High Concentrations Of Genistein Research Articles

The Effects of Genistein at Different Concentrations on MCF-7 Breast Cancer Cells and BJ Dermal Fibroblasts.

This study aimed to evaluate the safety and potential use of soy isoflavones in the treatment of skin problems, difficult-to-heal wounds and postoperative scars in women after the oncological treatment of breast cancer. The effects of different concentrations of genistein as a representative of soy isoflavonoids on MCF-7 tumor cells and BJ skin fibroblasts cultured in vitro were assessed. Genistein affects both healthy dermal BJ fibroblasts and cancerous MCF-7 cells. The effect of the tested isoflavonoid is closely related to its concentration. High concentrations of genistein destroy MCF-7 cancer cells, regardless of the exposure time, with a much greater effect on reducing cancer cell numbers at longer times (48 h). Lower concentrations of genistein (10 and 20 μM) increase the abundance of dermal fibroblasts. However, higher concentrations of genistein (50 μM and higher) are detrimental to fibroblasts at longer exposure times (48 h). Our studies indicate that although genistein shows high potential for use in the treatment of skin problems, wounds and surgical scars in women during and after breast cancer treatment, it is not completely safe. Introducing isoflavonoids to treatment requires further research into their mechanisms of action at the molecular level, taking into account genetic and immunological aspects. It is also necessary to conduct research in in vivo models, which will allow for eliminating adverse side effects of therapy.

High Concentrations of Genistein Decrease Cell Viability Depending on Oxidative Stress and Inflammation in Colon Cancer Cell Lines.

Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H2O2) production were studied. The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G2/M arrest, increased H2O2, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H2O2 production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.

Genistein inhibits chondrogenic differentiation and mineralization of ATDC5 cells

Isoflavones are phytoestrogens abundant in leguminous crops and are used to prevent a variety of hormonal disorders. In the present study, the effects of genistein and daidzein on the chondrogenic differentiation of ATDC5 cells were investigated. Genistein (10 μM) treatment markedly reduced production of sulfated proteoglycans and collagen fibers in the ATDC5 cells. Genistein suppressed the expression of genes involved in chondrocyte differentiation such as Sox9, Col2a1, Col10a1, Acan, and Tgfb1. Additionally, genistein significantly decreased calcium deposition in ATDC5 cells during chondrogenic differentiation; however, it increased calcification under non-chondrogenic mineralizing conditions. Daidzein exhibited a similar effect of suppressing chondrogenesis in ATDC5 cells, although its efficacy was 10-times lower than that of genistein. These findings suggest that a high concentration of genistein inhibits chondrogenesis and chondrogenic mineralization, whereas it enhances non-chondrogenic mineralization.

Inhibition of esophageal-carcinoma cell proliferation by genistein via suppression of JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways.

Esophageal carcinoma (EsC) is a clinically challenging neoplastic disease. Genistein, a natural isoflavone product, has anti-tumor properties. Through in vitro and in vivo studies, we found that genistein suppressed EsC cell proliferation in a time- and concentration-dependent manner. In addition, genistein markedly promoted apoptosis and arrested cell cycle at the G0/G1 phase in a concentration-dependent manner. Furthermore, high concentrations of genistein have no adverse effect on normal esophageal epithelial cells. Mechanistically, genistein treatment strikingly reduced the expression of cell cycle-associated genes, and up-regulated the expression of cell apoptosis-related genes in EsC cells. Additionally, genistein dramatically decreased epidermal growth factor receptor (EGFR) expression and attenuated its down-stream signaling molecules STAT3, MDM2, Akt and JAK1/2 phosphorylation, resulting in inhibited nuclear translocation of STAT3 and MDM2, thereby inhibiting the JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways. In xenograft nude mice, genistein administration strikingly impaired tumor growth in a dose-dependent manner. Moreover, similar disturbances in molecular mechanisms were observed in vivo. Taken together, genistein suppressed the JAK1/2-STAT3 and AKT/MDM2/p53 signaling pathways by decreasing EGFR expression, leading to cell apoptosis, cell cycle arrest, and proliferation inhibition in EsC cells. Our findings suggest that genistein may be a promising alternative adjuvant therapy for patients with EsC.

Achieving the balance: Biphasic effects of genistein on PC-3 cells.

This study examined the response of PC-3 cells to physiological (0.5, 2.5, 5, 10μM) and pharmacological (50μM) concentrations of genistein which is a main bioactive compound in soy. Following 48hr genistein treatment, cell-based assays and genome-wide microarray were performed. It was evidenced that maximal physiologically achievable concentrations of genistein (0.5-10μM) lead to significant increase in cell viability (p<0.05) and decrease in migration at 0.5μM (p=0.000) and 10μM (p=0.001). The highest percentage of apoptotic cells was obtained at 50μM. Microarray analysis gave the most critical pathways such as cell cycle regulation and proliferation, tumorigenesis, DNA damage and repair, stress response, and apoptosis. Physiological concentrations (≤10μM) induced activation of CDKs, MAPKs, and RPSKs, while high concentrations of genistein (>10μM) appeared to have a novel mechanism of action, specifically down-regulating TGF-β by decreasing specifically SMAD 2/3,4 which are in the downstream TGF-β signaling cascade. PRACTICAL APPLICATIONS: This study highlights for the first time that maximal physiologically achievable concentrations of genistein (0.5-10μM) have proliferative effects evidenced by alterations in global gene expression patterns of PC-3 cells. Our results particularly represent a closer examination of dietary genistein consumption for the prevention and/or treatment of cancer that maximal physiologically achievable concentrations of genistein could have detrimental effects on individuals with prostate cancer. Further studies as in vivo would be necessary to remove shadows on the effect of genistein on prostate cancer progression.

Potentiation of ABCA3 lipid transport function by ivacaftor and genistein.

ABCA3 is a phospholipid transporter implicated in pulmonary surfactant homoeostasis and localized at the limiting membrane of lamellar bodies, the storage compartment for surfactant in alveolar type II cells. Mutations in ABCA3 display a common genetic cause for diseases caused by surfactant deficiency like respiratory distress in neonates and interstitial lung disease in children and adults, for which currently no causal therapy exists. In this study, we investigated the effects of ivacaftor and genistein, two potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), on ABCA3‐specific lipid transport function. Wild‐type (WT) and functional ABCA3 mutations N568D, F629L, G667R, T1114M and L1580P were stably expressed in A549 cells. Three‐dimensional modelling predicted functional impairment for all five mutants that was confirmed by in vitro experiments (all <14% of WT functional activity). Treatment with potentiators rescued the mutants N568D (up to 114% of WT), F629L (up to 47% of WT), and G667R (up to 60% of WT), the latter variation needing higher concentrations of genistein, showing reduced affinity of the potentiator to the mutant protein. Our results present a first proof that functional ABCA3 mutations are rescued by CFTR potentiators, making them a potential therapeutical option for patients suffering from surfactant deficiency due to ABCA3 mutations.

Effects of genistein on insulin pathway-related genes in mouse differentiated myoblast C2C12 cell line: evidence for two independent modes of action.

Genistein (plant isoflavone) is a well-known anti-cancer drug with estrogenic-like properties. Genistein also regulates sugar and lipid metabolism; thus, it has anti-diabetic properties. The aim of the study was to evaluate in vitro effects of genistein on glucose transport, fatty acids oxidation, activation of PKB, and expression of genes related to insulin pathway in differentiated myoblast C2C12 mouse cell line. Differentiated myoblast C2C12 mouse cell line was used to assess the effects of different genistein concentrations on glucose transport and fatty acids oxidation measured by radioactivity technique, activation of PKB, and expression of selected genes related to insulin signaling pathway (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A, SH-PTP2) at the mRNA and protein levels. Cells were incubated with various concentrations of genistein under standard conditions for 0-48 hours. Genistein in low concentrations (0.1-1 μM) significantly increased glucose transport and decreased fatty acids oxidation in C2C12 cells after 48 h of incubation. High concentration of genistein (50 μM) had the opposite effect. Genistein stimulated PKB phosphorylation during the first 5-10 minutes of incubation. There was no significant impact on the protein expression of selected genes (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A-Ca, ER-a and ER-b) after 48 h treatment. We observed inverse correlation between genistein concentration and the expression of SH-PTP2 protein. Genistein affected the expression pattern of mRNAs for genes related to the insulin pathway, however, not the expression of the encoded proteins. The results of this study showed that depending on the concentration and time of incubation genistein significantly affects glucose and lipid metabolism and at low concentration modifies expression pattern of a few genes in C2C12 cells.

A Bivalent Role of Genistein in Sprouting Angiogenesis.

The effects of genistein on angiogenesis remain poorly understood. Some studies claim an antiangiogenic effect and others claim a pro-angiogenic one. Thus, the aim of this study was to determine if genistein may exhibit bivalent angiogenic effects. To address this question, genistein angiogenic modulatory effects were examined using an in vitro 3D angiogenesis model using human umbilical vein endothelial cells. In this model, a bivalent effect of genistein was demonstrated on sprouting angiogenesis, with angiogenic stimulation at low concentrations (0.001 - 1 µM) and inhibition at higher ones (25 - 100 µM). Enhancement of the endothelial tube formation correlated with an increase in human umbilical vein endothelial cell metabolic activity and proliferation. Inhibition of angiogenesis correlated with a decreased metabolic activity, proliferation, and migration. Moreover, high concentrations of genistein influenced human umbilical vein endothelial cell morphology. Expression of genes involved in the angiogenic process in response to genistein was measured to study the mechanism of action. Secretome profiling revealed that angiogenic regulators were modulated with genistein treatment. These results suggested a bivalent effect of genistein on human umbilical vein endothelial cell growth and angiogenesis, and further investigations on the benefit of genistein for cancer chemoprevention, cancer treatment, or pro-angiogenic therapies have to be carefully considered.

Genistein inhibits the growth and regulates the migration and invasion abilities of melanoma cells via the FAK/paxillin and MAPK pathways.

Genistein is one of the main components of soy-based foods, which are widely known for their many benefits, including anti-cancer, anti-inflammatory, and antioxidant effects. In this study, we investigated the anti-metastasis effects of genistein on B16F10 melanoma cells. Our results showed that genistein strongly inhibited B16F10 cell proliferation and induced apoptosis in time- and concentration-dependent manners. Genistein altered the morphology of B16F10 cells to an elongated shape with slim pseudopodia-like protrusions. Moreover, genistein inhibited the invasion and migration abilities of B16F10 cells in a dose-dependent manner. On one hand, a high concentration of genistein (100 μM) significantly inhibited cell adhesion and migration, as shown by wound healing assays and transwell-migration and invasion assays. Furthermore, the expression levels of p-FAK, p-paxillin, tensin-2, vinculin, and α-actinin were decreased by genistein. As a result, genistein is believed to strongly downregulate the migration and invasion abilities of B16F10 cells via the FAK/paxillin pathway. Moreover, p-p38, p-ERK, and p-JNK levels were also dramatically decreased by treatment with genistein. Finally, genistein significantly decreased the gene expression of FAK, paxillin, vimentin, and epithelial-to-mesenchymal transition-related transcription factor Snail, as shown by real-time PCR (qPCR) analysis. On the other hand, a lower concentration of genistein (12.5 μM) significantly promoted both invasion and migration by activating the FAK/paxillin and MAPK signaling cascades. Taken together, this study showed for the first time that genistein exerts dual functional effects on melanoma cells. Our findings suggest that genistein regulates the FAK/paxillin and MAPK signaling pathways in a highly concentration-dependent manner. Patients with melanoma should therefore be cautious of consuming soy-based foods in their diets.

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BackgroundPhytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts.MethodsThus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed.ResultsExposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells.ConclusionFrom these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

A High Concentration of Genistein Induces Cell Death in Human Uterine Leiomyoma Cells by Autophagy.

Genistein, an estrogenic, soy-derived isoflavone, may play a protective role against hormone-related cancers. We have reported that a high concentration of genistein inhibits cell proliferation and induces apoptosis in human uterine smooth muscle cells, but not in leiomyoma (fibroid) cells. To better understand the differential cell death responses of normal and tumor cells to a high concentration of genistein, we treated uterine smooth muscle cells and uterine leiomyoma cells with 50 μg/ml of genistein for 72 h and 168 h, and assessed for mediators of apoptosis, cytotoxicity and autophagy. We found that leiomyoma cells had increased protection from apoptosis by expressing an increased ratio of Bcl-2: bak at 72 h and 168 h; however, in smooth muscle cells, the Bcl-2: bak ratio was decreased at 72 h, but significantly rebounded by 168 h. The apoptosis extrinsic factors, Fas ligand and Fas receptor, were highly expressed in uterine smooth muscle cells following genistein treatment at both time points as evidenced by confocal microscopy. This was not seen in the uterine leiomyoma cells; however, cytotoxicity as indicated by elevated lactate dehydrogenase levels was significantly enhanced at 168 h. Increased immunoexpression of an autophagy/autophagosome marker was also observed in the leiomyoma cells, although minimally present in smooth muscle cells at 72 h. Ultrastructurally, there was evidence of autophagic vacuoles in the leiomyoma cells; whereas, the normal smooth muscle cells showed nuclear fragmentation indicative of apoptosis. In summary, our data show differential cell death pathways induced by genistein in tumor and normal uterine smooth muscle cells, and suggest novel cell death pathways that can be targeted for preventive and intervention strategies for inhibiting fibroid tumor cell growth in vivo.

Radioprotective effects of genistein on HL-7702 cells via the inhibition of apoptosis and DNA damage

Radiation induced normal tissue damage is the most important limitation for the delivery of a high potentially curative radiation dose. Genistein (GEN), one of the main soy isoflavone components, has drawn wide attention for its bioactivity in alleviating radiation damage. However, the effects and molecular mechanisms underlying the radioprotective effects of GEN remain unclear. In the present study, we showed that low concentration of GEN (1.5 µM) protected L-02 cells against radiation damage via inhibition of apoptosis, alleviation of DNA damage and chromosome aberration, down-regulation of GRP78 and up-regulation of HERP, HUS1 and hHR23A. In contrast, high concentration of GEN (20 µM) demonstrated radiosensitizing characteristics through the promotion of apoptosis and chromosome aberration, impairment of DNA repair, up-regulation of GRP78, and down-regulation of HUS1, SIRT1, RAD17, RAD51 and RNF8. These findings shed light on using low, but not high-concentration GEN, as a potential candidate for adjuvant therapy to alleviate radiation-induced injuries to human recipients of ionizing radiation.

In vitro and in vivo effects of phytoestrogens on protein turnover in rainbow trout (Oncorhynchus mykiss) white muscle

Soybeans and other legumes investigated as fishmeal replacements in aquafeeds contain phytoestrogens capable of binding to and activating estrogen receptors. Estradiol has catabolic effects in salmonid white muscle, partially through increases in protein turnover. The current study determines whether phytoestrogens promote similar effects. In rainbow trout (Oncorhynchus mykiss) primary myocyte cultures, the phytoestrogens genistein, daidzein, glycitein, and R- and S-equol reduced rates of protein synthesis and genistein, the phytoestrogen of greatest abundance in soy, also increased rates of protein degradation. Increased expression of the ubiquitin ligase fbxo32 and autophagy-related genes was observed with high concentrations of genistein (100μM), and R- and S-equol (100μM) also up-regulated autophagy-related genes. In contrast, low genistein concentrations in vitro (0.01–0.10μM) and in vivo (5μg/g body mass) decreased fbxo32 expression, suggesting a potential metabolic benefit for low levels of genistein exposure. Phytoestrogens reduced cell proliferation, indicating that effects of phytoestrogens extend from metabolic to mitogenic processes. Co-incubation of genistein with the estrogen receptor (ER) antagonist, ICI 182,780, ameliorated effects of genistein on protein degradation, but not protein synthesis or cell proliferation, indicating that effects of genistein are mediated through ER-dependent and ER-independent mechanisms. Collectively, these data warrant additional studies to determine the extent to which dietary phytoestrogens, especially genistein, affect physiological processes that impact growth and nutrient retention.

Genistein promotes a gene expression profile characteristic of brown rather than white adipocytes and increases Sirt1 expression in mouse NIH3T3‐L1 cells (372.7)

Various possible benefits have been attributed to the dietary isoflavone genistein. In common with resveratrol, which may act via sirtuin 1 (Sirt1), these benefits include protection from diet‐induced obesity. We thus aimed to determine if genistein affected adipogenesis and/or Sirt1 expression using a cell culture model.NIH3T3‐L1 cells were differentiated into adipocytes (from 48 h post‐confluence, taken as day 0) in the presence or absence of genistein. Gene expression was measured by RT‐qPCR.A low concentration of genistein (10 µM) and/or shorter exposure (days 0‐12) promoted differentiation to white adipocytes, indicated by large fat droplets and increased expression of adipocyte marker genes (Acaca (acetyl Co‐A carboxylase‐α), Fasn (fatty acid synthase), Fabp4 (fatty acid binding protein 4), Lipe (hormone sensitive lipase), Retn (resistin), Rarres2 (chemerin)). However, at higher concentrations of genistein (50‐100 µM) and/or after longer exposure (days 0‐12) cells had smaller fat droplets and lower expression of these genes, coupled with increased expression of Sirt1 and of genes characteristic of brown adipocytes (Ucp1 (uncoupling protein 1) and Cebpb (CCAAT/enhancer binding protein‐β)).Dietary genistein may thus protect against obesity by encouraging the development of brown, rather than white, adipose tissue, possibly through a mechanism involving Sirt1.Grant Funding Source: Kurdistan Regional Government

High concentrations of genistein exhibit pro-oxidant effects in primary muscle cells through mechanisms involving 5-lipoxygenase-mediated production of reactive oxygen species

Genistein, a typical soy isoflavone, is an important antioxidant for improving human health and animal production but the compound possesses some pro-oxidant potential. In order to explore the latter, the dose–response relationship of various concentrations of genistein on both cellular proliferation and the redox system were examined. The proliferation of primary muscle cells was promoted by a low concentration of genistein but was inhibited by high concentrations, which also enhanced lipid oxidation and suppressed membrane fluidity. By selecting a high concentration (200μM) as a pro-oxidant treatment, the mechanism underlying the pro-oxidant function of genistein was then explored. The generation of intracellular reactive oxygen species (ROS) was stimulated by 200μM genistein, with inhibited expression of NADPH oxidase 4 and cyclooxygenase 1 and 2 as well as increased activity of the glutathione redox system. The cellular expression of 5-lipoxygenase, however, was up-regulated by 200μM genistein and the addition of 5-lipoxygenase inhibitor (Zileuton) decreased genistein-induced intracellular ROS level, close to that from the addition of the ROS scavenger, N-acetylcysteine. It is concluded that higher concentrations of genistein exert pro-oxidant potential in the primary muscle cells through enhancing ROS production in a 5-lipoxygenase-dependent manner.

A high concentration of genistein down-regulates activin A, Smad3 and other TGF-β pathway genes in human uterine leiomyoma cells

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3, TGF-β2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real-time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that down-regulation of activin A and Smad3, both members of the TGF-β pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.

P3-04-07: Physiological Concentrations of Genistein and 17b-Estradiol Inhibit MDA-MB-231 Breast Cancer Cell Proliferation by Increasing Bax/Bcl2 Ratio and Decreasing pERK1/2 Expression.

Abstract Background: Our previous results were the first to show that physiological concentrations (1 μM) of genistein, a soy component, in the presence of17β-estradiol (1 nM) inhibited the cell proliferation of MDA-MB-231 (Estrogen receptor (ER) β positive) breast cancer cells. These results are relevant in premenopausal women with breast cancer of the ERα-negative and ERβ-positive type, especially given the increasing trends of soy intake among the US population in the past decade. The aim of the present study was to identify the mechanism by which genistein plus 17β-estradiol inhibits the cell proliferation of ERα-negative and ERb-positive breast cancer cells. Our hypothesis is that the balance of signaling actions by genistein plus 17β-estradiol from different signaling pathways is likely to lead to the cell's choice to either proliferate or enter the apoptotic pathway. For this purpose, the effect of low and high concentrations of genistein (1 μM and 100 μM) in the presence or absence of 17β-estradiol (1 nM) was studied on the expression of cell signaling proteins involved in cell proliferation, survival and apoptosis (pERK1/2, pAkt, Bax and Bcl2) and correlated to cell proliferation and apoptosis in MDA-MB-231 (ERβ positive and ERα negative) breast cancer cells. Methods: Cell proliferation was determined by the MTT assay, apoptosis determined microscopically by the use of acridine orange and ethidium bromide dyes and the expression of cell signaling proteins by western blotting. Results: Our results show that 1μM genistein plus 17β-estradiol significantly increased apoptosis (p&amp;lt;0.05) as compared to the control (12.47% vs 5.87% respectively). Increased expression of Bax/Bcl2 (2.5 fold) along with a decreased expression of phosphorylated ERK1/2 (2 fold) was observed in cells treated with 1μM genistein plus 17β-estradiol as compared to the control cells. Phosphorylated Akt did not show any differences in the treatment condition as compared to the control. High concentrations of genistein (100 μM) in the presence or absence of 17β-estradiol also increased apoptosis; however these changes could not be correlated to the expression of Bax/Bcl2, or pERK1/2. Conclusion: In conclusion, our results show that physiological concentrations of genistein in the presence of17β-estradiol inhibit cell growth through apoptosis via increased Bax/Bcl2 and a concomitant decrease in pERK1/2 expression. Our results also suggest that different concentrations of genistein elicit cell responses through different signaling mechanisms. These results are especially relevant to the cohort of premenopausal women with breast cancer of the ERβ positive and ERα negative type. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-04-07.

Developmental toxicity and brain aromatase induction by high genistein concentrations in zebrafish embryos

Genistein is a phytoestrogen found at a high level in soybeans. In vitro and in vivo studies showed that high concentrations of genistein caused toxic effects. This study was designed to test the feasibility of zebrafish embryos for evaluating developmental toxicity and estrogenic potential of high genistein concentrations. The zebrafish embryos at 24 h post-fertilization were exposed to genistein (1 3 10−4 M, 0.5 3 10−4 M, 0.25 3 10−4 M) or vehicle (ethanol, 0.1%) for 60 h. Genistein-treated embryos showed decreased heart rates, retarded hatching times, decreased body length, and increased mortality in a dose-dependent manner. After 0.25 3 10−4 M genistein treatment, malformations of survived embryos such as pericardial edema, yolk sac edema, and spinal kyphosis were also observed. TUNEL assay results showed apoptotic DNA fragments in brain. This study also confirmed the estrogenic potential of genistein by EGFP expression in the brain of the mosaic reporter zebrafish embryos. This study first demonstrated that high concentrations of genistein caused a teratogenic effect on zebrafish embryos and confirmed the estrogenic potential of genistein in mosaic reporter zebrafish embryos.

Decreased cell proliferation of breast cancer cells by genistein is caused by estrogen receptor related signaling molecules.

Abstract Abstract #6019 Background: Literature reports demonstrate that genistein, a soy phytoestrogen, shows a biphasic response to cell proliferation in breast cancer cells. Genistein increases cell proliferation at low concentrations and decreases cell proliferation at high concentrations. Our previous studies suggest that at low concentrations of genistein the cell proliferative response depends on the type of ER. At high concentrations genistein is believed to act as a tyrosine kinase inhibitor. Currently, the mechanisms of genistein's effect on breast cancer cell proliferation are unclear. The aim of this study was to evaluate the mechanisms of the biphasic response of genistein on breast cancer cell proliferation, specifically by determining the effect of genistein on ER-related cell signaling molecules involved in cell proliferation, cell survival and apoptosis. Based on literature reports, the ER-related cell signaling molecules chosen for this study were ERK1/2, p90RSK, JNK, Akt and NFκB. ERK1/2 and, p90RSK are involved in cell proliferation; JNK, Akt and NFκB are involved in cell survival and/or apoptosis.&amp;#x2028; Methods: The effect of genistein at 1 µM (low concentration) and 100 µM (high concentration) on cell signaling molecules was determined by a BioPlex Phosphoprotein detection kit. Results obtained from the BioPlex assay were confirmed by immunodetection. Cell proliferation was determined at 24, 48, and 72 hrs by the MTT (3-[4,5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium) assay. This study was carried out in T47D (ERα and ERβ) and MDA-MB-231 (ERβ) breast cancer cells.&amp;#x2028; Results: At 100 µM genistein exposure we observed a decrease in phosphorylated p90RSK (40%) in T47D cells and an increase in phosphorylated JNK (45%) in MDA-MB-231 cells in a statistically significant (p&amp;lt;0.05) manner. At 100 µM genistein exposure, a statistically significant (p&amp;lt;0.001) decrease in cell proliferation was observed in T47D cells (20-40%) and MDA-MB-231 cells (30-60%) as compared to the control. At 1 µM genistein exposure, there was a statistically significant (p&amp;lt;0.05) decrease (15%) of phosphorylated JNK in MDA-MB-231 cells. MDA-MB-231 cells exposed to 1 µM genistein did not show any statistically significant difference in cell proliferation as compared to the control. T47D cells exposed to 1 µM of genistein for 24 – 72 hours showed a statistically significant (p&amp;lt;0.001) increase (25 to 70%) in cell proliferation.&amp;#x2028; Conclusions: Our data suggests that in T47D cells which have both ERs, decreased cell proliferation due to high concentrations of genistein is probably due to a decrease in phosphorylated p90RSK, an ER-related cell proliferation protein. In MDA-MB-231 cells with only ERβ, the decrease in cell proliferation is probably due to an increase in phosphorylated JNK, an apoptotic protein. Our results suggest that at high concentrations of genistein, the cellular pathways of inhibition of breast cancer cell proliferation likely depend on ER status. This study reveals the importance of genistein as an effective chemotherapeutic agent in breast cancers that contain both ERs and only ERβ. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6019.

Genistein differentially modulates androgen-responsive gene expression and activates JNK in LNCaP cells

Genistein, the predominant isoflavone in soy, may be chemopreventive in prostate cancer (CaP). It down-regulates the prostate-specific antigen (PSA) and androgen receptor (AR) in androgen responsive cells. However, the extent of the down-regulation and whether genistein has a general effect on all androgen responsive genes (ARGs) are unclear. We investigated the ability of genistein to modulate ARG expression by the synthetic androgen R1881 in LNCaP cells. Given that there is important crosstalk between AR and mitogen activated protein kinase (MAPK) signaling, we also investigated whether genistein activates the MAPK end targets c-Jun N-terminal kinase (JNK) and c-Jun. Changes in ARG expression were determined by Western analysis and semi-quantitative RT-PCR. The activation of JNK and c-Jun was investigated by Western analysis and a solid phase kinase assay. The PSA protein and mRNA expression were both down-regulated by genistein. In contrast, KLK4 was up-regulated at the mRNA, but down-regulated at the protein level. NKX3.1 mRNA levels did not change significantly, but protein levels were significantly down-regulated. STAMP2 mRNA levels slightly increased whereas the protein expression was down-regulated. The AR mRNA expression changed significantly only at high concentrations of genistein when it was down-regulated, whereas AR protein levels were decreased at low concentrations of genistein. The solid phase kinase assay indicated a transient activation of JNK by genistein, which was supported by Western analysis. Thus genistein differentially modulates ARG mRNA expression, but has an inhibitory role on the ARG protein levels. The activation of the JNK pathway which inhibits AR signaling may provide a mechanism for the overall inhibition of protein levels.