Breast cancer*

Dietary genistein inhibits mammary tumor growth and metastasis of the highly metastatic MDA-MB-435 cancer cells in immunocompromised mice. The purpose of this study was to characterize the role of the novel oncogenic microRNA (miRNA) miR-155 in the anticancer effects of genistein in metastatic breast cancer. The effect of genistein was determined on breast cancer cell viability, apoptosis, and expression of miR-155 and its targets. At low physiologically relevant concentrations, genistein inhibits cell viability and induces apoptosis in metastatic MDA-MB-435 and Hs578t breast cancer cells, without affecting the viability of nonmetastatic MCF-7 breast cancer cells.*

In parallel with reduced cell viability, miR-155 is downregulated, whereas proapoptotic and anticell proliferative miR-155 targets FOXO3, PTEN, casein kinase, and p27 are upregulated in MDA-MB-435 and Hs578t cells in response to genistein treatment. However, miR-155 levels remain unchanged in response to genistein in the MCF-7 cells. Ectopic expression of miR-155 in MDA-MB-435 and Hs578t cells decreases the effects of genistein on cell viability and abrogates the effects of genistein on apoptosis and expression of proapoptotic genes.*

de la Parra et al., (2016) report a novel anticancer mechanism for genistein: downregulation of miR-155, a potent oncogene in breast cancer. This study suggests that decreased miR-155 in response to genistein may in parallel upregulate a large number of anticancer molecules, thus reducing breast cancer cell survival and proliferation, and inducing apoptosis. This mechanism is presumably the molecular basis for our previously reported reduced tumor growth and metastasis in response to dietary genistein, using the same MDA-MB-435 model (14). Therefore, genistein holds promise as a natural nontoxic miR-155 targeted anticancer therapeutic. Moreover, this study augments the body of knowledge on the role of dietary soy isoflavones in established breast cancer and has the potential to impact dietary and therapeutic decisions for breast cancer patients, survivors, and those at risk for breast cancer. Therefore, genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein in metastatic breast cancer.1*

Anticarcinogenic Effects of Dietary Phytoestrogens and Their Chemopreventive Mechanisms*

Hwang K-A, Choi K-C. Nutrition and Cancer. 9 Apr 2015 DOI:10.1080/01635581.2015.1040516

Phytoestrogens are phenolic compounds derived from plants and exert an estrogenic as well as an antiestrogenic effect and also various biological efficacies. Chemopreventive properties of phytoestrogens has emerged from epidemiological observations indicating that the incidence of some cancers including breast and prostate cancers is much lower in Asian people, who consume significantly higher amounts of phytoestrogens than Western people. There are 4 main classes of phytoestrogens: isoflavones, stilbenes, coumestans, and lignans.*

Currently, resveratrol is recognized as another major phytoestrogen present in grape and red wine and has been studied in many biological studies. Phytoestrogens have biologically diverse profitabilities and advantages such as low cytotoxicity to patients, lack of side effects in clinical trials, and pronounced benefits in a combined therapy. Hwang & Cho (2015) highlight the effects of genistein, daidzein, and resveratrol in relation with their anticarcinogenic activity.*

Besides well-known mechanisms such as antioxidant property and apoptosis, newly elucidated anticarcinogenic modes of action including epigenetic modifications and topoisomerase inhibition have been provided to examine the possibility of phytoestrogens as promising reagents for cancer chemoprevention and/or treatment and to suggest the importance of plant-based diet of phytoestrogens.*

Genistein and Gut Inflammation: Role of Nitric Oxide*

Sadowska-Krowicka H, et al. Exp Biol Med (Maywood) March 1998 vol. 217 no. 3 351-357. doi: 10.3181/00379727-217-44244

Genistein at low doses (0.1 mg/kg) had mild anti-inflammatory effects in ileitis. Therapeutic benefit included a reduction in nitric oxide production, granulocyte infiltration and improved mucosal architecture. Genistein, at low doses, also appeared to attenuate immunohistochemical staining for inducible nitric oxide synthase (iNOS) and nitrotyrosine. The beneficial effects of genistein were not apparent at doses above 0.1 mg/kg. We found that genistein also inhibited LPS-induced nitrite production by cultured macrophages and protected against LPS-induced necrosis despite its ability to cause apoptosis. These results indicate that genistein displayed mild antiinflammatory properties which may, in part, involve an attenuation of nitric oxide release via inducible nitric oxide synthase, and the formation of peroxynitrite.*

Genistein

Genistein is the aglycone (aglucon) of genistein. The isoflavone is found naturally as the glycoside genistin and as the glycosides 6"-O-malonylgenistin and 6"-O-acetylgenistin. Genistein and its glycosides are mainly found in legumes, such as soybeans and chickpeas. Soybeans and soy foods are the major dietary sources of these substances. Non-fermented soy foods, such as tofu, contain higher levels of the genistein glycosides, while fermented soy foods, such as tempeh and miso, contain higher levels of the aglycone.*

Recent research suggests that the isoflavonoid genistein, a phytoestrogen found in abundance in soyfoods, may be one of the principal molecular components responsible for health benefits such as the prevention of cancer as well as diseases and symptoms associated with estrogen deficiency. It is a potent estrogen agonist and has cell growth inhibitory actions over a physiologically relevant concentration range.*

In in vitro models, genistein has been found to inhibit the proliferation of various human tumor cell lines in culture (including skin and colon cancers). Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritionally relevant concentrations.2*

Genistein has oestrogenic and antioxidant activities. It may also have anticarcinogenic, anti-atherogenic and anti-osteoporotic activities. For women with an increased risk of breast cancer due to higher oestrogen levels, a diet rich in soy isoflavones may offer a modest breast-protective effect.3*

The chemopreventive effect of the soy isoflavone, genistein, has been observed through the suppression of cell proliferation, inhibition of angiogenesis and stimulation of apoptosis in breast carcinoma cells. Cancer metastasis consists of interdependent processes, including cell adhesion, migration and invasion. In the present study, we compare the effect of soy isoflavones in the form of aglycones (genistein, daidzein and glycitein) and glucosides (genistin, daidzin and glycitin) on the behavior of highly invasive breast cancer cells. Here we demonstrate that genistein suppresses cell adhesion and migration by inhibiting the constitutively active transcription factors NF-kappaB and AP-1, resulting in the suppression of secretion of urokinase-type plasminogen activator (uPA) in breast cancer cells. In addition, we show that all tested soy isoflavone aglycones (genistein, daidzein, glycitein) and glucosides (genistin, daidzin, glycitin) markedly reduced motility of MDA-MB-231 cells. However, only genistein and daidzein inhibited constitutively active NF-kappaB and AP-1 and suppressed secretion of uPA from breast cancer cells.4*

Genistein inhibits protein tyrosine kinase (PTK), which is involved in phosphorylation of tyrosyl residues of membrane-bound receptors leading to signal transduction, and it inhibits topoisomerase II, which participates in DNA replication, transcription and repair. By blocking the activities of PTK, topoisomerase II and matrix metalloprotein (MMP9) and by down-regulating the expression of about 11 genes, including that of vascular endothelial growth factor (VEGF), genistein can arrest cell growth and proliferation, cell cycle at G2/M, invasion and angiogenesis. Furthermore, genistein can alter the expression of gangliosides and other carbohydrate antigens to facilitate their immune recognition. Genistein acts synergistically with drugs such as tamoxifen, cisplatin, 1,3-bis 2-chloroethyl-1-nitrosourea (BCNU), dexamethasone, daunorubicin and tiazofurin, and with bioflavonoid food supplements such as quercetin, green-tea catechins and black-tea thearubigins.5 6*

Synergistic inhibitory effects have been found between genistein and tamoxifen on human dysplastic and malignant epithelial breast cells in vitro.7*

Genistein & Cancer

Evidence that low-dose, long-term genistein treatment inhibits oestradiol-stimulated growth in MCF-7 cells by down-regulation of the PI3-kinase/Akt signalling pathway*

Anastasius N, Boston S, Lacey M, Storing N, Whitehead SA. J Steroid Biochem Mol Biol. 2009 May 3

The reduced incidence of breast cancer in certain Eastern countries has been attributed to high soy diets although this evidence is simply epidemiological. One of the major constituents of soy is genistein, but paradoxically this phytoestrogen binds to oestrogen receptors and stimulates growth at concentrations that would be achieved by a high soy diet, but inhibits growth at high experimental concentrations.To determine the effects of low-dose, long-term genistein exposure we have cultured MCF-7 breast cancer cells in 10nM genistein for 10-12 weeks and investigated whether or not this long-term genistein treatment (LTGT) altered the expression of oestrogen receptor alpha (ERalpha) and the activity of the PI3-K/Akt signalling pathway. This is known to be pivotal in the signalling of mitogens such as oestradiol (E(2)), insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). LTGT significantly reduced the growth promoting effects of E(2) and increased the dose-dependent growth-inhibitory effect of the PI3-K inhibitor, LY 294002, compared to untreated control MCF-7 cells. This was associated with a significant decreased protein expression of total Akt and phosphorylated Akt but not ERalpha. Rapamycin, an inhibitor of one of the down-stream targets of Akt, mammalian target of rapamycin (mTOR), also dose-dependently inhibited growth but the response to this drug was similar in LTGT and control MCF-7 cells. The protein expression of liver receptor homologue-1 (LRH1), an orphan nuclear receptor implicated in tumourigenesis was not affected by LTGT.*

The results show that LTGT results in a down-regulation of the PI3-K/Akt signalling pathway and may be a mechanism through which genistein could offer protection against breast cancer.*

Effects of EGF and Genistein on Proliferation of Human Breast Cancer Cell*

Wang Li, et al. Zhong Guo Gong Gong Wei Sheng. 2005, 21(6):656-657

Objective: To observe the effect of genistein and epidermal growth factor (EGF) on the proliferation of human breast cancer cell MCF-7 in vitro.*

Methods: Cell growth status was observed with methyl thiazolyl tetrazolium(MTT)when it was cultured with different doses of genistein or EGF for different time respectively, or with definite dose of genistein and EGF.*

Results: The growth of MCF-7 was inhibited when it was exposed to genistein over 25∞ mol/L seemed to be an optional dose. Cell growth was greatly promoted when it was exposed to EGF over 4.15nmol/L, the more the dose and time became, the greater the promoting effect was, and 8.30nmol/L seemed to be an optional dose. When MCF-7 was cultured with 50∞ mol/L genistein and 8.30nmol/L EGF simultaneously, an interaction effect was observed. The promoting effect of EGF was reversed by genistein.*

Conclusion Genistein can inhibit the proliferation of human breast Cancer cell MCF-7 induced by EGF.*

Progression of renal cell carcinoma is inhibited by genistein and radiation in an orthotopic model*

Gilda G Hillman, Yu Wang, Mingxin Che, Julian J Raffoul, Mark Yudelev, Omer Kucuk and Fazlul H Sarkar. BMC Cancer 2007, 7:4doi:10.1186/1471-2407-7-4

We have previously reported the potentiation of radiotherapy by the soy isoflavone genistein for prostate cancer using prostate tumour cells in vitro and orthotopic prostate tumour models in vivo. However, when genistein was used as single therapy in animal models, it promoted metastasis to regional para-aortic lymph nodes. To clarify whether these intriguing adverse effects of genistein are intrinsic to the orthotopic prostate tumour model, or these results could also be recapitulated in another model, we used the orthotopic metastatic KCI-18 renal cell carcinoma (RCC) model established in our laboratory.*

Methods: The KCI-18 RCC cell line was generated from a patient with papillary renal cell carcinoma. Following orthotopic renal implantation of KCI-18 RCC cells and serial in vivo kidney passages in nude mice, we have established a reliable and predictable metastatic RCC tumour model. Mice bearing established kidney tumours were treated with genistein combined with kidney tumour irradiation. The effect of the therapy was assessed on the primary tumour and metastases to various organs.*

Results: In this experimental model, the karyotype and histological characteristics of the human primary tumour are preserved. Tumour cells metastasize from the primary renal tumour to the lungs, liver and mesentery mimicking the progression of RCC in humans. Treatment of established kidney tumours with genistein demonstrated a tendency to stimulate the growth of the primary kidney tumour and increase the incidence of metastasis to the mesentery lining the bowel. In contrast, when given in conjunction with kidney tumour irradiation, genistein significantly inhibited the growth and progression of established kidney tumours. These findings confirm the potentiation of radiotherapy by genistein in the orthotopic RCC model as previously shown in orthotopic models of prostate cancer.*

Conclusion: Our studies in both RCC and prostate tumour models demonstrate that the combination of genistein with primary tumour irradiation is a more effective and safer therapeutic approach as the tumour growth and progression are inhibited both in the primary and metastatic sites.*

Dosage research

Positive

zero or 250 mg/kg - Prostate cancer

Genistein mechanisms and timing of prostate cancer chemoprevention in lobund-wistar rats.*

Wang J, Eltoum IE, Carpenter M, Lamartiniere CA. Asian Pac J Cancer Prev. 2009 Jan-Mar;10(1):143-50.

The objective of the present study was to determine if a specific window of development (neonatal/ prepubertal only, adult only, or life-time) is effective for genistein chemoprevention of prostate cancer, and the potential mechanisms of genistein chemoprevention in vivo. Male Lobund-Wistar (L-W) rats were fed zero or 250 mg genistein/kg AIN-76A diet at designated periods of time and then injected with N-methylnitrosourea (NMU) into the dorsolateral prostate (DLP) on day 70 for cancer initiation. Rats were necropsied at 11 months. The incidence of poorly differentiated (PD) carcinomas was 43.5% in rats fed a phytoestrogen-free AIN-76A diet only, 29.6% in rats provided genistein in the diet from postnatal days 1-35, 28.6% in rats fed genistein from months 3-11, and 20% in rats provided genistein from birth through 11 months. Genistein induces cell apoptosis and inhibits cell proliferation in both prostate cancerous and nontumorigenic DLP. These actions are accompanied with the regulation of PTEN/Akt-AR axis. Our data demonstrate that dietary genistein reduces the incidence of advanced prostate cancer induced by NMU in L-W rats during adult and lifetime exposure, the latter being more effective. The regulation of AR/Akt/PTEN axis by genistein may be one of the molecular mechanisms by which it inhibits cell proliferation and induces apoptosis, thus providing evidence of roles of genistein in prostate cancer prevention and treatment.*

2 g/kg / epatic mRNA and activity levels of NQO1

Effect of Dietary Genistein on Phase II and Antioxidant Enzymes in Rat Liver*

Wiegand H, Wagner AE, Boesch-Saadatmandi C, Kruse HP, Kulling S, Rimbach G. Cancer Genomics Proteomics. 2009 Mar-Apr;6(2):85-92.

Isoflavones are thought to be biologically active components in soy that play a role in the prevention of chronic diseases including cancer. How isoflavones may mediate their beneficial effects has not yet been fully established. Potential mechanisms of cellular action of isoflavones may include their ability to modulate gene expression and the activity levels of enzymes involved in antioxidant defence and the metabolism of xenobiotics including NAD(P)H (Nicotinamide-adenine-dinucleotide-phosphate) quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST).*

Although there is increasing evidence from cell culture studies that genistein, the major isoflavone present in soy, may regulate the expression of genes encoding for phase II and antioxidant enzymes, little is known about its effect in vivo.*

Feeding rats over 3 weeks with semisynthetic diets enriched with genistein (2 g/kg) significantly increased both the hepatic mRNA and activity levels of NQO1. The total GST activity did not change in response to dietary genistein supplementation, whereas the mRNA levels of individual GST isoenzymes were differentially modulated. The hepatic mRNA level of Gsta2 (class alpha 2) was significantly increased whereas the mRNA levels of Gstm2 (class mu 2) and Gstp1 (class pi 1) were significantly lowered due to genistein supplementation. The protein level of Nrf2 (Nuclear factor E2-related factor 2), a transcription factor involved in the regulation of phase II enzymes, was not altered by dietary genistein.*

Furthermore, genistein did not affect the hepatic enzyme activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) or liver lipid peroxidation and glutathione levels. The induction of NQO1 may be one mechanism by which dietary genistein improves the capacity of the liver to detoxify carcinogens.*

100 mg/kg / chronic colitis

Oral treatment with genistein reduces the expression of molecular and biochemical markers of inflammation in a rat model of chronic TNBS-induced colitis.*

Seibel J, Molzberger AF, Hertrampf T, Laudenbach-Leschowski U, Diel P. Eur J Nutr. 2009 Feb 21.

Background: Inflammatory bowel disease (IBD) in humans has a high incidence in Europe and the USA, whereas in East Asia, incidence has been historically low. The risk of IBD appears to increase in Asian immigrants adopting western lifestyles, suggesting a strong link of environmental/dietary factors in the development of IBD. Exposure to high levels of isoflavones such as genistein (Gen) in traditional East Asian diets has been associated with a decreased risk of developing breast cancer and may also be beneficial for the prevention of IBD. AIM: In this study, the effect of orally administered genistein on the inflammatory response in the TNBS-induced chronic colitis rat model was investigated.*

Methods: Eighteen male Wistar rats, aged 12 weeks, were randomized to one of three groups (n = 6). Two groups received a 2,4,6-trinitrobenzenesulfonic acid (TNBS) enema, then were treated daily by oral gavage with either Gen (100 mg/kg b.w.) or vehicle, for 14 days. The last group served as a control group, not receiving the TNBS enema. At the end of the 14 days, animals were killed and tissues collected. Molecular and biochemical inflammatory markers in the colon, specifically cyclooxygenase-2 (COX-2) and myeloperoxidase (MPO), were analyzed. In addition, to assess the efficacy of Gen treatment, relative wet weights of the accessory sexual organs, specifically prostate and the seminal vesicle, were compared between the groups treated or not with Gen.*

Results: Wet weights of both prostates and seminal vesicles were significantly (P < 0.01) reduced upon Gen administration. In the colon, expression of COX-2 mRNA and protein was reduced (P < 0.05) in the Gen treatment group, as compared to the control group, whereas there was no significant inhibitory effect of Gen on the expression of proliferating cell nuclear antigen. In Gen treated animals colon wet weight was not altered, however a decrease in MPO activity (P < 0.01) was seen.*

Conclusion: These results may provide evidence that oral administration of Gen exerts beneficial anti-inflammatory effects in a rodent model of TNBS-induced chronic colitis. While the sample size of this study was small, it nevertheless might encourage the realization of larger blinded randomized controlled studies for the proof of concept.*

100 and 200 mg/kg / mammary gland proliferation

Effects of genistein on the mammary gland proliferation of adult ovariectomised Wistar rats*

Hertrampf T, Schmidt S, Seibel J, Laudenbach-Leschowsky U, Degen GH, Diel P. Planta Med. 2006 Mar;72(4):304-10.

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E(2), does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.*

250 and 1000 mg/kg / testicular development

Dietary diethylstilbestrol but not genistein adversely affects rat testicular development

Fritz WA, Cotroneo MS, Wang J, Eltoum IE, Lamartiniere CA. J Nutr. 2003 Jul;133(7):2287-93.

Isoflavones, including genistein, contribute to the health benefits of a soy diet. However, the estrogenic activity of genistein suggests that there may be adverse effects on reproductive tract development. We investigated the potential of exposure to genistein (250 and 1000 mg/kg diet) and the synthetic estrogen and known male reproductive toxicant, diethylstilbestrol (DES, 75 micro g/kg diet) from d 21 to d 35 to alter rat testicular development. These dietary genistein concentrations resulted in serum concentrations that approximate or exceed concentrations in Asian men on a soy-containing diet. DES exposure reduced testicular weights, altered morphology and increased apoptosis in the seminiferous tubules. The effects of DES were accompanied by a reduction in androgen receptor (AR) protein concentrations, predominantly localized to Sertoli cells. Increased expression and immunostaining for the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK) 1 and 2 in spermatagonia and spermatocytes were also observed. Immunohistochemical analysis of serial sections demonstrated that greater EGFR expression correlated with increased cellular proliferation, rather than apoptosis, and reflected impaired testicular development in DES-treated rats. Genistein in the diet did not significantly alter testicular weights, morphology, AR, EGFR and ERK expression or apoptosis. However, the higher concentration significantly reduced testicular aromatase activity, an effect that may contribute to reduced estrogen concentrations and suppression of prostate cancer development. These data suggest that exposure to genistein in the diet at concentrations that result in serum concentrations at the upper limit of humans consuming soy products does not adversely affect testicular development, but may provide health benefits.*

zero, 20, 150 and 1000 mg/kg / Reproductive safety

Reproductive safety studies with genistein in rats

McClain RM, Wolz E, Davidovich A, Edwards J, Bausch J. Food Chem Toxicol. 2007 Aug;45(8):1319-32. Epub 2007 Jan 21.

Genistein is a phytoestrogen that occurs naturally in the diet and is found in a wide variety of plant-derived foods especially in soybeans and soy-based foods. There is wide spread interest in genistein and related phytoestrogens as chemopreventive agents for a variety of human diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Soy, and hence its constituents, such as genistein, have been consumed at high levels in several Asian populations for many centuries without any apparent adverse effects and to the contrary, many health benefits have been associated with the ingestion of soy based foods. Concern has been raised, however, of potential adverse effects due to the estrogenic and other activities of the isoflavones and thus a comprehensive series of safety studies was performed with genistein. To assess the teratogenic and fetal toxic potential of genistein, several studies were conducted. Genistein was tested in an in vitro rat whole embryo culture assay (WEC), which is a preliminary screen, for fetotoxic and teratogenic potential, over a concentration range of from 1 to 100 microg/mL. Treatment related anomalies were observed at concentrations of >or= 10 microg and at 100 microg/mL, all embryos were malformed. Two in vivo embryo fetal developmental safety studies were conducted with genistein by oral administration (gavage and dietary admix) in which there was no evidence for a teratogenic effect. In an oral (gavage) embryonic and fetal development pilot study, genistein was administered to rats at dose levels of 0, 20, 150 and 1000 mg/kg/day from days 6-20 of gestation to females that were allowed to litter and rear their offspring up to day 7 of lactation. A slight maternal toxicity at 1000 mg/kg/day was observed as indicated by decreased body weight and food consumption and at this dose, adverse effects in the pups were observed including increased pup mortality, poor general condition, reduced pup body weights, and reduced pup milk uptake. At the high dose of 1000 mg/kg, no external malformations were noted, however some minor visceral and skeletal variations were observed. At the low dose of 20 mg/kg/day, an increased mortality, reduced milk uptake, a decreased % male sex ratio, and decreased body weights during lactation were observed. Due to lack of effects at the mid dose and the small number of animals, a relationship to treatment was considered unlikely. In an oral (dietary admix) Prenatal developmental safety study, genistein was administered to rats at dose levels of 0, 5, 50, 100 and 500 mg/kg/day from day 5-21 of gestation. At 500 mg/kg, maternal body weight and food consumption were markedly reduced. The incidence of resorptions was markedly increased with a corresponding decrease in the number of live fetuses per dam. Fetal body weights were also reduced. No treatment-related teratogenic effects were noted during external, visceral and skeletal examination of fetuses or in body weight normalized anogenital distance. On the basis of these studies, it is concluded that genistein has no teratogenic potential in vivo at very high doses of up to 1000 mg/kg/day by oral gavage in the embryo-fetal toxicity pilot study or up to 500 mg/kg/day by dietary admix in the Prenatal developmental study even though these doses were maternally toxic and fetal-toxic. In vitro, genistein had teratogenic potential at high concentrations in the WEC screening assay, however this was not predictive of the in vivo findings. On the basis of the definitive Prenatal development study, the NOAEL for maternal toxicity and adverse effects on embryonic development was considered to be 100 mg/kg/day when administered orally by dietary admix.*

20 mg/kg / DMBA-induced genotoxicity

Antigenotoxic effect of genistein against 7,12-dimethylbenz[a]anthracene induced genotoxicity in bone marrow cells of female Wistar rats*

Pugalendhi P, Manoharan S, Panjamurthy K, Balakrishnan S, Nirmal MR. Pharmacol Rep. 2009 Mar-Apr;61(2):296-303.

Carcinogen induced mutation in somatic cells leads to genetic instability, which is considered as an important facet of carcinogenesis. Agents that inhibit DNA adduct formation, stimulate DNA repair mechanisms, and possess antioxidant functions are considered as antigenotoxic agents. Genistein, the major isoflavone of soy products, protects animals against experimentally induced mammary and prostate cancers. 7,12-Dimethylbenz[a]anthracene (DMBA), a potent site-specific carcinogen, induce mutations in DNA through its active metabolite, dihydrodiol epoxide, what is a crucial step in cancer initiation. The antigenotoxic effect of genistein against DMBA-induced genotoxicity has been investigated in the present study by analyzing the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and chromosomal aberrations as cytogenetic end-points. The status of lipid peroxidation, antioxidants and detoxication agents were used as biochemical end-points to assess the antigenotoxic effect of genistein. Elevated MnPCEs frequency, marked chromosomal aberrations and enhanced status of lipid peroxidation, antioxidants and detoxication agents were observed in DMBA-treated animals. Oral pretreatment of genistein (20 mg/kg b.w.) for 5 days to DMBA-treated animals significantly reduced the frequency of micronucleus formation and chromosomal abnormalities as well as reversed the status of biochemical variables. Our results suggest that genistein has potent antigenotoxic effect against DMBA-induced genotoxicity.*

up to 2000 mg/kg / Genetic toxicity

Genetic toxicity studies with genistein

Michael McClain R, Wolz E, Davidovich A, Bausch J. Food Chem Toxicol. 2006 Jan;44(1):42-55. Epub 2005 Sep 28.

Genistein is a phytoestrogen that occurs naturally in the diet especially in soybeans and soy-based foods. Genistein and related phytoestrogens are of interest as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence of reduced cancer rates in populations with a high intake of soy. Although soy and its constituents have been consumed at high levels in Asian populations without apparent adverse effects, concern has been raised of potential adverse effects due to estrogenic and other activities of the isoflavones. In these studies, genistein was evaluated for mutagenicity and clastogenicity in vitro in the S. typhimurium assay (Ames Test), the mouse lymphoma assay and in vivo in the micronucleus test in mice and rats. There was no evidence for a mutagenic effect in the in vitro S. typhimurium assay with and without metabolic activation (S9). In the in vitro mouse lymphoma assay, genistein increased resistant mutants with and without metabolic activation (S9), which were predominantly small colonies indicating that genistein acts as a clastogen. Three independent in vivo micronucleus tests were performed in Moro mice, RAIf rats and Wistar rats. MORO male and female mice were treated orally for 14 days at doses up to 20 mg/kg/day. RAIf and Wistar male and female rats were treated orally at doses up to 2000 mg/kg without an increase in micronuclei in treated mice or rats. It is concluded that genistein was not mutagenic in the S. typhimurium assay or mutagenic or clastogenic in vivo in the mouse and rat micronucleus test. In the mouse lymphoma assay, genistein induced an increase of predominantly small colonies indicating that genistein acts as a clastogen. This observation is in agreement with published data on the inhibitory action of genistein on topoisomerase II, which is known to lead to chromosomal damage with a threshold dose response.*

Negative

750 p.p.m. / mammary tumors

Dietary genistein results in larger MNU-induced, oestrogen-dependent mammary tumours following ovariectomy of Sprague-Dawley rats*

Allred CD, Allred KF, Ju YH, Clausen LM, Doerge DR, Schantz SL, Korol DL, Wallig MA, Helferich WG. Carcinogenesis. 2004 Feb;25(2):211-8. Epub 2003 Oct 24.

Due to the estrogenic properties of soy-derived isoflavones, many postmenopausal women are using these compounds as a natural alternative to hormone replacement therapy (HRT). How isoflavones impact breast cancer in postmenopausal women is important, because a majority of breast cancer cases occur in this age group. Chemical induction of mammary tumours in female rats has been used to determine that exposure of the mammary gland to soy isoflavones prior to tumour induction is protective against tumour formation. Here we investigate the effect of dietary genistein on mammary tumours that have already formed. The study was designed to determine the action of dietary genistein in a low endogenous oestrogen environment as is observed in postmenopausal women.*

Animals were ovariectomised (OVX) after mammary tumour development and were then placed into one of three treatment groups: positive-control (OVX+ estradiol implant), genistein (OVX+ 750 p.p.m. genistein) and negative-control (OVX alone). Tumours were distinguished as malignant or benign by histopathological examination and were further characterized as either oestrogen-dependent or oestrogen-independent using immunohistochemistry to identify the presence of both oestrogen receptor (ER) alpha and the progesterone receptor (PR).*

Genistein at 750 p.p.m. increased the weight of oestrogen-dependent adenocarcinomas in ovariectomised rats compared with the negative-control animals. Genistein treatment also resulted in a higher percentage of proliferative cells in tumours and increased uterine weights when compared with negative-control animals. Collectively, these effects are probably due to the estrogenic activity of genistein. Plasma genistein concentrations in animals fed the isoflavone-containing diet were at physiological levels relevant to human exposure. Estradiol concentrations in ovariectomised animals not receiving an estradiol supplement were similar to those observed in postmenopausal women.*

The data suggest that in an endogenous oestrogen environment similar to that of a postmenopausal woman, dietary genistein can stimulate the growth of a mammary carcinogen MNU-induced oestrogen-dependent mammary tumours.*

Biological effects of a diet of soy protein rich in isoflavones on the menstrual cycle of premenopausal women.*

Cassidy A, Bingham S, Setchell KD. Am J Clin Nutr. 1994 Sep;60(3):333-40.

Isoflavones, in the form of a diet rich in soy protein, were studied for their effect on the menstrual cycle of premenopausal women (Cassidy et al., 1994). Mid-cycle increases of luteinizing hormone and follicle-stimulating hormone were significantly reduced during the dietary intervention. Isoflavones such as genistein could, because of their antiestrogen effects, be useful especially in the management of women at high risk for breast cancer and may also help explain the relatively low incidence in Japanese and Chinese women with a high soy intake. Extracts of some plants contain antihormonal components, explaining some long-standing uses in traditional medicine. Miksicek (1995) surveyed the structural features of polycyclic phenols associated with estrogenic activity. Natural estrogens belong to several chemically related classes: chalcones, flavanones, flavones, flavonols, and isoflavones. Auf’mkolk et al. (1986) noted the action of aurones from plant extracts to inhibit rat liver iodothyronine deiodinase, the regulator of extrathyroidal thyroxine metabolism. Some aurones produced potent, concentration-dependent inhibition of three different metabolic monodeiodination pathways catalyzed by rat liver microsomal type I iodothyronine deiodinase. The most potent plant-derived inhibitors of the deiodinase system (IC50, 0.50 mM) were the 39,49,4,6-(tetra)trihydroxyaurones. Computer graphic modeling studies were used to confirm aurone conformations with the conformation of the thyroid hormones and suggested the possibility of using this procedure to design other deiodinase inhibitors (Koehrle et al., 1986). Genistein strongly inhibited the effect of an A1-adenosine receptor agonist on thyroid-stimulating hormoneinduced PLC activation in FRTL-5 thyroid cells. Genistein also competitively inhibited adenosine-induced cAMP accumulation in pertussis toxin-treated cells (Okajima et al., 1994).*

Quercetin proved to be an effective inhibitor of insulin receptor tyrosine kinase-catalyzed phosphorylation of a glutamic acid-tyrosine random copolymer, while insulin stimulated autophosphorylation of the receptor itself. In rat adipocytes, quercetin inhibited glucose transport, oxidation, and incorporation into lipids (Shisheva and Shechter, 1992). With respect to alteration of transmembrane transport systems, it is worth noting that hexose transport in a human diploid fibroblast cell line was inhibited by quercetin (Salter et al., 1978). Vera et al. (1996) also showed that genistein was an inhibitor of hexose and dehydroascorbic acid transport through the glucose transporter GLUT.*

Genistein caused 50% inhibition of [3H]estradiol binding to the estrogen receptor. However, this compound had a bimodal effect on the growth of human mammary cancer cells (MCF-7); low concentrations (1028–1026 M) stimulated growth, while 1025 M or greater caused inhibition. Genistein potently inhibited the growth of human breast carcinoma cell lines MDA-468 (estrogen receptor negative) and MCF-7 and MCF-7-D40 (estrogen receptor positive) with IC50 values of 6.5 to 12 mg/ml (Peterson and Barnes, 1991). Biochanin A and daidzein were less effective, and the glycosides of genistein and daidzein were essentially inactive. The activity of the isoflavones was not dependent on the presence of the estrogen receptor.*

Of interest also was the observation that the growth-inhibitory activity of genistein and biochanin A was not affected in the cell line MCF-7-D40, which overexpresses gp 170, the gene product responsible for multidrug resistance. The low rate of breast cancer in Oriental women may be related to the high isoflavonecontaining soy content of their diet. Catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of polyphenolic compounds in red wine, were shown to inhibit groeth of human breast cancer cells at picomolar concentrations (Damianaki et al., 2000). The same compounds were also shown to potently inhibit human prostate cancer cells (Kampa et al., 2000). Retinoids and carotenoids also have inhibitory activity on breast cancer cell proliferation in vitro (Prakash et al., 2000).*

3-Methoxyquercetin, quercetin, and ipriflavone (a synthetic flavanone), but not rutin or hesperidin, induced type II EBS in both ER-positive and ER-negative human breast cancer cell lines (Scambia et al., 1993). The quercetin effect was concentration-related and required synthesis of mRNA and protein. The flavonoid stimulated enhancement of type II EBS correlated well with increased sensitivity of the tumor cells to the inhibitory effects of low concentrations of quercetin. This same group of investigators also reported that meningiomas possessed type II EBS to which quercetin bound, but not rutin or hesperidin. Genistein inhibited the in vitro growth of human T cell leukemia (Jurkat) and L-929 mouse transformed fibroblast cells (Pagliacci et al., 1993). Cell cycle analysis revealed a G2/M cell cycle arrest after genistein treatment.*

Synergistic inhibitory effects of genistein and tamoxifen on human dysplastic and malignant epithelial breast cells in vitro*

Tanos V, Brzezinski A, Drize O, et al. European Journal of Obstetrics & Gynecology and Reproductive Biology 102 (2002) 188–194

Objective: Genistein is a phytoestrogen with in vitro anticancerogenic activity. We examined in vitro the effects of genistein alone, or in combination with estradiol and tamoxifen, on the growth of human dysplastic and malignant epithelial breast cell lines.*

Methods: Dysplastic breast cell lines (MCF-10A1, MCF-ANeoT, MCF-T63B) and cell lines of breast cancer (MCF-7, MDA-231, MDA-435) were cultured as monolayers in RPMI 1640 medium supplemented with 10% fetal bovine serum, and L-glutamine. After preincubation of 20 h, genistein (1, 2.5, 5, 7.5 and 10 mg/ml) alone or in combination with estrogen or tamoxifen was added to the cultured cells. The cells were treated continuously for 72 h and then the growth rate was assessed colorimetrically. Stepwise multiple linear regression analysis was used to evaluate the effect of genistein, tamoxifen, and estradiol on cell proliferation.*

Results: Genistein had a significant (dose-dependent) inhibitory effect on the proliferation of both dysplastic (P < 0:0001) and malignant (P < 0:0001) cells. The growth inhibition was significantly higher P < 0:0001 in dysplastic cells compared to the cancer cells. Addition of tamoxifen to genistein further inhibited the proliferation of both cell types, reflecting a synergistic antiproliferative effect on dysplastic cells P < 0:0001 and an additive growth inhibition effect P < 0:0003 on malignant cells. Estradiol significantly (P ¼ 0:005) stimulated the growth of dysplastic cell lines while a significant (P ¼ 0:003) antiproliferative effect on growth of the malignant cells was observed. The concentration of estrogen receptor (ER) had no significant effect on growth rates and did not modulate the effects of genistein or tamoxifen.*

Conclusions: Genistein (1–10 mg/ml) inhibits the growth of dysplastic and malignant epithelial breast cancer cells in vitro and the addition of tamoxifen (106, 107 M) has a synergistic/additive inhibitory effect. These effects are not modulated by the presence of ER.*

The combined genistein and tamoxifen treatment was of particular interest since a synergistic effect on dysplastic cells growth inhibition and an additive antiproliferative effect on malignant cells were evident. These synergistic or additive effects were evident in all types of cells and they were independent of ER expression. The combination might also be effective as an adjuvant therapy in women with breast cancer. The fact that genistein has inhibitory growth effect on breast cells independent of the ER presence, gives the opportunity to be used as first line treatment in cases with ER negative tumors. Even patients that have been taking tamoxifen for many years and they have to stop preventively they could use genistein.*

Molecular effects of genistein on estrogen receptor mediated pathways*

Wang TTY, Sathyamoorthy N, Phang JM. Carcinogenesls vol.17 no.2 pp.271-275, 1996

Genistein, a component of soy products, may play a role in the prevention of breast and prostate cancer. However, little is known about the molecular mechanisms involved. In the present study, we examined the effects of genistein on the estrogen receptor positive human breast cancer cell line MCF-7. We observed that genistein stimulated estrogen-responsive pS2 mRNA expression at concentrations as low as 10−8 M and these effects can be inhibited by tamoxifen. We also showed that genistein competed with (3H)estradiol binding to the estrogen receptor with 50% inhibition at 5 × 10−7 M. Thus, the estrogenic effect of genistein would appear to be a result of an interaction with the estrogen receptor. The effect of genistein on growth of MCF-7 cells was also examined. Genistein produceda concentration-dependent effect on the growth of MCF-7 cells. At lower concentrations (10−8-10−6 M) genistein stimulated growth, but at higher concentrations (>10−5M) genistein inhibited growth. The effects of genistein on growth at lower concentrations appeared to be via the estrogen receptor pathway, while the effects at higher concentrations were independent of the estrogen receptor. We also found that genistein, thoughestrogenic, can interfere with the effects of estradiol. In addition, prolonged exposure to genistein resulted in a decrease in estrogen receptor mRNA level as well as a decreased response to stimulation by estradiol.*

Results from the present study provide evidence for the anti-estrogenic effects of genistein. We observed that not only does genistein compete with estradiol for binding to the ER, but it also interferes with the effects of estrogen. When genistein and estradiol were added together in the pS2 expression assay, the expression of pS2 mRNA was lower than when these compounds were added individually. In addition, the duration of exposure to genistein also appeared to be important. MCF- 7 cells treated with genistein showed a decrease in the response to estrogen as well as a decrease in ER mRNA levels. This is similar to cells treated with estradiol, as has been described by others (1). Taken together, the results support the idea that genistein at physiologically achievable concentrations may, in fact, interfere with the action of estrogen through direct competition for binding to ER and by reducing ER expression. These effects could be considered anti-estrogenic, consistent with the proposed cancer-preventive effects of soy diet.*

In summary, our findings provide support for a model in which phyto-estrogens such as genistein, at physiological concentrations, may exert their effect by modulating estrogenic pathways. This interaction may occur directly through competition with estrogen for binding to ER. In addition, prolonged exposure may result in the reduction of ER expression and hence lead to decreased responsiveness to endogenous estrogens.*

Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells.*

Wang C, Kurzer MS.Nutr Cancer. 1997;28(3):236-47.

Thirteen isoflavonoids, flavonoids, and lignans, including some known phytoestrogens, were evaluated for their effects on DNA synthesis in estrogen-dependent (MCF-7) and -independent (MDA-MB-231) human breast cancer cells. Treatment for 24 hours with most of the compounds at 20-80 microM sharply inhibited DNA synthesis in MDA-MB-231 cells. In MCF-7 cells, on the other hand, biphasic effects were seen. At 0.1-10 microM, coumestrol, genistein, biochanin A, apigenin, luteolin, kaempferol, and enterolactone induced DNA synthesis 150-235% and, at 20-90 microM, inhibited DNA synthesis by 50%. Treatment of MCF-7 cells for 10 days with genistein or coumestrol showed continuous stimulation of DNA synthesis at low concentrations. Time-course experiments with genistein in MCF-7 cells showed effects to be reversed by 48-hour withdrawal of genistein at most concentrations. Induction of DNA synthesis in MCF-7 cells, but not in MDA-MB-231 cells, is consistent with an estrogenic effect of these compounds. Inhibition of estrogen-dependent and -independent breast cancer cells at high concentrations suggests additional mechanisms independent of the estrogen receptor. The current focus on the role of phytoestrogens in cancer prevention must take into account the biphasic effects observed in this study, showing inhibition of DNA synthesis at high concentrations but induction at concentrations close to probable levels in humans.*

Effects of phytoestrogens on DNA synthesis in MCF-7 cells in the presence of estradiol or growth factors.*

Wang C, Kurzer MS. Nutr Cancer. 1998;31(2):90-100.

Phytoestrogen effects on estrogen action and tyrosine kinase activity have been proposed to contribute to cancer prevention. To study these mechanisms, a number of phytoestrogens and related compounds were evaluated for their effects on DNA synthesis (estimated by thymidine incorporation analysis) in estrogen-dependent MCF-7 cells in the presence of estradiol (E2), tamoxifen, insulin, or epidermal growth factor. We observed that 1) at 0.01-10 microM, genistein and coumestrol enhanced E2-induced DNA synthesis, as did 10 microM enterolactone. Chrysin at 1.0-10 microM and 10 microM luteolin or apigenin inhibited E2-induced DNA synthesis, as did all compounds at > 10 microM, 2) tamoxifen enhanced genistein-induced DNA synthesis but inhibited DNA synthesis induced by all other compounds, and 3) genistein enhanced insulin- and epidermal growth factor-induced DNA synthesis at 0.1-1.0 and 0.1-10 microM, respectively. At higher concentrations, inhibition was observed. Similar effects were seen with coumestrol. In conclusion, the effects of phytoestrogens in the presence of E2 or growth factors are concentration dependent and variable. At low concentrations, genistein and coumestrol significantly enhanced E2-induced and tyrosine kinase-mediated DNA synthesis; at high concentrations, inhibition was observed. Differing effects were observed with the other compounds. The variable effects of phytoestrogens on DNA synthesis must be considered when their roles in cancer prevention or treatment are evaluated.*

Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro*

Zava DT, Duwe G. Nutr Cancer. 1997;27(1):31-40.

Experimental and epidemiologic studies support the view that soyfoods prevent cancer as well as diseases and symptoms associated with estrogen deficiency. Recent research suggests that the isoflavonoid genistein, a phytoestrogen found in abundance in soyfoods, may be one of the principal molecular components responsible for these health benefits. In this study we investigated the effects of a broad physiologically relevant concentration range of genistein on estrogen receptor (ER) binding, induction of the estrogen-regulated antigen pS2, and cell proliferation rate in ER(+) and ER(-) human breast cancer cells grown in vitro. Dose response to genistein was compared with that of estradiol, tamoxifen, and several other structurally similar iso- and bioflavonoids (e.g., equol, kaempferol, and quercetin). Our results revealed that genistein has potent estrogen agonist and cell growth-inhibitory actions over a physiologically achievable concentration range (10 nM-20 microM). Other flavonoids over the same concentration range were good estrogen agonists and poor cell growth inhibitors (equol) or poor estrogen agonists and potent growth inhibitors (kaempferol and quercetin). The growth-inhibitory actions of flavonoids were distinctly different from those of triphenyl antiestrogens like tamoxifen. In summary, our results reveal that genistein is unique among the flavonoids tested, in that it has potent estrogen agonist and cell growth-inhibitory actions over a physiologically relevant concentration range.*

Effects of genistein and structurally related phytoestrogens on cell cycle kinetics and apoptosis in MDA-MB-468 human breast cancer cells*

Balabhadrapathruni S, Thomas TJ, Yurkow EJ, Amenta PS, Thomas T. Oncol Rep. 2000 Jan-Feb;7(1):3-12.

We have studied the effects of phytoestrogens (genistein, quercetin, daidzein, biochanin A and kaempferol) on proliferation, cell cycle kinetics, and apoptosis of MDA-MB-468 breast cancer cells. Genistein and quercetin inhibited cell growth with IC50 values of 8.8 and 18.1 muM, respectively, while the other phytoestrogens were less effective. Flow cytometric analysis showed G2/M cell cycle arrest with 25 muM and higher concentrations of genistein. At 100 muM, genistein, quercetin and kaempferol caused accumulation of 70, 60 and 35% of cells, respectively, in G2/M phase by 24 h. In contrast, biochanin A and daidzein were ineffective. APO-BRDU analysis revealed apoptosis with 10 muM genistein (19.5%), reaching 86% at 100 muM. Apoptosis by genistein was confirmed by Hoechst 33342 staining and fluorescence microscopy. With 100 muM quercetin, 47% of the cells were apoptotic, while the other bioflavonoids had little effect. Genistein treatment resulted in a biphasic response on cyclin B1: 70% increase in cyclin B1 level at 25 muM, and 50 and 70% decrease at 50 and 100 muM, respectively. In contrast, the action of quercetin involved an increase in cyclin B1 level. Genistein had no effect on cdc2 level up to 50 muM concentration; however, there was a decrease in the phosphorylated form of the protein at 100 muM. Quercetin had no effect on cdc2 levels. Our results suggest that the action of genistein and quercetin involves G2/M arrest and apoptosis in MDA-MB-468 cells. Biochanin A and daidzein, although structurally related to genistein, did not share this mechanism. Thus, structurally related phytoestrogens have discrete target sites and mechanisms in their growth inhibitory action on breast cancer cells.*

Soy isoflavones have an antiestrogenic effect and alter mammary promoter hypermethylation in healthy premenopausal women*

Qin W, Zhu W, Shi H, Hewett JE, Ruhlen RL, MacDonald RS, Rottinghaus GE, Chen YC, Sauter ER. Nutr Cancer. 2009;61(2):238-44.

We determined if soy isoflavones have dose-related estrogenic and methylation effects. Thirty-four healthy premenopausal women were randomized to 40 mg or 140 mg isoflavones daily through one menstrual cycle. Breast specific and systemic estrogenic effects were assessed measuring the estrogenic marker complement (C)3 and changes in cytology, whereas methylation assessment of 5 cancer related genes (p16, RASSF1A, RARbeta2, ER, and CCND2) was performed on intraductal specimens. Serum genistein significantly increased after consuming both isoflavone doses. Cytology did not significantly change at either isoflavone dose. Serum C3 levels posttreatment were inversely related to change in serum genistein (r =-0.76, P = 0.0045) in women consuming low but not high dose isoflavones. The RAR beta 2 hypermethylation increase posttreatment correlated with the posttreatment genistein level considering the entire group (r = 0.67, P = 0.0017) and those receiving high-dose isoflavones (r = 0.68, P = 0.021). At the low but not the high isoflavone dose, CCND2 hypermethylation increase correlated with posttreatment genistein levels (r = 0.79, P = 0.011). In summary, the inverse correlation between C3 and genistein suggests an antiestrogenic effect. Isoflavones induced dose-specific changes in RARbeta2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.*

Estrogen Receptor α Mediates the Proliferative but Not the Cytotoxic Dose-Dependent Effects of Two Major Phytoestrogens on Human Breast Cancer Cells*

Maggiolini M, Bonofiglio D, Marsico S, et al. Molecular Pharmacology September 1, 2001 vol. 60 no. 3 595-602

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ERα by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ERα mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ERα and ERβ fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ERα and ERβ confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ERα-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.*