Addition Of Genistein Research Articles

Genistein abrogates G2 arrest induced by curcumin in p53 deficient T47D cells

BackgroundThe high cost and low level of cancer survival urge the finding of new drugs having better mechanisms. There is a high trend of patients to be “back to nature” and use natural products as an alternative way to cure cancer. The fact is that some of available anticancer drugs are originated from plants, such as taxane, vincristine, vinblastine, pacitaxel. Curcumin (diferuloylmethane), a dietary pigment present in Curcuma longa rizhome is reported to induce cell cycle arrest in some cell lines. Other study reported that genistein isolated from Glycine max seed inhibited phosphorylation of cdk1, gene involved during G2/M transition and thus could function as G2 checkpoint abrogator. The inhibition of cdk1 phosphorylation is one of alternative strategy which could selectively kill cancer cells and potentially be combined with DNA damaging agent such as curcumin.MethodsT47D cell line was treated with different concentrations of curcumin and genistein, alone or in combination; added together or with interval time. Flow Cytometry and MTT assay were used to evaluate cell cycle distribution and viability, respectively. The presence of apoptotic cells was determined using acridine orange-ethidium bromide staining.ResultsIn this study curcumin induced G2 arrest on p53 deficient T47D cells at the concentration of 10 μM. Increasing concentration up to 30 μM increased the number of cell death. Whilst genistein alone at low concentration (≤10 μM) induced cell proliferation, addition of genistein (20 μM) 16 h after curcumin resulted in more cell death (89%), 34% higher than that administered at the same time (56%). The combination treatment resulted in apoptotic cell death. Combining curcumin with high dose of genistein (50 μM) induced necrotic cells.ConclusionsGenistein increased the death of curcumin treated T47D cells. Appropriate timing of administration and concentration of genistein determine the outcome of treatment and this method could potentially be developed as an alternative strategy for treatment of p53 defective cancer cells.

Characterization of the Flavonoid-Responsive Regulator FrrA and Its Binding Sites

Previous microarray analyses revealed that in Bradyrhizobium japonicum, about 100 genes are induced by genistein, an isoflavonoid secreted by soybean. This includes the three genes freC, freA, and freB (systematic designations bll4319, bll4320, and bll4321), which are likely to form a genistein-, daidzein-, and coumestrol-inducible operon and to encode a multidrug efflux system. Upstream of freCAB and in the opposite orientation, FrrA (systematic designation Blr4322), which has similarity to TetR-type regulators, is encoded. A deletion of frrA leads to increased expression of freB in the absence of an inducer. We identified the correct translational start codon of frrA and showed that the gene is inducible by genistein and daidzein. The protein, which was heterologously expressed and purified from Escherichia coli, binds to two palindrome-like DNA elements (operator A and operator B), which are located in the intergenic region between freC and frrA. The replacement of several nucleotides or the insertion of additional spacer nucleotides prevented binding. Binding of FrrA was also affected by the addition of genistein. By mapping the transcription start sites, we found that operator A covers the transcriptional start site of freC and operator B is probably located between the -35 regions of the two divergently oriented genes. Operator A seems to be conserved in a few similar gene constellations in other proteobacteria. Our data indicate that in B. japonicum, besides NodD1 (the LysR family) and NodVW (a two-component response regulator), a third regulator type (a TetR family member) which responds to the plant signal molecules genistein and daidzein exists.

Structure and Elasticity of Lipid Membranes with Genistein and Daidzein Bioflavinoids Using X-ray Scattering and MD Simulations

This work reports the effects of the bioflavinoids genistein and daidzein on lipid bilayers as determined by volume measurements, X-ray scattering, and molecular dynamics simulations. The experimental and simulated total molecular volumes were found to be in outstanding agreement with each other before the addition of genistein and daidzein and also after their addition. Both bioflavinoids inserted into the hydrocarbon region of both DOPC and diphytanoylPC near the carbonyls of the lipids and both decreased the bilayer thicknesses. The long axes of both bioflavinoids were oriented nearly parallel to the plane of the bilayer with their carbonyl groups preferentially pointed toward the proximal surface. A difference is that daidzein had a solubility limit of ∼0.14 mol fraction in DOPC (∼0.12 mol fraction in diphytanoylPC), whereas genistein was soluble at least to 0.20 mol fraction in both lipid membranes. Measurements of bending modulus K(C) and simulation results for area compressibility modulus K(A) indicate that both bioflavinoids soften bilayers.

Influence of aggregation on the antioxidative capacity of milk peptides

This study investigated the effects of pH and addition of genistein prior to freeze-drying on the aggregation of reconstituted antioxidative milk peptides. The peptides having molecular masses below 6.5kDa aggregated into higher mass aggregates of 16.9kDa after tryptic hydrolysis for 30min. Subsequent freeze-drying at pH 2.0 further promoted the aggregation of peptides to aggregates of 26.6kDa, both in the absence and presence of genistein. The reconstituted peptides clustered into macroaggregates of 6μm, having oxygen radical absorbance capacity (ORACFL) between 0.07 and 0.22μmol Trolox equivalent (TE)mg−1 protein. Although freeze-drying at pH 6.5 promoted the aggregation of peptides, the reconstituted peptides retained a size of 150–170nm and had an ORACFL of 0.59–0.88μmol TEmg−1 protein. The addition of genistein synergistically increased Trolox equivalent antioxidant capacity, compared with the reconstituted milk peptide alone (P<0.05).

Morphological and physiological response of soybean treated with the microsymbiont Bradyrhizobium japonicum pre-incubated with genistein

Genistein, a major root-secreted isoflavone of soybean (Glycine max (L.) Merr), is critical for the legume-Bradyrhizobium symbiosis as it induces several bacterial nod-gene systems. An experiment with soybean grown under salt stress was conducted to evaluate the effect of exogenous genistein addition to the Bradyrhizobium culture medium on subsequent nodulation, nitrogen fixation and selected plant physiological attributes. Five day-old plants (in pots) were inoculated with a liquid B. japonicum broth culture and irrigated with B&D solution containing either 0, 25, 50 and 100mM NaCl. Four weeks after inoculation, maximum photochemical efficiency of PSII (Fv/Fm), photosynthetic rate, stomatal conductance, and transpiration rate were measured. Number of nodules per plant and apparent nitrogen fixation (as acetylene reduction activity) were determined. Salt stress decreased nodule number/plant and nitrogenase activity/plant and induced large changes of both photosynthetic parameters and antioxidant enzyme activity, compared to the control, genistein reversed the effect in each level of salinity tested. Moreover, pre-treatment of the microsymbiont with genistein enhanced maximum photochemical efficiency, photosynthetic rate, stomatal conductance and transpiration rate, while the enzymatic activities of catalase, superoxide dismutase and peroxidase in leaves and roots were not affected. It can be concluded that preincubation of the B. japonicum inoculant with genistein probably contributed towards growth in soybean via enhancement of nodulation and nitrogen fixation under both normal and salt stress conditions.

In this issue

AbstractAssociations meet on a rarely traveled/trampled road: Corner of Yellow Brick Road and Appian WayNo room number or hotel specified. Ising required. This is not cake frosting in a can. So, what is the Ising model? Originally, a mathematical model devised to study magnetic fields, it was soon recognized as a powerful model for studies of inheritance. Unlike on/off models of genes, Ising genes are two‐state: +1 or −1 and describe the spin states of a magnetic dipole or the state of an allele that might or might not be shared by siblings. I'll save you the 14 equations that Li et al. apply to the model – they are not impenetrable. Working with an Affymetrix data set, 286 patients were lymph node negative to start. A Markov Random Field model identified 107 Differentially Expressed (DE) genes and the Ising model found 125. These included a number of previously unknown and unclassified networks and subnetworks.pp. 3845–3852Taxol, you've been a wonderful drugBut you've let me down, lately. Breast cancer is still the no. 1 killer of women. Prognosis is poor due to late tumor detection or rapid drug resistance. Some multiple drug combinations fail too frequently but Taxanes in combination with anthracyclines or Herceptin give some improvement, depending on the genetics of the patient (“personalized medicine”). The mechanism of action of the taxane resistance involves interactions with microtubules including β‐tubulin mutations, insufficient cyclin‐dependent kinase I, modulation of ERB‐B2, p53, or Bcl‐2. On the other hand, protein kinase phosphatase MKP‐1 has been nominated to mediate chemoresistance by inhibiting c‐Jun N kinase‐dependent apoptosis. Choices, choices, choices....pp. 3877–3886His and hers: Cardiovascular eventsSex‐change operations do not help: cardiovascular disease is the leading cause of morbidity and mortality among both men and women if you adjust for age and menopause, inferring that both androgens and estrogens are contributors to the disease. Estrogen replacement has proven to have some protective value but results appear to be conflicting in human trials. Phytoestrogens are a group of potential candidates derived from plant materials. A particularly interesting compound is genistein (as long as the experimenter controls for variable levels in common lab chow). Schwab et al. took environmental age, sex, and heart condition into consideration in the proteomic study reported here. Addition of genistein as an oral dietary estrogen supplement had effects on both male and female mice at the levels of transcription and translation. Mitochondrial enzymes were affected, including fatty‐acid metabolism, oxidative phosphorylation, and cytoskeletal components. Clearly obscure...pp. 3887–3904

Cover Picture: Proteomics 19'11

AbstractAssociations meet on a rarely traveled/trampled road: Corner of Yellow Brick Road and Appian WayNo room number or hotel specified. Ising required. This is not cake frosting in a can. So, what is the Ising model? Originally, a mathematical model devised to study magnetic fields, it was soon recognized as a powerful model for studies of inheritance. Unlike on/off models of genes, Ising genes are two‐state: +1 or −1 and describe the spin states of a magnetic dipole or the state of an allele that might or might not be shared by siblings. I'll save you the 14 equations that Li et al. apply to the model – they are not impenetrable. Working with an Affymetrix data set, 286 patients were lymph node negative to start. A Markov Random Field model identified 107 Differentially Expressed (DE) genes and the Ising model found 125. These included a number of previously unknown and unclassified networks and subnetworks.Li, X. et al., Proteomics 2011, 11, 3845–3852.Taxol, you've been a wonderful drugBut you've let me down, lately. Breast cancer is still the no. 1 killer of women. Prognosis is poor due to late tumor detection or rapid drug resistance. Some multiple drug combinations fail too frequently but Taxanes in combination with anthracyclines or Herceptin give some improvement, depending on the genetics of the patient (“personalized medicine”). The mechanism of action of the taxane resistance involves interactions with microtubules including β‐tubulin mutations, insufficient cyclin‐dependent kinase I, modulation of ERB‐B2, p53, or Bcl‐2. On the other hand, protein kinase phosphatase MKP‐1 has been nominated to mediate chemoresistance by inhibiting c‐Jun N kinase‐dependent apoptosis. Choices, choices, choices....Froidevaux‐Klipfel, L. et al., Proteomics 2011, 11, 3877–3886.His and hers: Cardiovascular eventsSex‐change operations do not help: cardiovascular disease is the leading cause of morbidity and mortality among both men and women if you adjust for age and menopause, inferring that both androgens and estrogens are contributors to the disease. Estrogen replacement has proven to have some protective value but results appear to be conflicting in human trials. Phytoestrogens are a group of potential candidates derived from plant materials. A particularly interesting compound is genistein (as long as the experimenter controls for variable levels in common lab chow). Schwab et al. took environmental age, sex, and heart condition into consideration in the proteomic study reported here. Addition of genistein as an oral dietary estrogen supplement had effects on both male and female mice at the levels of transcription and translation. Mitochondrial enzymes were affected, including fatty‐acid metabolism, oxidative phosphorylation, and cytoskeletal components. Clearly obscure... Schwab, K. et al., Proteomics 2011, 11, 3887–3904.

Effects of genistein modification on miscibility and hydrogen bonding interactions in poly(amide)/poly(vinyl pyrrolidone) blends and membrane morphology development during coagulation

Miscibility characteristics of poly(amide):poly(vinyl pyrrolidone) (PA:PVP) blends containing a soybean-derived phytochemical called “genistein” have been investigated using differential scanning calorimetry (DSC) and polarized optical microscopy (POM). The occurrence of hydrogen bonding in the binary PA/genistein (PA/G) and PVP/genistein (PVP/G) pairs as well as their ternary blends has been confirmed by Fourier transformed infrared spectroscopy (FTIR). On the basis of DSC and POM data, the morphology phase diagram of PA:PVP/G blends is mapped out, which consisted of various coexistence regions such as isotropic, liquid + liquid, liquid + crystal, liquid + liquid + crystal, and solid crystal regions. Subsequently, PA:PVP membranes modified with genistein were prepared by coagulation via solvent (dimethyl sulfoxide, DMSO) and non-solvent (water) exchange. Addition of genistein reduced the miscibility gap of the PA/DMSO/water system. The actual amounts of genistein in the final membranes have been quantified as a function of the genistein in feed. Of particular interest is the development of the gradient cross-sectional porous channels, showing the progressively larger diameters from the surface to the bottom substrate with the progression of solvent/non-solvent exchange or solvent power. Scanning electron microscopy (SEM) investigation of the morphologies of the modified membranes revealed that genistein crystals were embedded on the membrane surface as well as in the cross-section even at a very low feed concentration of genistein. A schematic of a coagulation pathway was inscribed inside a prism phase diagram in order to comprehensively illustrate the formation of genistein modified PA:PVP membranes through the solvent/non-solvent exchange process followed by drying.

Transplacental Transfer and Biotransformation of Genistein in Human Placenta

ObjectiveTo study transplacental transfer and biotransformation of genistein in the human placenta. Study design and outcomesHuman placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180min following addition of genistein (10ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC–MS). ResultsThe transfer percentage for antipyrine and genistein was 25.6±1.40% and 22.1±1.61% respectively and the transfer index for genistein was 0.90±0.04 after 180min of perfusion. 12.0±2.40% of genistein in the fetal compartment and 7.36±4.73% of genistein in the maternal compartment were in the conjugated form. ConclusionsGenistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action.

Collagen‐derived dipeptide, proline‐hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fibroblasts

Orally ingested collagen undergoes degradation to small di- or tripeptides, which are detected in circulating blood 2 h after ingestion. The influence of collagen-derived peptides on dermal extracellular matrix components and cell proliferation was studied using cultured human dermal fibroblasts. Of the various collagenous peptides tested here, the dipeptide proline-hydroxyproline (Pro-Hyp) enhanced cell proliferation (1.5-fold) and hyaluronic acid synthesis (3.8-fold) at a dose of 200 nmol/mL. This was concomitant with a 2.3-fold elevation of hyaluronan synthase 2 (HAS2) mRNA levels. Small interfering RNA (siRNA)-mediated knockdown of the HAS2 gene in human dermal fibroblasts inhibited Pro-Hyp-induced HAS2 mRNA transcription and cell mitotic activity. Addition of genistein or H7, a protein kinase inhibitor, abolished the Pro-Hyp-induced HAS2 mRNA stimulation. Pro-Hyp elevated phosphorylation of signal transducer and activator of transcription 3 (STAT3) within a short time period (60 min). These results suggest that Pro-Hyp stimulates both cell mitotic activity and hyaluronic acid synthesis, which is mediated by activation of HAS2 transcription.

Cell Viability of Normal Human Skin Fibroblast and Fibroblasts Derived from Granulation Tissue: Effects of Nutraceuticals

The effects of lycopene, genistein, and epigallocatechin-3-gallate (EGCG) on cell viability were tested in vitro using a normal human skin fibroblast (NHSF) cell line (CRL-1474) and granulation tissue fibroblasts (GTFs) obtained from a patient with middle ear cholesteatoma. Cell cultures were added with lycopene (1, 5, and 10 microM), genistein (1, 5, 10, 25, and 50 microM), and EGCG (1, 5, 10, 25, and 50 microM) and their respective control cultures were established by adding 5 mL/L tetrahydrofuran (THF), 5 mL/L dimethyl sulfoxide (DMSO), and 5 mL/L DMSO. A colorimetric assay was employed for determining cell viability using thiazolyl blue tetrazolium bromide. Cell viability was expressed as a percentage of the control. Data were analyzed using two-way analysis of variance separately for each compound. Lycopene addition decreased viability of NHSFs and GTFs compared with THF addition (64.1%, 60.5%, and 100%, respectively, P < .0001). Genistein addition also increased viability of both NHSFs and GTFs compared with DMSO addition (P < .02). Increasing EGCG concentration tended to cause a linear increase in viability of NHSFs but did not alter viability of GTFs (P < .10). Our data suggest that genistein and EGCG but not lycopene could help maintaining or improving skin health through enhancing viability of skin fibroblasts.

Alleviating salt stress on soybean ( Glycine max (L.) Merr.) – Bradyrhizobium japonicum symbiosis, using signal molecule genistein

For the onset of symbiosis process between soybean ( Glycine max (L.) Merr.) and Bradyrhizobium japonicum, signals should be exchanged. Salinity has inhibitory effects on the symbiosis between the two partners. Hence, a greenhouse experiment was planned to: (1) determine the stressful effects of salinity on soybean and B. japonicum symbiosis, hypothesizing that they can inhibit the signal exchange process between the two partners, and (2) determine if the addition of genistein (a nod gene inducer) to B. japonicum (strain 532C) inocula could overcome the stressful effects of salinity on the Bradyrhizobium – soybean symbiosis. Three levels of salinity (control, 36 and 61 mmolar or 3.6 and 6.1 mmhos/cm) and three levels of genistein (0, 5 and 20 μM) were combined in a factorial fashion in four replicates. Soybean plants were harvested at three different times including 20, 40 and 60 days after inoculation (DAI). Genistein enhanced soybean nodulation and growth, and such effects became greater with time under high salinity levels. For example, at 60 DAI the enhancing effects of genistein on the symbiosis process in soybean was more pronounced at the highest level of salinity. The significant interaction effect between genistein 5 μM and salinity 61 mmolar may reveal the direct role of genistein 5 μM in overcoming the stressful effects of salinity on the symbiosis between B. japonicum and soybean, and hence, plant growth. This novel finding may be very useful to increase soybean yields in salty croplands.

Analysis of the secretome of the soybean symbiont Bradyrhizobium japonicum

Proteins from the supernatant of Bradyrhizobium japonicum were separated by two-dimensional gel electrophoresis and stained with Coomassie. This revealed more than 100 protein spots. Sixty-eight proteins were identified by mass spectrometry. Thirty-five are predicted to contain an N-terminal signal peptide characteristic for proteins transported by the general secretory pathway. Most of these appear to be substrate-binding proteins of the ABC transporter family. Ten proteins were categorized as unclassified conserved or hypothetical. None of the proteins has similarity to proteins transported by a type I secretion system or to autotransporters. Three of the proteins might be located in the outer membrane. The addition of genistein led to changes in the spot pattern of three flagellar proteins and resulted in the identification of the nodulation outer protein Pgl. Moreover, the application of shot-gun mass spectrometry resulted in the first-time identification of NopB, NopH and NopT, which were present only after genistein induction. Replacing genistein with daidzein or coumestrol reduced the amount of the type III-secreted protein GunA2.

A novel anti-cancer effect of genistein: reversal of epithelial mesenchymal transition in prostate cancer cells1

The aim of the present study was to investigate whether low dose genistein affects the invasion and epithelial mesenchymal transition (EMT) of prostate cancer (PCa) cells. Human PCa cell lines, IA8-ARCaP and LNCaP/ HIF-1a, were used in this study. The cell lines were found to process EMT in our previous study. The PCa cells were treated with increasing concentrations, from 0.1 to 75 micromol/L. Proliferation was assessed with 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. EMT was proven by cell morphological transition and the expression changes of EMT-related markers, which were confirmed by RT-PCR, Western blotting, and indirect immunofluorescence labeling. Transwell invasion assay was used to analyze the invasive potency. The addition of genistein to the medium reduced the IA8-ARCaP and LNCaP/HIF-1a viable cell number in a dose-dependent manner (with increasing concentrations from 15 to 75 micromol/L). Less than 15 micromol/L genistein was selected as the low dose concentration, which did not affect cell proliferation. The treatment of cells with low-dose genistein induced the reversal of EMT, which was confirmed by cell morphological transition and the expression changes of EMT-related markers. The reversal of EMT in the PCa cells by low-dose genistein was in a dose-dependent manner. Moreover, low-dose genistein effectively inhibited invasion of the PCa cells in vitro. These results showed that treatment with low-dose genistein may be a potential strategy for the suppression of invasive growth through the reversal of EMT in cancer cells, which justifies the potential use of soybean foods as a practical chemopreventive approach for patients with PCa.

Role of estrogen receptors and aromatase on brain protein synthesis rates in ovariectomized female rats fed genistein

We have reported that the dietary addition of genistein, a phytoestrogen found abundantly in soy products, stimulates brain protein synthesis rates of ovariectomized female rats. In the present study, we determine whether stimulation of brain protein synthesis rates in ovariectomized female rats by the dietary addition of genistein was conducted via estrogen receptors and aromatase-mediating actions. After ovariectomy, Wistar female rats were treated with genistein, the estrogen receptor antagonist ICI 182,780, and/or fadrozole a systemic aromatase inhibitor. In the cerebral cortex, the cerebellum and the hypothalamus, the fractional (Ks) rates of protein synthesis were increased by the dietary addition of genistein. These effects of genistein were inhibited by the administration of ICI 182,780 and fadrozole. However, the degrees to which ICI 182,780 and fadrozole inhibited the effects of genistein differed depending on the brain region. This result suggests that dietary genistein elevates the rate of protein synthesis in the brain of ovariectomized female rats. In addition, the estrogen receptors of the brain and the aromatase of the peripheral tissue and brain are, at least partly, related to the rate of brain protein synthesis caused by genistein.

Apoptotic activity of genistein on human lung adenocarcinoma SPC-A-1 cells and preliminary exploration of its mechanisms using microarray

Soy isoflavone genistein is active against certain solid malignancies, but its direct effect on lung adenocarcinoma and its mechanisms of action remain to be elucidated. In the present study, using the human lung adenocarcinoma cell line SPC-A-1, we found that genistein decreased SPC-A-1 cell viability in both a dose and time dependent manner. Flow cytometry analysis revealed that genistein significantly induced arrest of SPC-A-1 cells at the G2/M phase of the cell cycle. Furthermore, through DNA fragmentation and TUNEL assays, we demonstrated that the addition of genistein led to SPC-A-1 apoptosis in both a dose and time dependent manner. Finally, the apoptosis pathway-related gene expression profile affected by genistein was investigated using the oligonucleotide microarray method. The result showed that the expression profile of 20 genes (ratio of genistein group/control group >2 or <0.5) related to the apoptotic pathways changed. These genes, mainly consisting of the Bcl-2 family and TNF ligand and receptor family, are involved in regulation of the apoptosis process.

Overcoming the Stressful Effects of Salinity and Acidity on Soybean Nodulation and Yields Using Signal Molecule Genistein Under Field Conditions

ABSTRACT Soil stresses such as salinity and acidity may adversely affect nitrogen (N)2-fixation. The hypothesis of this study is that soil salinity and acidity inhibit soybean [Glycine max (L.) Merr.] nodulation and N2-fixation due to, at least in part, disruption of the signal exchange process. The objectives were: 1) to determine the effects of stressful soil salinity and acidity on the signal exchange processes between soybean and Bradyrhizobium japonicum, and 2) to determine whether or not the addition of signal molecule genistein to B. japonicum can overcome at least part of the inhibition of nodulation, caused by stressful soil salinity and acidity. Salt (sodium chloride) and sulfur (S) were applied. Genistein (0, 5, and 20μ M) was tested. Genistein addition could partially overcome the salt and acidity stresses by increasing soybean yields up to 21% and 23%, respectively. These novel findings may be very useful for planting soybean under salinity and acidity stresses.

Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca(2+). We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca(2+) influx through L- or T-type voltage-gated Ca(2+) channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetin-plus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca(2+) efflux by sodium orthovanadate, a Ca(2+)-pump inhibitor, blocked quercetin- plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.

Identification of genistein-inducible and type III-secreted proteins of Bradyrhizobium japonicum

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium-genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.

Phytoestrogens Induce Differential Estrogen Receptor Alpha- or Beta-Mediated Responses in Transfected Breast Cancer Cells

Increased intake of phytoestrogens may be associated with a lower risk of cancer in the breast and several other sites, although there is controversy surrounding this activity. One of the mechanisms proposed to explain the activity of phytoestrogens is their ability to bind and activate human estrogen receptor alpha (ERalpha) and human estrogen receptor beta (ERbeta). Nine phytoestrogens were tested for their ability to transactivate ERalpha or ERbeta at a range of doses. Mammary adenocarcinoma (MCF-7) cells were co-transfected with either ERalpha or ERbeta, and an estrogen-response element was linked to a luciferase reporter gene. Dose-dependent responses were compared with the endogenous ligand 17beta-estradiol. Purified genistein, daidzein, apigenin, and coumestrol showed differential and robust transactivation of ERalpha- and ERbeta-induced transcription, with an up to 100-fold stronger activation of ERbeta. Equol, naringenin, and kaempferol were weaker agonists. When activity was evaluated against a background of 0.5 nM 17beta-estradiol, the addition of genistein, daidzein, and resveratrol superstimulated the system, while kaempferol and quercetin were antagonists at the highest doses. This transfection assay provides an excellent model to evaluate the activation of ERalpha and ERbeta by different phytoestrogens in a breast cancer context and can be used as a screening bioassay tool to evaluate the estrogenic activity of extracts of herbs and foods.