In-vivo and in-silico toxicity studies of daidzein: an isoflavone from soy.

Daidzein is a naturally occurring compound belonging to the class isoflavones and found in soya beans and other legumes. Acute oral toxicity was performed as per OECD guideline (TG 423) with slight modifications. A repeated dose toxicity study was carried out as per OECD guideline (TG 407). In-silico toxicity such as AMES toxicity, carcinogenicity, mutagenicity, immunotoxicity, hepatotoxicity, skin irritation, reproductive effect, rat and mouse toxicity, LD50, hERG I, II inhibitor and minnow toxicity were predicted using online servers and tools. In an acute oral toxicity study, daidzein did not show any mortality in experimental animals. The No Observed Adverse Effect Level (NOAEL) of daidzein was found to be above 5000 mg/kg. 28 days treatment of diadzein at all doses did not show changes in hematology parameters, clinical biochemistry and kidney function parameters. Gross necropsy or histopathology of important organs showed no signs of toxicity. In-silico predicted parameters also demonstrated risks ranging from low to a nontoxic level. Thus, daidzein was found to be safe in acute and repeated oral dose toxicity studies at all selected doses. In-silico study also indicated that daidzein is safe.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization.

ETHNOPHARMACOLOGICAL RELEVANCE: Glabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. AIM OF THE STUDY: The study identified the toxic impurities of glabridin and their toxicological mechanism. MATERIALS AND METHODS: In total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. RESULTS: Although glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 µM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. CONCLUSION: Glabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.

Dihydroisocoumarins and Dihydroisoflavones from the Rhizomes of Dioscorea collettii with Cytotoxic Activity and Structural Revision of 2,2'-Oxybis(1,4-di-tert-butylbenzene).

The investigation of the constituents of the rhizomes of Dioscorea collettii afforded one new dihydroisocoumarin, named (-)-montroumarin (1a), along with five known compounds-montroumarin (1b), 1,1'-oxybis(2,4-di-tert-butylbenzene) (2), (3R)-3'-O-methylviolanone (3a), (3S)-3'-O-methylviolanone (3b), and (RS)-sativanone (4). Their structures were elucidated using extensive spectroscopic methods. To the best of our knowledge, compound 1a is a new enantiomer of compound 1b. The NMR data of compound 2 had been reported but its structure was erroneous. The structure of compound 2 was revised on the basis of a reinterpretation of its NMR data (1D and 2D) and the assignment of the 1H and 13C NMR data was given rightly for the first time. Compounds 3a-4, three dihydroisoflavones, were reported from the Dioscoreaceae family for the first time. The cytotoxic activities of all the compounds were tested against the NCI-H460 cell line. Two dihydroisocoumarins, compounds 1a and 1b, displayed moderate cytotoxic activities, while the other compounds showed no cytotoxicity.

Puerarin suppresses MPP+/MPTP-induced oxidative stress through an Nrf2-dependent mechanism.

In this study, we hypothesized that anti-parkinsonian effect of puerarin is attributable to its antioxidant properties via Nrf2-dependent glutathione (GSH) biosynthesis mechanism. Experimentally, we found that puerarin attenuated 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress through elevating biosynthetic capacity of GSH in PC12 cells. Mechanistically, puerarin suppressed Fyn phosphorylation by GSK-3ß-dependent mechanism in MPP+-challenged PC12 cells. Furthermore, puerarin induced accumulation of Nrf2 in the nucleus via inhibiting its nuclear exclusion. In parallel, puerarin up-regulated antioxidant response element (ARE)-driven catalytic subunits from glutamate cysteine ligase (GCLc) expression at levels of transcription and translation. Most interestingly, pharmacological inhibitor of GSK-3ß or Fyn shRNA blocked puerarin-induced Nrf2 activation in MPP+-challenged PC12 cells. Concomitantly, puerarin ameliorated motor deficits and inhibited oxidative stress in the ventral midbrain in MPTP-intoxicated wild-type (WT) mice, but failed to attenuate MPTP neurotoxicity and up-regulate GCLc gene in Nrf2-knockout (Nrf2-/-) mice, suggesting that anti-parkinsonian effect of puerarin was dependent on Nrf2. Additionally, puerarin regulated Fyn and GSK-3ß phosphorylation in the ventral midbrain in MPTP-intoxicated WT mice. Collectively, the results of the study provide molecular insights into the potential therapeutic action of puerarin in Parkinson's disease, suggesting that puerarin may be a promising candidate for the treatment of Parkinson's disease.

Lupiwighteone induces caspase-dependent and -independent apoptosis on human breast cancer cells via inhibiting PI3K/Akt/mTOR pathway.

Breast cancer is one of the most common causes of mortality in women. Lupiwighteone has anticancer effects in prostate cancer cells and neuroblastoma cells. However, the molecular and cellular mechanisms of lupiwighteone effects on human breast cancer cells are not as well known. In the present study, we investigated the effects of lupiwighteone on the proliferation and apoptosis of two different human cancer cells; MCF-7, an estrogen receptor (ER)-positive human breast cancer cell, and MDA-MB-231, a triple negative human breast cancer cell. Lupiwighteone treatment decreased the viability of MCF-7 and MDA-MB-231 cells. Lupiwighteone treatment resulted in apoptotic cell death in breast cancer cells, which was characterized by DNA fragmentation, accumulation of apoptotic cells, and nuclear condensation. We also showed that treatment with lupiwighteone induced caspase-dependent apoptosis (up-regulation of caspase-3, -7, -8, -9, PARP, and Bax or down-regulation of Bid, Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), and inhibition of the PI3K/Akt/mTOR signaling pathway (down-regulation of PI3K, p-Akt, and p-mTOR) in both MCF-7 and MDA-MB-231 cells. These results suggest that lupiwighteone induces caspase-dependent and -independent apoptosis in both breast cancer cell lines via inhibiting PI3K/Akt/mTOR pathway.

In Vitro Metabolism of Auriculasin and Its Inhibitory Effects on Human Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

Auriculasin has a wide range of pharmacological effects, including anticancer and anti-inflammatory effects. In this work, we explored the metabolic characteristics and inhibitory effect of auriculasin against cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes in vitro. Auriculasin inhibited UGT1A6, UGT1A8, UGT1A10, UGT2B7, CYP2C9, and CYP3A4 strongly at a concentration of 100 µM. Different species showed significant differences in auriculasin metabolism, and metabolic characteristics were similar between pig and human. We identified seven metabolites, and hydroxylated auriculasin was the main metabolite. In addition, CYP2D6, CYP2C9, CYP2C19, and CYP2C8 were the major CYP isoforms involved in the metabolism of auriculasin. Molecular docking studies showed that noncovalent interactions between auriculasin and the CYPs are dominated by hydrogen bonding, π-π stacking, and hydrophobic interactions. Our in vitro study provides insights into the pharmacological and toxicological mechanisms of auriculasin.

Effects of the isoflavone daidzein in Senegalese sole, Solea senegalensis: Modulation of the oestrogen receptor-ß, apoptosis and enzymatic signalling pathways.

Phytochemicals are widely present in the aquatic environment and they are derived from many anthropogenic activities. The isoflavone daidzein is a natural compound that is found in the soya products used as habitual constituents of aquafeeds. Nevertheless, this isoflavone possesses oestrogenic and apoptotic properties. The present study determined the effects of daidzein (at 20 mg/L) during the first month and a half of life (from 7 to 44 days post-hatching -dph-) of the flatfish Senegalese sole, Solea senegalensis, focusing at the metamorphosis. We have analysed different gene expression levels and immunohistochemical protein patterns implicated in some oestrogenic, apoptosis and enzymatic pathways. In general, the oestrogen receptor (ERß) and stimulating apoptosis death receptor factor (Fas) transcript levels showed similar baseline patterns and transcriptional responses induced by daidzein. Both ERß and Fas were up-regulated by this isoflavone at the pre-metamorphosis and metamorphosis, and they were down-regulated in post-metamorphosed stages. The expression pattern of the apoptotic effector caspase (Casp6) was exclusively up-regulated at the pre-metamorphic phase. The Birc5 transcripts (i.e. anti-apoptosis, Survivin) were down-regulated by daidzein during certain metamorphic and post-metamorphosed stages. Besides, daidzein showed an up-regulating effect on both enzymatic complexes, the haemoprotein CYP1A and the acetylcholinesterase (AChE), except for a temporary AChE down-regulation in some post-metamorphosed stages. Immunostaining analysis only showed increased CYP1A signals in the liver of daidzein exposed fish. Overall, a majority of the transcriptional oestrogenic and apoptotic imbalances could be gradually and/or temporarily stabilised. Most controls and exposed larvae (70-80%) developed and grew following normal ontogenetic developmental patterns.

Polygonatone H, a new homoisoflavanone with cytotoxicity from Polygonatum Cyrtonema Hua.

A new homoisoflavonoid, (3R)-5,7-dihydroxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)-chroman-4-one (1), namely polygonatone H, in addition to fourteen known homoisoflavones (2-15) were isolated from the rhizome of Polygonatum Cyrtonema Hua. The structures were identified with the aid of 1D/2D NMR spectroscopic technologies. Compounds 2, 6, 8, 10, 11, 13, and 15 were isolated from P. Cyrtonema for the first time. Compound 1 showed cytotoxicities to human cancer cell lines with IC50 values to comparable those of cisplatin.

Tectorigenin Promotes Osteoblast Differentiation and in vivo Bone Healing, but Suppresses Osteoclast Differentiation and in vivo Bone Resorption.

Although tectorigenin (TG), a major compound in the rhizome of Belamcanda chinensis, is conventionally used for the treatment of inflammatory diseases, its effects on osteogenesis and osteoclastogenesis have not been reported. The objective of this study was to investigate the effects and possible underlying mechanism of TG on in vitro osteoblastic differentiation and in vivo bone formation, as well as in vitro osteoclast differentiation and in vivo bone resorption. TG promoted the osteogenic differentiation of primary osteoblasts and periodontal ligament cells. Moreover, TG upregulated the expression of the BMP2, BMP4, and Smad-4 genes, and enhanced the expression of Runx2 and Osterix. In vivo studies involving mouse calvarial bone defects with µCT and histologic analysis revealed that TG significantly increased new bone formation. Furthermore, TG treatment inhibited osteoclast differentiation and the mRNA levels of osteoclast markers. In vivo studies of mice demonstrated that TG caused the marked attenuation of bone resorption. These results collectively demonstrated that TG stimulated osteogenic differentiation in vitro, increased in vivo bone regeneration, inhibited osteoclast differentiation in vitro, and suppressed inflammatory bone loss in vivo. These novel findings suggest that TG may be useful for bone regeneration and treatment of bone diseases.

Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-ß/smad signaling.

Oral submucous fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis, and it causes chronic inflammation and persistent activation of myofibroblasts. As yet, existing treatments only provide temporary symptomatic relief and there is a lack of an effective intervention to cure OSF. Therefore, development of approaches to ameliorate myofibroblast activities becomes a crucial objective to prevent the malignant progression of OSF. In this study, we examined the inhibitory effect of glabridin, an isoflavane extracted from licorice root, on the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results showed that myofibroblast activities, including collagen gel contractility, migration, invasion and wound healing abilities were reduced after exposure of glabridin in a dose-dependent manner. Most importantly, we demonstrated that the arecoline-induced myofiroblast activities were abolished by glabridin treatment. Additionally, the expression of the myofibroblast marker α-smooth muscle actin and other fibrogenic marker, type I collagen, in fBMFs were dose-dependently downregulated. Moreover, we showed that the production of TGF-ß was suppressed by glabridin in fBMFs and the protein expression of phospho-Smad2 was decreased as well. In summary, our data suggested that glabridin repressed the myofibroblast features in fBMFs via TGF-ß/Smad2 signaling pathway. Glabridin also prevented the arecoline-increased myofibroblast activities, and could serve as a natural anti-fibrosis compound for OSF.

Calycosin Inhibits the Migration and Invasion of Human Breast Cancer Cells by Down-Regulation of Foxp3 Expression.

BACKGROUND/AIMS: Calycosin, a phytoestrogenic compound, has recently emerged as a promising antitumor drug. It has been shown that calycosin suppresses growth and induces apoptosis of breast cancer cells. However, the effect of calycosin on migration and invasion of breast cancer cells and the underlying molecular mechanisms have not been elucidated. METHODS: Human breast cancer cells MCF-7 and T47D were treated with, or without, different doses (0, 6.25, 12.5, 25, 50, 100 or 150 µM) of calycosin, and the viability of different groups was determined by MTT assay. Next, the inhibitory effect of higher doses (50, 100 or 150 µM) of calycosin on migration and invasion of the two cell lines was determined by wound healing and transwell assay. The relative expression levels of forkhead box P3 (Foxp3), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in MCF-7 and T47D cells were determined by quantitative RT-PCR and Western blot. RESULTS: Treatment with lower doses (6.25 or 12.5 µM) promoted proliferation of breast cancer cells, but with higher doses significantly reduced the viability of MCF-7 and T47D cells. Furthermore, higher doses of calycosin were found to inhibit migration and invasion of the two cell lines in a dose-dependent manner. Additionally, treatment with a higher dose of calycosin significantly reduced the expression levels of Foxp3, followed by down-regulation of VEGF and MMP-9 in both MCF-7 and T47D breast cancer cells. CONCLUSION: Treatment with a higher dose of calycosin tends to reduce migration and invasion capacity of human breast cancer cells, by targeting Foxp3-mediated VEGF and MMP-9 expression.

Synthesis of isoflavene-thiosemicarbazone hybrids and evaluation of their anti-tumor activity.

Phenoxodiol is an isoflavene with potent anti-tumor activity. In this study, a series of novel mono- and di-substituted phenoxodiol-thiosemicarbazone hybrids were synthesized via the condensation reaction between phenoxodiol with thiosemicarbazides. The in vitro anti-proliferative activities of the hybrids were evaluated against the neuroblastoma SKN-BE(2)C, the triple negative breast cancer MDA-MB-231, and the glioblastoma U87 cancer cell lines. The mono-substituted hybrids exhibited potent anti-proliferative activity against all three cancer cell lines, while the di-substituted hybrids were less active. Selected mono-substituted hybrids were further investigated for their cytotoxicity against normal MRC-5 human lung fibroblast cells, which identified two hybrids with superior selectivity for cancer cells over normal cells as compared to phenoxodiol. This suggests that mono-substituted phenoxodiol-thiosemicarbazone hybrids have promising potential for further development as anti-cancer agents.

Soy isoflavone exposure through all life stages accelerates 17ß-estradiol-induced mammary tumor onset and growth, yet reduces tumor burden, in ACI rats.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17ß-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

Bioassay-Guided Isolation of Two Flavonoids from Derris scandens with Topoisomerase II Poison Activity.

Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4'-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment.

Prenatal Exposure to Soy Isoflavones Altered the Immunological Parameters in Female Rats.

Information on the effects of phytoestrogens on animals has increased recently; however, there were only few studies on prenatal exposure on cellular immune response. Pregnant rats were assigned to 3 groups (12 rats per group), the first was fed control diet, the second was fed low-dose (6.5 g/100 g of diet) soy isoflavones, while the third was fed high-dose (26 g/100 g of diet) soy isoflavones. The female offspring cell-mediated immune response was determined using phytohemagglutinin (PHA) injection, and intumesce index was calculated on postnatal day 50. After 24 hours of PHA injection, blood samples were collected for tumor necrosis factor α, interferon γ (IFN-γ), and interleukin (IL)-12 determination. Spleen, thymus, and PHA-injected footpads were fixed for histopathology. Intumesce index was significantly (P < 0.05) reduced in rats' offspring born from dams fed low- and high-dietary soy isoflavones than that in control groups. Thymic relative weights in offspring of rats fed high-dietary soy isoflavones showed a significant (P < 0.05) decrease compared to that in the control group. Female offspring where low and high-dietary soy isoflavones were fed to their dams showed a significant (P < 0.05) decrease in IFN-γ and IL-12 than that in control ones. Spleen of rats born from dams fed high dose of dietary soy isoflavones showed lymphocytic depletion in white pulp. Taking together, it is clear that dietary soy isoflavones at prenatal period had immunosuppressive effect on female offspring after PHA stimulation. This effect was mediated through reduced IFN-γ that interplayed in IL-12 production pathway thus reducing its level.

Evaluation of kudzu root extract-induced hepatotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Kudzu root, the root of Pueraria lobata (Willd.) Ohwi, has been used as food and medicine for centuries, but few studies indicate that kudzu root may cause liver damage. AIM OF STUDY: We studied the hepatotoxicity of kudzu root extract in mice, HepG2 cells and mice hepatocytes. MATERIALS AND METHODS: Mice were administrated with kudzu root extract (10mg/day) for 4 weeks, and then the biochemical analysis and histopathological changes were carried out. To explore the potential mechanism by which kudzu root extract-induced hepatotoxicity, HepG2 cells and mice hepatocytes were co-cultured with kudzu root extract or puerarin, which is a kudzu root isoflavone, for 2h. RESULTS: The increase of serum ALT and AST and histopathological changes in treated mice revealed that kudzu root extract was hepatotoxic. The increase of LDH leakage for HepG2 cells and mice hepatocytes further confirmed hepatotoxicity of kudzu root extract. Kudzu root extract and puerarin significantly up-regulated Mt1 mRNA involved in the acute phase response and Bax which is crucial for apoptosis. Gclc, Nrf2 and Ho-1 mRNA expressions did not change in treatment group. CONCLUSIONS: Kudzu root extract may be hepatotoxic and caution may be required for its use.

Synthesis, biological evaluation and structure-activity relationship studies of isoflavene based Mannich bases with potent anti-cancer activity.

Phenoxodiol, an analogue of the isoflavone natural product daidzein, is a potent anti-cancer agent that has been investigated for the treatment of hormone dependent cancers. This molecular scaffold was reacted with different primary amines and secondary amines under different Mannich conditions to yield either benzoxazine or aminomethyl substituted analogues. These processes enabled the generation of a diverse range of analogues that were required for structure-activity relationship (SAR) studies. The resulting Mannich bases exhibited prominent anti-proliferative effects against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines. Further cytotoxicity studies against MRC-5 normal lung fibroblast cells showed that the isoflavene analogues were selective towards cancer cells.

Prenatal exposure to the phytoestrogen daidzein resulted in persistent changes in ovarian surface epithelial cell height, folliculogenesis, and estrus phase length in adult Sprague-Dawley rat offspring.

Daidzein (DZ), an isoflavone with the potential to interfere with estrogen signaling, is found in soy products, which have gained popularity due to purported beneficial effects on the cardiovascular and skeletal systems and potential antineoplastic properties. However, the ingestion of phytoestrogens has been associated with impaired reproductive function in many species. The aim of this study was to determine the long-term effects on the ovaries of rat offspring exposed to DZ or ethinyl estradiol (EE) during prenatal development. Gravid rats were administered either vehicle or 5 or 60 mg DZ/kg body weight/d or 0.002 mg 17-α EE /kg body weight/d on gestational days 6-21. Ovarian-related endpoints were investigated during adulthood in female offspring. The mean cell height of the ovarian surface epithelium was significantly reduced in all treated groups. Alterations in folliculogenesis included increased follicular atresia, a reduction in secondary and tertiary follicle numbers, and cyst formation. An elevated prevalence of a slightly prolonged estrus phase was also observed. The morphological changes to the ovarian surface epithelium are consistent with an antiproliferative effect, while ovarian folliculogenesis was adversely affected. The effects of the high dose DZ were similar to those observed with 17-α EE.

Formononetin inhibits migration and invasion of MDA-MB-231 and 4T1 breast cancer cells by suppressing MMP-2 and MMP-9 through PI3K/AKT signaling pathways.

Formononetin is a naturally existing isoflavone, which can be found in the roots of Astragalus membranaceus, Trifolium pratense, Glycyrrhiza glabra, and Pueraria lobata. It was found to be associated with inhibition of cell proliferation and cell cycle progression, as well as induction of apoptosis in various cancer cell lines. However, the effect of formononetin on breast cancer cell metastasis remains unclear. In this study, we examined the effect of formononetin on the migration and invasion of breast cancer cells MDA-MB-231 and 4T1 in vitro and in vivo. Our data demonstrated that formononetin did not effectively inhibit the cell viability of MDA-MB-231 and 4T1 in 24 h with the concentration lower than 160 µmol/l. When treated with nontoxic concentration of formononetin, the migration and invasion of MDA-MB-231 and 4T1 cells were markedly suppressed by wound healing assay, chamber invasion assay, and in vivo mouse metastasis model. In vitro, formononetin reduced the expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Furthermore, the immunofluorescence and immunoblotting assays indicated that formononetin was very effective in suppressing the phosphorylation of Akt and PI3K. Collectively, these results suggest that formononetin inhibited breast cancer cell migration and invasion by reducing the expression of MMP-2 and MMP-9 through the PI3K/AKT signaling pathway. These findings demonstrate a potentially new therapeutic strategy of formononetin as anti-invasive agent for breast cancer.

Coexposure to phytoestrogens and bisphenol a mimics estrogenic effects in an additive manner.

Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors (ER), ERα and ERß, and may contribute to estrogen-linked diseases such as breast cancer. Early exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of, eg, BPA. In addition, infants are frequently fed soy-based formula (SF) that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein, and an extract of infant SF mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERß reporter cells; on proliferation, genome-wide gene regulation and non-ER-mediated effects in MCF7 breast cancer cells; and how coexposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that coexposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling, we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17ß-estradiol, and are abolished by cotreatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that coexposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.