative miR‐155 targets FOXO3, PTEN, casein kinase, and p27 were upregulated in response to genistein in the ER (‐) cells. Ectopic expression of miR‐155 in the MDA‐MB‐435 and Hs578t cells decreased the effects of genistein on cell viability, and abrogated the effects of genistein on apoptosis and expression of pro‐apoptotic genes. We conclude that genistein‐mediated downregulation of miR‐155, a critical suppressor of apoptosis, contributes to the anticancer effects of genistein in ER (‐) breast cancer.

Genistein Induces Cytochrome P450 1B1 Gene Expression and Cell Proliferation in Human Breast Cancer MCF-7 Cells.

Cytochrome P450 1B1 (CYP1B1) plays an important role in breast cancer development and progression by bioactivating endogenous estrogens and environmental carcinogens. In this study, we examined the effect of the soy isoflavone genistein on CYP1B1 gene expression, reactive oxygen species (ROS) production, and cell proliferation in human breast cancer MCF-7 cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed a dose-dependent induction of CYP1B1 gene expression in the cells treated with 5 and 25 μM of genistein. Genistein at 5 μM exhibited a synergistic effect on the CYP1B1 mRNA level induced by the environmental carcinogen 7,12-dimethylbenz[a]anthracene. We also found that genistein at 5 μM increased cellular levels of ROS and stimulated cell proliferation starting from the second day of culture. This study suggests that physiological concentrations of genistein stimulate ROS production and breast cancer cell proliferation through the induction of CYP1B1 gene expression. More extensive studies are warranted to further characterize the role of genistein and the CYP1B1 enzyme in human breast carcinogenesis.

Soy and the soy isoflavone genistein promote adipose tissue development in male mice on a low-fat diet.

Several nutrients act as phytoestrogens, being anti-adipogenic when consumed with a fat-rich diet. Their effect on a low-fat diet (LFD) background is unknown. We tested soy and genistein effects on adipose tissue in LFD-fed mice and genistein activity in the 3T3-L1 adipogenesis model. C57BL/6 J male mice were fed an 8.5% soy-supplemented LFD (SS-LFD) or a soy-free LFD (SF-LFD) for 147 days. Groups of 3-week-old (pubertal) and 6-week-old (adult) mice on the SF-LFD were also treated with 17ß-estradiol (E2, 5 µg/kg/day) ip or pure genistein (5 mg/kg/day) by gavage for 15 days. Body fat deposition and gene expression profiles were evaluated. E2 and genistein effects on ERα, ERβ and PPARγ transcriptional activities were characterized in ERα- or ERβ-transfected 3T3L1 cells during differentiation, by the use of reporter plasmids. The SS-LFD group increased fat mass compared with the SF-LFD group. Genistein alone increased while E2 decreased fat pads in the 15-day-treated mice. In visceral fat, genistein differentially regulated 13 metabolic pathways compared to E2. PPARγ-controlled genes were downregulated by E2, while they were upregulated by genistein. In 3T3-L1 cells, genistein activated ERβ-driven transcription, differentiation and lipid accumulation, while inhibited ERα-driven transcription, without effects on lipid accumulation. E2 activated both ERs only in preadipocytes. In differentiated untransfected cells, genistein inhibited PPARγ, while activated PPARγ in the presence of ERβ. Soy and genistein at nutritional doses induce fat development in LFD-fed mice and adipogenesis in 3T3-L1 cells, with a mechanism that involves, at least in vitro, ERβ and is dependent on cell differentiation stage.

Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies.

The biotransformation of isoflavones by gut microbiota and by drug metabolizing enzymes plays a crucial role in the understanding of their potential health-promoting effects. The purpose of our work was to develop a simultaneous, sensitive, and robust automated ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify the soy isoflavones daidzein and genistein, their conjugative metabolites, as well as their major microbial degradation products in order to provide a method for use in large clinical trials or animal studies. An automated, 96-well solid-phase extraction method was used to extract the isoflavone analytes from plasma and urine. Separation of genistein, daidzein, and 19 of its metabolites, including five glucuronides, seven sulfates, and two sulfoglucuronides, as well as five microbial metabolites, was achieved in less than 25min using a sub-2μm particle column and a gradient elution with acetonitrile/methanol/water as mobile phases. Analysis was performed under negative ionization electrospray MS via the multiple reaction monitoring (MRM). Validation was performed according to the analytical method validation guidelines of Food and Drug Administration (FDA) and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) consisting of selectivity, accuracy, precision, linearity, limit of detection, recovery, matrix effect, and robustness. All validated parameters essentially matched the FDA and ICH requirements. The application of this method to a pharmacokinetic study in postmenopausal women showed that isoflavones are extensively metabolized in vivo. A robust automated analytical approach was developed, which allows the handling of large sample sizes but nevertheless provides detailed information on the isoflavone metabolite profile leading to a better understanding and interpretation of clinical and animal studies.

Refractory wound healing and FoxO1/iNOS pathway in type 1 diabetes (1142.3)

Aims/hypothesis Refractory wounds in diabetic patients constitute a serious complication that often leads to amputation with limited treatment regimens. The present study was designed to determine the role of FoxO1/iNOS pathway and effect of the soy isoflavone genistein on diabetic wound healing.Methods Streptozotocin (STZ)‐induced type 1 diabetic mice with full‐thickness excisional wounds given intragastrically 1 and 3 mg/kg/day of iNOS selective inhibitor (L‐NIL) or received 0.2, 1 and 5 mg/kg/day of genistein via subcutaneous injection.Results In diabetic wound tissues, the inducible nitric oxide synthase (iNOS) was activated, silent information regulator 1 (SIRT1) and forkhead box O transcription factor 1 (FoxO1) were elevated. Genistein administration prevented increased iNOS activity and attenuated diabetic cutaneous SIRT1 and FoxO1 levels in a dose‐dependent manner. Diabetic conditions significantly increased superoxide anion (O2.‐) production, nitrotyrosine formation, and decreased nitrite levels in wound tissues. L‐NIL and genistein treatments normalized the elevated O2.‐ production, nitrotyrosine formation and reversed the attenuated nitrite level. L‐NIL and genistein dose‐dependently rescued the delay of wound closure in diabetic mice. Five mg/kg/day of genistein treatment significantly increased the mean perfusion rate, and in vitro treatment with genistein protected against high glucose‐induced impairment of capillary tube formation in cultured endothelial cells.Conclusions/interpretation FoxO1/iNOS signaling may contribute to the delayed wound healing in type 1 diabetes. Genistein could rescue the impaired wound healing and improved wound angiogenesis in type 1 diabetic mice, at least in part, by suppression of FoxO1, iNOS activity and oxidative stress.Grant Funding Source: This work was supported by the NSFC and Beijing Higher Education Young Elite Teacher Project

Glycation is regulated by isoflavones.

The effect of soy isoflavones on the Maillard reaction (MR) was investigated. Model systems composed of the soy protein glycinin (10 mg mL(-1)) and fructose (40 mg mL(-1)) under basic pH (∼12) conditions were employed for testing the anti-glycative effect of the major antioxidant soy isoflavones (genistin and genistein at 10 μg mL(-1)) and a soy isoflavone-rich extract. The contents of total phenols (TPCs) and total flavonoids (TFCs) of the isoflavone-rich extract were determined. Glycinin was pre-incubated with isoflavones for 1 h and 16 h at 60 °C prior to MR. The progress of MR was estimated by analysis of free amino groups by OPA assay; carbohydrate covalently bound to the protein backbone using phenol-sulfuric acid assay, protein-bound N(ε)-(carboxymethyl)lysine (CML) by UPLC-MS and spectral analysis of fluorescent protein-bound AGEs. Genistin (10 μg mL(-1), 23 μM) and its aglycone genistein (10 μg mL(-1), 37 μM) did not prevent protein glycation (p > 0.05). The soy isoflavone-rich extract containing 2.5 mg mL(-1) of TFC efficiently decreased the amount of carbohydrate bound to the protein skeleton (20%) (p < 0.05) and formation of advanced glycation end products (AGEs) (>80%) (p < 0.05). The anti-glycative mechanism of isoflavones may be related to its conjugation to glycation sites of the protein structure (free amino groups), their antioxidant character and trapping of dicarbonyl intermediates. Extracts based on mixtures of isoflavones may be useful for producing glycated conjugates avoiding the substantial formation of AGEs bound to protein.

Cooperative effects of soy isoflavones and carotenoids on osteoclast formation

Osteoclasts play a major role in bone resorption. Several functional food components, such as soy isoflavones and carotenoids, are reported to inhibit osteoclast formation. However, the cooperative effect of functional foods or their constituents on bone metabolism has not been clarified. This study aimed to investigate the cooperative effect of soy isoflavones and carotenoids on osteoclast formation in vitro using cultures of RAW264 and bone marrow cells in the presence of receptor activator of nuclear factor κ-B ligand. In RAW264 cells, treatment with soy isoflavones (genistein or equol) or carotenoids (β-carotene) suppressed osteoclast formation. At 10 µM, genistein and equol inhibited RAW264 cell proliferation but did not affect cell viability. When 10 µM genistein or equol was combined with 0.1 µM β-carotene, we observed an additive suppressive effect on osteoclast differentiation. Similar results were observed with bone marrow cell cultures. We found that 10 µM of zeaxanthin or lutein suppressed osteoclast formation singly, and further enhanced the suppressive effects of daidzein or genistein when administered in combination. These results suggest that the combination of soy isoflavones and carotenoids have an enhanced suppressive effect on osteoclast formation. This knowledge might be important in planning diet for bone health.

Transcriptomic analysis identifies gene networks regulated by estrogen receptor α (ERα) and ERβ that control distinct effects of different botanical estrogens.

The estrogen receptors (ERs) ERα and ERβ mediate the actions of endogenous estrogens as well as those of botanical estrogens (BEs) present in plants. BEs are ingested in the diet and also widely consumed by postmenopausal women as dietary supplements, often as a substitute for the loss of endogenous estrogens at menopause. However, their activities and efficacies, and similarities and differences in gene expression programs with respect to endogenous estrogens such as estradiol (E2) are not fully understood. Because gene expression patterns underlie and control the broad physiological effects of estrogens, we have investigated and compared the gene networks that are regulated by different BEs and by E2. Our aim was to determine if the soy and licorice BEs control similar or different gene expression programs and to compare their gene regulations with that of E2. Gene expression was examined by RNA-Seq in human breast cancer (MCF7) cells treated with control vehicle, BE or E2. These cells contained three different complements of ERs, ERα only, ERα+ERβ, or ERβ only, reflecting the different ratios of these two receptors in different human breast cancers and in different estrogen target cells. Using principal component, hierarchical clustering, and gene ontology and interactome analyses, we found that BEs regulated many of the same genes as did E2. The genes regulated by each BE, however, were somewhat different from one another, with some genes being regulated uniquely by each compound. The overlap with E2 in regulated genes was greatest for the soy isoflavones genistein and S-equol, while the greatest difference from E2 in gene expression pattern was observed for the licorice root BE liquiritigenin. The gene expression pattern of each ligand depended greatly on the cell background of ERs present. Despite similarities in gene expression pattern with E2, the BEs were generally less stimulatory of genes promoting proliferation and were more pro-apoptotic in their gene regulations than E2. The distinctive patterns of gene regulation by the individual BEs and E2 may underlie differences in the activities of these soy and licorice-derived BEs in estrogen target cells containing different levels of the two ERs.

Inhibitory effects of a major soy isoflavone, genistein, on human DNA topoisomerase II activity and cancer cell proliferation

The inhibitory activity of 3 soy isoflavones (daidzein, genistein and glycitein) and their glycosides (daidzin, genistin and glycitin) on mammalian DNA polymerases (pols) and topoisomerases (topos) was investigated. Of the compounds tested, only genistein selectively inhibited human topo II activity and had an IC50 value of 37.5 µM. These isoflavones had no effect on the activity of human topo I; mammalian pols α, β, γ and κ; or on any other DNA metabolic enzyme tested. Thermal transition analysis indicated that genistein did not influence the direct binding to double-stranded DNA. Genistein prevented the proliferation of HCT116 human colon carcinoma cells with an LD50 of 94.0 µM and it halted the cell cycle in G2/M phase. These results suggest that decreases in cell proliferation due to genistein may result from the inhibition of cellular topo II and that genistein, a major soy isoflavone, may be an anticancer food component. The relationship between the structures and these bioactivities of soy isoflavones is discussed.

Interactive effects of 1, 25-dihydroxyvitamin D3 and soy protein extract (SPE) on oral cancer growth in vitro: evidence for potential functional relationships.

Background: Previous studies have found specific soy isoflavones (Genistein, Daidzein, Glycitein) demonstrate anti-tumor properties against several cancer types, including oral cancer. Few studies have evaluated whole soy extract, containing a combination of these isoflavones and other bioreactive compounds, which may function synergistically and more effectively against oral cancers. Preliminary work by this group has now demonstrated whole soy protein extract (SPE) inhibits oral cancer cell growth specifically and selectively, through independent cell-cycle and apoptotic pathways. However, more recent evidence now suggests that ingestion of vitamin D3, either in dietary foods or supplements may potentiate the activity of soy components and their anti-tumor effects.Objective: The primary goal of this study was to investigate the interactive and inter-connected effects of 1, 25-dihydroxyvitamin D3 administration with the anti-proliferative effects of whole soy protein extract (SPE) on oral cancer and normal cell lines in vitro.Methods: Three oral squamous cell carcinoma cell lines (SCC15, SCC25, and CAL27) were treated with 1, 25-dihydroxy Vitamin D3 at physiological concentrations (10-125 nmol). Cell growth was then compared with cell treatment using soy protein extract (SPE) within the normal physiologic range (0 - 10 μM/L). Interactive effects were then evaluated using co-administration of SPE and 1, 25-dihydroxy Vitamin D3. Quantitative RT-PCR was performed at various time points to determine any changes in mRNA expression for key cell cycle and apoptotic signaling pathway regulators, including p53, c-myc, ornithine decarboxylase (ODC), caspase-2, caspase-8, and bax. Results: Administration of 1, 25-dihydroxy Vitamin D3 induced distinct dose-dependent, growth-inhibitory effects in all three oral cancer cell lines examined. These inhibitory effects were comparable to the overall range of growth inhibition induced by SPE. However, the combined effects of co-administration were far greater, suggesting the presence of synergistic relationships between these components. In addition, these results indicate that either treatment alone appeared to modulate mRNA expression of oral cancer cell-cycle promoters c-myc and ODC, as well as the caspase-dependent apoptosis pathway, while only 1, 25-dihydroxy Vitamin D3 administration appeared to influence the bax pathway. Conclusion: These results suggest that co-administration with 1, 25-dihydroxy Vitamin D3 and SPE may enhance their anti-tumor effects. This study may help to explain, in part, why balanced diets rich in fruits, vegetables, and soy protein, are associated with protection against development and progression of oral cancers, although further study is needed to develop specific public health recommendations for oral cancer treatment and prevention.Key words: vitamin D, soy extract, whole soy protein, oral cancer, growth inhibition.

Abstract 4873: Genistein and soy isoflavone isolate decreased tumor incidence and multiplicity in a Sprague-Dawley ER-positive breast cancer model.

Abstract This study evaluated the chemopreventive efficacy of the pure soy isoflavone genistein and of a soy isoflavone mixture (PTI G-4660) containing roughly 90% genistein in an ER-positive breast cancer model in rats. Female Sprague Dawley rats were injected i.v. with N-nitroso-N -methylurea (MNU) at 50 days of age. In a model examining the effects on cancer initiation, promotion, and progression, rats were fed either a standard Teklad diet or a diet supplemented with genistein (200 or 2000 mg/kg diet) beginning at 43 d of age. Supplemented or standard diets were administered continuously thereafter until the end of the study. In a separate study testing the effects on progression, rats were administered standard Teklad diet or diet supplemented with PTI G-4660 (2000 mg/kg diet) beginning at 55 d of age. All rats were weighed weekly, palpated for mammary tumors twice per week, and checked daily for signs of toxicity. The studies were terminated 126 d after MNU treatment and tumors were weighed. After 126 days, genistein at 200 and 2000 mg/kg diet decreased tumor incidence in a dose-dependent fashion by 15 and 20% (p&amp;lt;0.05), respectively, and significantly decreased tumor multiplicity by 49.6 and 60.3% (p&amp;lt;0.05), respectively. PTI non-significantly decreased tumor incidence by 14% and tumor multiplicity by 24%. In addition, gene expression profiling of livers from rats treated with standard Teklad diet or genistein revealed pathways modified by genistein which may be useful as biomarkers. Citation Format: Holly L. Nicastro, Ronald A. Lubet, Barbara K. Dunn, Clinton J. Grubbs. Genistein and soy isoflavone isolate decreased tumor incidence and multiplicity in a Sprague-Dawley ER-positive breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4873. doi:10.1158/1538-7445.AM2013-4873

Self-reported menopausal symptoms in a racially diverse population and soy food consumption

ObjectivesEvaluate the association of self-reported vasomotor symptom (VMS) frequency with race/ethnicity among a diverse midlife US population and explore menopause symptom differences by dietary soy isoflavone (genistein+daidzein) consumption. Study designCross-sectional population-based study of peri- and postmenopausal women, ages 45–58. OutcomesRecent VMS frequency, VMS ever; recent symptom bother (hot flashes, night sweats, headache and joint-ache). ResultsOf 18,500 potentially eligible women, 9325 returned questionnaires (50.4% response); 3691 were excluded (premenopausal, missing data, taking hormones). Of 5634 remaining women, 82.1% reported hot flashes ever, 73.1% reported night sweats ever; 48.8% and 38.6% reported recent hot flashes or night sweats, respectively. Compared with White women, Chinese, Japanese, Vietnamese, other Asian (each p<0.001) and Filipino (p<0.01) women less commonly reported ever having hot flashes; Asian women less commonly reported recent VMS bother (p<0.001). Black women more commonly reported hot flashes ever (p<0.05) and recent VMS bother (p<0.05). Compared with non-Hispanic White women, Hispanic women were less likely to report hot flashes (p<0.05) or night sweats (p<0.001) ever. Women were classified by isoflavone consumption: (1) none (n=1819), (2) 0.01–4.30mg/day (n=1931), (3) 4.31–24.99mg/day (n=1347) and (4) ≥25mg/day (n=537). There were no group differences in recent VMS number/day: (1) 7.0 (95% CI 6.5, 7.5); (2) 6.4 (95% CI 6.0, 7.1); (3) 7.0 (95% CI 6.3, 8.2); and (4) 6.8 (95% CI 6.1, 7.7). ConclusionsMenopausal symptoms, independent of isoflavone intake, varied considerably by race/ethnicity and were least common among Asian races.

Production of ortho-hydroxydaidzein derivatives by a recombinant strain of Pichia pastoris harboring a cytochrome P450 fusion gene

CYP57B3 from Aspergillus oryzae was recently discovered to catalyze the ortho-hydroxylation of the soyisoflavone genistein. In the present study, the gene encoding CYP57B3 was fused with the reductase domain of the CYP102A1 gene (BM3R) from Bacillus megaterium, and recombinant Pichia pastoris harboring the P450 fusion gene was evaluated for its ability to produce ortho-hydroxydaidzein derivatives from daidzein. The results showed that 8-hydroxydaidzein (8-OHDe), 3′-hydroxydaidzein (3′-OHDe), and 6-hydroxydaidzein (6-OHDe) were produced during fermentation with a maximal conversion of 2.4, 0.9, and 36.3%, respectively. The maximal yield of 6-OHDe by the recombinant strain was 9.1mg/l. To our knowledge, both the maximal yield and the conversion efficiency of 6-OHDe from daidzein in the present study are the highest among those reported in the literatures to date. The present study is also the first to demonstrate production of ortho-hydroxydaidzein derivatives using a fusion fungus cytochrome P450 enzyme.

Evaluation of the Estrogenic Activity of Pueraria (Kudzu) Flower Extract and Its Major Isoflavones Using ER-Binding and Uterotrophic Bioassays

Pueraria flower extract (PFE) is a hot water extract of the Kudzu flower (Pueraria thomsonii). Tea made from dried Kudzu flower is widely used inChina, and PFE is utilized as a nutritional supplement inJapan. PFE contains unique isoflavones such as 6-hydroxygenistein 6,7-di-O-glucoside (6HGDG), tectorigenin 7-O-xylosylglucoside (TGXG), and tectoridin. 6HGDG is known to be metabolized into 6-hydroxygenistein, and TGXG and tectoridin are known to be metabolized into tectorigenin in the digestive tract. Isoflavones typically mimic the effects β-estradiol has on estrogen receptors (ERs) and may influence the female genital system in the case of excessive intake. As a result, the upper limit of safe daily consumption of soy isoflavones has been enforced inJapan. In the present study, ER-binding assays were performed using the EnBio estrogen receptor/cofactor assay system to compare the estrogenic activity of 6-hydroxygenistein and tectorigenin to that of the soy isoflavone genistein. In addition, uterotrophic bioassays were performed to investigate the estrogenic effects of PFE in vivo. The ER-binding assays revealed that the ER-binding affinities of 6-hydroxygenistein and tectorigenin were approximately 0.01 - 0.04 that of genistein. Soy isoflavone products also induced an increase in uterine wet and blotted weight at doses of 500 mg/kg and 1000 mg/kg, whereas PFE did not cause adverse estrogenic effects, even at a dose of 1000 mg/kg. Based on these results, PFE does not appear to contain compounds with strong estrogenic activity or cause adverse estrogenic effects in vivo. Importantly, the results of this study confirm the safety of PFE as a food supplement.

Anti-diabetic functions of soy isoflavone genistein: mechanisms underlying its effects on pancreatic β-cell function

Type 2 diabetes is a result of chronic insulin resistance and loss of functional pancreatic β-cell mass. Strategies to preserve β-cell mass and a greater understanding of the mechanisms underlying β-cell turnover are needed to prevent and treat this devastating disease. Genistein, a naturally occurring soy isoflavone, is reported to have numerous health benefits attributed to multiple biological functions. Over the past 10 years, numerous studies have demonstrated that genistein has anti-diabetic effects, in particular, direct effects on β-cell proliferation, glucose-stimulated insulin secretion and protection against apoptosis, independent of its functions as an estrogen receptor agonist, antioxidant, or tyrosine kinase inhibitor. Effects are structure-specific and not common to all flavonoids. While there are limited data on the effects of genistein consumption in humans with diabetes, there are a plethora of animal and cell-culture studies that demonstrate a direct effect of genistein on β-cells at physiologically relevant concentrations (<10 μM). The effects appear to involve cAMP/PKA signaling and there are some studies that suggest an effect on epigenetic regulation of gene expression. This review focuses on the anti-diabetic effects of genistein in both in vitro and in vivo models and potential mechanisms underlying its direct effects on β-cells.

The Soy Isoflavone Equol May Increase Cancer Malignancy via Up-regulation of Eukaryotic Protein Synthesis Initiation Factor eIF4G

Dietary soy is thought to be cancer-preventive; however, the beneficial effects of soy on established breast cancer is controversial. We recently demonstrated that dietary daidzein or combined soy isoflavones (genistein, daidzein, and glycitein) increased primary mammary tumor growth and metastasis. Cancer-promoting molecules, including eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E, were up-regulated in mammary tumors from mice that received dietary daidzein. Herein, we show that increased eIF expression in tumor extracts of mice after daidzein diets is associated with protein expression of mRNAs with internal ribosome entry sites (IRES) that are sensitive to eIF4E and eIF4G levels. Results with metastatic cancer cell lines show that some of the effects of daidzein in vivo can be recapitulated by the daidzein metabolite equol. In vitro, equol, but not daidzein, up-regulated eIF4G without affecting eIF4E or its regulator, 4E-binding protein (4E-BP), levels. Equol also increased metastatic cancer cell viability. Equol specifically increased the protein expression of IRES containing cell survival and proliferation-promoting molecules and up-regulated gene and protein expression of the transcription factor c-Myc. Moreover, equol increased the polysomal association of mRNAs for p 120 catenin and eIF4G. The elevated eIF4G in response to equol was not associated with eIF4E or 4E-binding protein in 5' cap co-capture assays or co-immunoprecipitations. In dual luciferase assays, IRES-dependent protein synthesis was increased by equol. Therefore, up-regulation of eIF4G by equol may result in increased translation of pro-cancer mRNAs with IRESs and, thus, promote cancer malignancy.

Mechanisms underlying the dualistic mode of action of major soy isoflavones in relation to cell proliferation and cancer risks

Isoflavones are phytoestrogens that have been linked to both beneficial as well as adverse effects in relation to cell proliferation and cancer risks. The present article presents an overview of these seemingly contradicting health effects and of mechanisms that could be involved in this dualistic mode of action. One mechanism relates to the different ultimate cellular effects of activation of estrogen receptor (ER) α, promoting cell proliferation, and of ERβ, promoting apoptosis, with the major soy isoflavones genistein and daidzein activating especially ERβ. A second mode of action includes the role of epigenetics, including effects of isoflavones on DNA methylation, histone modification and miRNA expression patterns. The overview presented reveals that we are only at the start of unraveling the complex underlying mode of action for effects of isoflavones, both beneficial or adverse, on cell proliferation and cancer risks. It is evident that whatever model system will be applied, its relevance to human tissues with respect to ERα and ERβ levels, co-repressor and co-activator characteristics as well as its relevance to human exposure regimens, needs to be considered and defined.