Doses Of Genistein Research Articles

Perinatal xenohormone exposure impacts sweet preference and submandibular development in male rats

To determine the effect of perinatal exposure to low doses of genistein and/or vinclozolin on submandibular salivary gland (SSG) development in juvenile and adult male rats and to establish a link with sweet preference. Female rats received orally (1mgkg(-1) body weight/day) genistein and vinclozolin, alone or in combination, from the first gestational day up to weaning. Sweet preference was assessed at weaning and in adulthood in male offspring; submandibular glands were then collected to study the morphogenesis and mRNA expression of steroid receptors, growth factors and taste related proteins. Exposure to genistein and/or vinclozolin resulted in a higher saccharin intake on postnatal day 25 (P<0.05) linked to a higher number of pro-acinar cells (P<0.01) and mRNA expression of progesterone receptor, growth factors and gustine (P<0.01). These increases disappeared in adulthood, but mRNA expressions of sex hormone receptors and growth factors were strongly repressed in all treated groups (P<0.01). Our findings confirm that the SSG are target for xenohormones and provide evidence that perinatal exposure to low doses of genistein and/or vinclozolin could simultaneously disrupt not only the salivary gland prepubertal development and sweet intake but also endocrine gene mRNA expression later in life.

English

Genistein (GEN), the primary isoflavone in legumes, has a well known weak estrogenic effect by binding to estrogen receptors, and widely used for the treatment of ovary disease induced by chemotherapeutics, however, the details of the exact mechanisms was unclear so far, thus, the aim of our study was to find the effect on granulosa cells of ovary induced by cisplatin (CDDP) after using GEN treatment by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) method and flow cytometry. The results demonstrated that CDDP could inhibit the proliferation of granulosa cells of ovary by affecting cell cycle S stage. Moreover, CDDP also could arrest cell cycle in G1-M stage, which would evidently increase the number of apoptotic cell. Genistein has the potential to prevent the damaging effect of CDDP and improve the differentiation and proliferations of the cells to make the blockage of the cell cycle disappear, which is related to the dose of GEN and time. The present study provides improvement in understanding the molecular pathogenic mechanism of premature ovarian failure (POF) induced by chemotherapeutics and development of GEN as effective treatment drugs. Key words: Genistein, Premature ovarian failure, Granulosa cells of ovary, Flow Cytometry.

Protective effect of short-term genistein supplementation on the early stage in diabetes-induced renal damage.

Hyperglycemia-induced oxidative stress has been concerned in the development of diabetic nephropathy (DN), which may cause kidney damage associated with inflammation and fibrosis. This study has been conducted to investigate the role of genistein supplementation in an acute DN state. Mice with FBG levels more than 250 mg/dL after alloxan injection (single i.p., 150 mg/kg) were considered as diabetic. Diabetic mice (DM) were further subdivided according to their FBG levels, medium-high FBG (DMMH < 450 mg/dL) and high FBG (DMH; 450 mg/dL) and were administrated by an AIG-93G diet supplemented with different doses of genistein (0, 0.025 or 0.1%). After 2 weeks' treatment, the levels of kidney malondialdehyde (MDA), blood urea nitrogen (BUN), and plasma creatinine and lipid profiles, as well as oxidative stress and inflammation-related markers, were measured (P < 0.05). Genistein supplementation improved levels of FBG in the DMMH groups, but not in the DMH group, regardless of the treatment dose. Moreover, the supplementation attenuated kidney oxidative stress indicated by MDA, BUN, and plasma creatinine. In addition, genistein treatment decreased inflammatory markers such as nuclear factor kappa B (p65), phosphorylated inhibitory kappa B alpha, C-reactive protein, monocyte chemotactic protein-1, cyclooxygenase-2, and tumor necrosis factor-alpha and improved oxidative stress markers (nuclear-related factor E2, heme oxygenase-1, glutathione peroxidase, and superoxide dismutase isoforms) in treatment groups, regardless of the genistein treatment dose. Furthermore, genistein supplementation inhibited the fibrosis-related markers (protein kinase C, protein kinase C-beta II, and transforming growth factor-beta I) in the DN state. However, 0.1% genistein supplementation in diabetes with high FBG levels selectively showed a preventive effect on kidney damage. These results suggest that genistein might be a good protective substance for DN through regulation of oxidative stress and inflammation. In particular, genistein is more efficient in diabetes patients with medium-high blood glucose levels. Finally, it is required to establish the beneficial dosage of genistein according to blood glucose levels.

Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling.

To investigate the effects of Genistein on the osteogenic related gene expression profiles during osteoblastic differentiation of human bone marrow mesenchymal stem cell (hBMSC) cultures, the hBMSCs were cultured under osteogenic differentiation medium with the addition of Genistein (10-8∼10-5 M) for 12 days. The cell proliferation was measured by BrdU incorporation, while the osteoblastic differentiation in hBMSC cultures was assessed by cellular alkaline phosphatase (ALP) activity. The cell apoptosis was determined by caspase 3/7 activation. GEArray Q series human osteogenesis gene array was used to analyze large-scale gene expression in Genistein-treated hBMSC cultures compared to the control group. Quantitative real-time RT-PCR, small interfering RNA (siRNA), and western blot analysis were used to confirm the microarray data in five representative transcripts. Genistein (10-8∼10-6 M) dose- and time-dependently increased cell proliferation and cellular ALP activity, but had no significant effect on cell apoptosis in hBMSC cultures. The 96-gene array analysis indicated that 22 genes were upregulated more than 2-fold and 7 genes were downregulated at least 1.5-fold. The expressions of bone morphogenetic proteins (BMPs), small mothers against decapentaplegic homologs (SMADs), and Runt-related transcription factor 2 (RUNX2) were concomitantly increased under Genistein treatment while insulin-like growth factor 2 and inhibitory SMADs 6 and 7 expressions were significantly decreased. The results of the real-time RT-PCR had a correlation with the results of microarray analysis and were estrogen-receptor dependent. Specific gene siRNAs knock-down further confirmed the osteogenic effects of Genistein on BMP2, SMAD5 and RUNX2 protein expression. Genistein enhanced osteogenic differentiation in cultured hBMSCs mainly through the BMP-dependent SMADs and RUNX2 signaling.

Effects of the phytoestrogen genistein on the development of the reproductive system of Sprague Dawley rats

OBJECTIVES:Genistein is known to influence reproductive system development through its binding affinity for estrogen receptors. The present study aimed to further explore the effect of Genistein on the development of the reproductive system of experimental rats.METHODS:Eighteen post-weaning female Sprague Dawley rats were divided into the following groups: (i) a control group that received vehicle (distilled water and Tween 80); (ii) a group treated with 10 mg/kg body weight (BW) of Genistein (Gen 10); and (iii) a group treated with a higher dose of Genistein (Gen 100). The rats were treated daily for three weeks from postnatal day 22 (P22) to P42. After the animals were sacrificed, blood samples were collected, and the uteri and ovaries were harvested and subjected to light microscopy and immunohistochemical study.RESULTS:A reduction of the mean weekly BW gain and organ weights (uteri and ovaries) were observed in the Gen 10 group compared to the control group; these findings were reversed in the Gen 100 group. Follicle stimulating hormone and estrogen levels were increased in the Gen 10 group and reduced in the Gen 100 group. Luteinizing hormone was reduced in both groups of Genistein-treated animals, and there was a significant difference between the Gen 10 and control groups (p<0.05). These findings were consistent with increased atretic follicular count, a decreased number of corpus luteum and down-regulation of estrogen receptors-α in the uterine tissues of the Genistein-treated animals compared to the control animals.CONCLUSION:Post-weaning exposure to Genistein could affect the development of the reproductive system of ovarian-intact experimental rats because of its action on the hypothalamic-pituitary-gonadal axis by regulating hormones and estrogen receptors.

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.

Genistein Protects Hematopoietic Stem Cells Against G-CSF Induced DNA Damage

Abstract 3486Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. A particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. Our results suggest that genistein’s effects are mediated primarily through inhibition of HSC proliferation. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, we demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support. Disclosures:No relevant conflicts of interest to declare.

Dose-dependent effects of a genistein-enriched diet in the heart of ovariectomized mice

The isoflavone genistein is used as a pharmacological compound and as a food supplement. The duration and the level of exposure of humans to genistein are considerable. However, the magnitude of genistein-supplemented dietary interventions necessary to induce any changes in the heart has not been studied so far. The aim of this study was to investigate the dose-dependent effects of dietary genistein in the disease- and stress-free mouse heart. Female C57BL/6J mice at the age of 2 months were ovariectomized and randomly assigned to feed on diets with seven different genistein doses (0.01, 0.03, 0.1, 0.3, 1, 3 and 10 g genistein/kg food) for 3 months. Mice with intact ovaries or ovariectomized fed on soy-free diets were used as controls. Ovariectomy led to an increase in body weight, while the two highest genistein doses prevented this increase. Absolute uterus weight was decreased in the ovariectomized group and all genistein groups except for the 10 g/kg food group compared with the intact ovaries/soy-free group. Considering cardiac mass, although the 3 and 10 g/kg food groups had significantly lower absolute heart weight than all other groups, heart-to-body-weight ratios did not differ between these two groups and the intact ovaries/soy-free group, while all remaining groups had smaller ratios. Next, we observed dose-dependent effects of genistein on cardiac gene expression. The present findings indicate that exposure of female mice to the soy isoflavone genistein influences body weight and cardiac mass and gene expression in a dose-dependent manner. Human exposure to dietary genistein supplements may influence cardiac function.

Out of Field Cancer Risk in Mice Following Exposure to a Clinical Proton Beam

(37 C, 5% CO2) for 7 days before being assayed for survival using a standard clonogenic assay. Results: Data indicated that both miso and 10mM genistein treatments resulted in a reduction in PC3 cell colony number of w20%, suggesting a marked growth inhibitory action for these compounds. Treatment with 30mM genistein enhanced PC3 cell growth inhibition even more substantially, resulting in an 85% reduction in cell colony number compared to untreated controls. Additionally, data from these studies suggested that both miso and genistein continued to exert their growth inhibitory effect on the PC3 cells over a range of radiation exposures up to 300cGy. However, a differential response was seen between the misotreated and 10mM genistein-treated radiation cell survival curves and the 30mM genistein-treated radiation cell survival curve. Specifically, both the miso and 10mM genistein treatments interacted with radiation treatments in an additive manner, resulting in enhanced tumor cell kill, but producing radiation cell survival curves whose shape was almost identical to the untreated irradiated control. In contrast, the reduction in colony number in PC3 cells treated with 30mM genistein plus radiation resulted in a highly supra-additive reduction in colony number, especially at doses greater than 50cGy, thereby, producing a markedly steeper radiation cell survival curve than observed for the untreated irradiated control. Conclusions: Data suggest that administration of either miso or genistein may potentiate radiation-induced prostate tumor cell kill. This is particularly true when combining higher doses of genistein (30mM) with radiation, where synergistic cell kill appears to occur. These results may have consequence as a way to improve the outcome of radiation therapy regimens for prostate cancer patients. Author Disclosure: J. Sattler: None. C.R. Kempf: None. R.M. Johnke: None.

Effects of diets containing genistein and diadzein in a long-term study on sex steroid dynamics of goldfish (Carassius auratus)

The effect of long-term exposure of goldfish to dietary genistein and diadzein on the concentrations of plasma sex steroids (testosterone (T), 17β-estradiol (E2)) and the gonadosomatic index (GSI) was assessed. The study was conducted on four groups for a period of 2 years, from the age of 20 weeks to first spawning. Four doses of genistein and diadzein were applied in the feed: genistein: 0 µg/g, diadzein: 0 µg/g (control group); genistein: 24.26 µg/g, diadzein: 21.7 µg/g (diet 1); genistein: 51.55 µg/g, diadzein: 46.13 µg/g (diet 2); and genistein: 75.83 µg/g, diadzein: 67.82 µg/g (diet 3). Throughout the experiment, there were no significant dose- or time-related effects of genistein and diadzein contents on the T level in both sexes. Furthermore, at the highest genistein and diadzein contents, there was an elevating plasma concentration of E2 at all sampling points (p < 0.05) and a time-related effect occurred (p < 0.05). Although the E2 concentrations in the plasma of female, throughout the experiment, were higher than in males, at the last sampling, the plasma concentrations of E2 reduced among females and became lower than that in males. The effects of isoflavone content were found on GSI of females at the fourth and fifth sampling among the treatments. Isoflavone contents also affect GSI of males at the second, fourth and the last sampling. Our findings suggest that overall genistein and diadzein exposure in early life stages can cause alterations in the reproductive organs and influence sex steroidogenesis.

Protective effects of genistein and estradiol on PAHs-induced developmental toxicity in zebrafish embryos

The toxicity of exposure to polycyclic aromatic hydrocarbons (PAHs) or phytoestrogen is relatively well characterized. However, the toxicity of combined exposure to PAHs and phytoestrogen is not well investigated. In the present study, benzo(a)pyrene (B(a)P) and benzo(k)fluorathene (B(k)F), genistein, along with 17β-estradiol (E2), were investigated for their single and combined developmental toxicity using zebrafish embryos as model system. We demonstrated that two representative PAHs, both B(a)P (≥1 μM) and B(k)F (≥10 μM), can cause significant malformation and mortality in developing zebrafish embryos. The toxicity effect of B(a)P was in general higher than that of B(k)F. Developmental exposure to high level of genistein (>20 μM) or E2 (>10 μM), also caused significant malformation and mortality in zebrafish larvae at 120 hours post fertilization (hpf). However, different toxic effects were observed for the combined exposure to PAHs and phytoestrogen in zebrafish. Lower doses of genistein (1 and 10 μM) and E2 (0.1 and 1 μM), when used in combination with high concentration of B(a)P (1 μM) or B(k)F (20 μM), can significantly suppress the toxicity effect of B(a)P and B(k)F in developing zebrafish embryos. The beneficial effect of genistein may be due to the inhibition of cytochrome P450 enzymes via directly interacting with aryl-hydrocarbon receptor (AhR) pathway, or disturbing the AhR pathway through interacting with estrogen receptor pathway.

Alternative methods for the treatment of post-menopausal troubles.

BackgroundMenopause is described as the transition from the reproductive phase of a women to the non reproductive. Changes in hormone levels might lead to complaints and health consequences especially during peri- and postmenopause. Hormone therapy has a potential damaging health risk profile and is recommended for temporal limited therapy for acute vasomotor symptoms only.ObjectiveThe present HTA-report aims to assess the effectiveness and the cost-effectiveness of alternative treatment methods for women with postmenopausal symptoms in Germany regarding patient relevant endpoints (reduction of symptoms and frequency of adverse events and improvement of quality of life).MethodsA systematic literature search was carried out in 33 relevant databases in September 2010. Citations were selected according to pre-defined criteria and were extracted and evaluated.ResultsIn the systematic research 22 studies are identified for the effectiveness evaluation, 22 primary studies and one review.High doses of isolated genistein reduce the frequency/intensity of hot flashes while low doses of genistein show no significant effect. Intake of isoflavone extract such as genistein, daidzein, glycitein in various combinations does not have an effect on improvement of cognitive function or vaginal dryness. The effect of black cohosh and hop extract for menopausal complaints cannot be determined since results are heterogenous. The combination of isoflavone, black cohosh, monk’s pepper, valerian and vitamin E has a positive effect on menopause symptoms. Ginkgo biloba shows no significant effect on menopause symptoms and cognitive improvement beside mental flexibility. Acupuncture has a significant influence on hot flashes especially in severe cases.Discussion/ConclusionNo final statement can be drawn regarding the effectiveness of alternative treatment methods due to qualitative shortcomings of included studies and a general limited availability of studies in this field. Furthermore, the generalization of the present HTA is limited due to the inclusion of only postmenopausal women.

Abstract 4113: Combination of LC3 II shRNA and genistein completely inhibited rapamycin-induced autophagy and promoted apoptosis in human malignant neuroblastoma cell lines

Abstract Neuroblastoma is an extracranial solid tumor that mainly occurs in children. It is derived from embryonic neural crest cells of the peripheral sympathetic nervous system. The overall survival rate of malignant neuroblastoma patients is very poor despite multi-modal therapy including surgery, radiotherapy, and chemotherapy. Efficacy of chemotherapy is often compromised due to presence of autophagy, which is a survival mechanism in solid tumors. Autophagy is a catablolic process for lysosomal degradation of cytoplasmic contents for recycling and it is activated during stress such as nutrient starvation and growth factor deprivation. The hallmark of autophagy is generation of double membrane structure called autophagosome that contains the microtubule-associated protein light chain 3 form II (LC3 II). In this study, we used rapamycin to mimic starvation-induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR32 cells and then investigated capability of the combination of LC3 II knockdown and genistein (GST) treatment for inhibition of autophagy and promotion of apoptosis. First, we treated both cell lines with 200 nM rapamycin for 6, 12, and 24 h and performed acridine orange (AO) staining, flow cytometric analysis, RT-PCR, and Western blotting to establish time-dependent increases in autophagic features such as formation of acidic vesicular organelles (AVO) and LC3 II. Then, we carried out dose-response studies by transfecting the rapamycin-exposed cell lines with various concentrations of LC3 II short hairpin RNA (shRNA) plasmid and treating them with several doses of GST alone and in combination. MTT assays showed the highest synergistic efficacy in inhibiting cell viability using the combination of 50 nM LC3 II shRNA and 25 µM GST in SK-N-BE2 cells while the combination of 100 nM LC3 II shRNA and 25 µM GST in IMR32 cells. Our AO staining and flow cytometric quantitation of AVO confirmed that combination of LC3 II shRNA and GST completely inhibited rapamycin-induced autophagy to promote apoptotic death. In situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy most effectively induced apoptosis in both cell lines when compared with control shRNA, LC3 II shRNA, or GST alone. We unraveled the molecular mechanisms for inhibition of autopphagy and induction of apoptosis by Western blotting of the proteins involved in these processes. Down regulation of LC3 II and upregulation of mTOR in both cell lines showed inhibition of autophagy. Combination therapy promoted mitochondrial pathway leading to activation of caspase-3 and degradation of PARP for inducing apoptosis. Collectively, our results clearly show that combination of LC3 II knockdown and GST treatment serves as promising therapeutic strategy for inhibition of autophagy and induction of apoptosis in different human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4113. doi:1538-7445.AM2012-4113

Gene expression‐targeted isoflavone therapy

Lysosomal storage diseases (LSD) form a group of inherited metabolic disorders caused by dysfunction of one of the lysosomal proteins, resulting in the accumulation of certain compounds. Although these disorders are among first genetic diseases for which specific treatments were proposed, there are still serious unsolved problems that require development of novel therapeutic procedures. An example is neuronopathy, which develops in most of LSD and cannot be treated efficiently by currently approved therapies. Recently, a new potential therapy, called gene expression-targeted isoflavone therapy (GET IT), has been proposed for a group of LSD named mucopolysaccharidoses (MPS), in which storage of incompletely degraded glycosaminoglycans (GAGs) results in severe symptoms of virtually all tissues and organs, including central nervous system. The idea of this therapy is to inhibit synthesis of GAGs by modulating expression of genes coding for enzymes involved in synthesis of these compounds. Such a modulation is possible by using isoflavones, particularly genistein, which interfere with a signal transduction process necessary for stimulation of expression of certain genes. Results of in vitro experiments and studies on animal models indicated a high efficiency of GET IT, including correction of behavior of affected mice. However, clinical trials, performed with soy isoflavone extracts, revealed only limited efficacy. This caused a controversy about GET IT as a potential, effective treatment of patients suffering from MPS, especially neuronopathic forms of these diseases. It this critical review, I present possible molecular mechanisms of therapeutic action of isoflavones (particularly genistein) and suggest that efficacy of GET IT might be sufficiently high when using relatively high doses of synthetic genistein (which was employed in experiments on cell cultures and mouse models) rather than low doses of soy isoflavone extracts (which were used in clinical trials). This proposal can be tested in double-blinded, placebo-controlled clinical trials.

In Utero and Lactational Exposure to Low-Dose Genistein-Vinclozolin Mixture Affects the Development and Growth Factor mRNA Expression of the Submandibular Salivary Gland in Immature Female Rats

It has been suggested that hormonally controlled submandibular salivary gland (SSG) development and secretions may be affected by endocrine disruptor compounds. We investigated the effects of oral gestation-lactation exposure to 1 mg/kg body weight daily dose of the estrogenic soy-isoflavone genistein and/or the anti-androgenic food contaminant vinclozolin in female rats. The SSGs of female offspring were collected at postnatal day 35 to study gland morphogenesis and mRNA expression of sex-hormone receptors and endocrine growth factors as sex-dependent biomarkers. Because of high expression in neonatal SSG, mRNA expression of transforming growth factor α was also studied. Exposure to genistein, vinclozolin, or a genistein+vinclozolin mixture resulted in significantly lower numbers of striated ducts linked to an increase in their area and lower acinar proliferation (Ki-67-positive nuclei). Exposure to the mixture had the highest significant effects, which were particularly associated with repression of epidermal growth factor, nerve growth factor, and transforming growth factor α expression. In conclusion, early exposure to low doses of genistein and vinclozolin can affect glandular structure and endocrine gene mRNA expression in prepubertal SSG in female rats, and the effects are potentialized by the genistein+vinclozolin mixture. Our study provides the first evidence that SSG are targeted by both estrogenic and anti-androgenic disrupting compounds and are more sensitive to mixtures.

Low-dose dietary genistein negates the therapeutic effect of tamoxifen in athymic nude mice

The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10-13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.

Short term effects of genistein on the testes of quail (Coturnix coturnix)

Although there have been a number of studies on the effects of phytoestrogens, especially genistein, on testes, its effects on the testis tissue of birds have not been studied yet. The present study aims to reveal the effects of genistein on quail testes through the use of histological and immunohistochemical methods. In the study, a total of 64 male quails (Coturnix coturnix) were used, each of which was 50 days old. Three different doses of genistein were administered by gavage to the separate groups at intervals of 15 and 30 days. According to the results of the histological analysis and DNA in situ nick end labeling (TUNEL), it was detected that high doses of genistein, especially in 30-day applications, caused more damage compared to the control group and the group receiving 15-day applications in terms of histology and sperm number. Moreover, it was also detected in the 15-day and 30-day application results that both low and high doses of genistein induced apoptosis.

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.

Genistein in Sanfilippo disease: A randomized controlled crossover trial

Sanfilippo disease (mucopolysaccharidosis type III [MPS III]) is a rare neurodegenerative metabolic disease caused by a deficiency of 1 of the 4 enzymes involved in the degradation of heparan sulfate (HS), a glycosaminoglycan (GAG). Genistein has been proposed as potential therapy but its efficacy remains uncertain. We aimed to determine the efficacy of genistein in MPS III. Thirty patients were enrolled. Effects of genistein were determined in a randomized, crossover, placebo-controlled intervention with a genistein-rich soy isoflavone extract (10mg/kg/day of genistein) followed by an open-label extension study for patients who were on genistein during the last part of the crossover. Genistein resulted in a significant decrease in urinary excretion of total GAGs (p = 0.02, slope -0.68 mg GAGs/mmol creatinine/mo) and in plasma concentrations of HS (p = 0.01, slope -15.85 ng HS/ml/mo). No effects on total behavior scores or on hair morphology were observed. Parents or caregivers could not predict correctly during which period of the crossover a patient was on genistein. Genistein at 10mg/kg/day effectively reduces urinary excretion of GAGs and plasma HS concentration in patients with MPS III. However, the absolute reduction in GAGs and in HS is small and values after 12 months of treatment remain within the range as observed in untreated patients. No clinical efficacy was detected. Substantially higher doses of genistein might be more effective as suggested by recent studies in animal models.

Physiological levels of soy isoflavones reduce proliferation and promote apoptosis in the ERβ-positive breast cancer cell line MDA-MB-231

Dietary soy, particularly the high levels consumed as part of traditional Eastern-Asian diets, may be protective against certain subtypes of breast cancer(1). This effect is partly related its high isoflavone phytoestrogen content (genistein and daidzein). However much in vitro evidence suggests a contradictory, growth promoting impact of physiological (serum) levels of isoflavones in oestrogen receptor alpha positive (ERa+) breast cancer cell lines(2). This has led to the contraindication of isoflavone supplement use in post menopausal breast cancer survivors. The inconsistency between epidemiological and in vitro evidence may relate to the relative expression levels of ERa and its counterpart ERb. Unlike in cell lines predominantly expressing ERa, the proliferation of ERb-dominant cell lines can be inhibited by concentrations of genistein as low as 1 nM(3,4). This information is relevant as the ERa - /b+ subgroup represent approximately 18% of all breast tumours(5) but the beta-receptor is not routinely tested for upon diagnosis. We assessed the impact of physiologically relevant concentrations (serum levels achievable through diet: 0.01nM to 31.6 mM) of the soy isoflavones genistein and daidzein on proliferation (MTT assay) and apoptosis with the Annexin V-Cy32 Apoptosis Detection Kit and DAPI staining/Nuclear Area Factor (NAF) calculation in the ERa - /b+ breast cancer cell line MDA-MB-231. Low doses of genistein or daidzein (‡ 0.01 nM) inhibited MDA-MB-231 proliferation by around 20%, although this frequently failed to achieve statistical significance. The highest concentrations used (31.6 mM) resulted in a dramatic decrease in percent proliferation compared with a vehicle only control (meanSD : 48.512.6, p = 0.004 and 48.413.1, p<0.001 for genistein and daidzein respectively; n = 3). 17b-oestradiol (E2; the major circulating oestrogen) also slightly reduced proliferation at its pre- and post-menopausal serum concentrations (1nM and 1 pM respectively). This reduction in proliferation was associated with an observed increase in cell death at 10 and 31.6 mM genistein and daidzein, which was at least partly explained by an increase in the percentage of apoptotic cells. While the combination of E2 and isoflavones failed to show any synergistic growth inhibitory, or pro-apoptotic effects, some inhibition of proliferation was still documented. This implies that the growth inhibitory effects of genistein, daidzein and E2 may not be regulated by the same mechanisms. However, more importantly, the apoptotic cell death observed with soy isoflavone treatment was not abrogated by the addition of physiological E2 concentrations. Overall, our data suggests that at concentrations achievable through the diet (£ 10 mM) soy isoflavones may be potentially beneficial as chemotherapeutic agents against ERa - /b+ breast cancers, through their ability to reduce proliferation and promote apoptosis, even in the presence of physiological E2 levels. Routine determination of the expression levels of ERb in addition to ERa in breast cancer would be an invaluable biomarker to indicate the potential efficacy of this cheap and readily available treatment, and indicates a possible pharmacological target. 1. Wu AH, Yu MC, Tseng CC, Pike MC (2008) Brit J Cancer 98:9–14. 2. Hwang CS, Kwak HS, Lim HJ, et al. (2006) J Steroid Biochem Mol Bio 101:246–53. 3. Rajah TT, Du N, Drews N, Cohn R (2009) Pharmacology 84:68–73. 4. Kang X, Jin S, Zhang Q (2009) J Food Sci 74:H237–H242. 5. Skliris GP, Leygue E, Watson PH, Murphy LC (2008) J Steroid Biochem Mol Bio 109:1–10.