Genistein 99%



Mousavi Y, Adlercreutz H. Steroids. 1993 Jul;58(7):301-4.

Studies have indicated a correlation between a high level of urinary lignans and isoflavonoid phytoestrogens, particularly genistein, and a low incidence of hormone-dependent cancers, such as breast and prostate cancer. Previously it has been observed that a vegetarian diet is associated with high plasma levels of sex hormone-binding globulin (SHBG), reducing clearance of sex hormones and probably risk of breast and prostate cancer. In the present study we investigated the in vitro effect of genistein on the production of SHBG by human hepatocarcinoma (Hep-G2) cells in culture and its effect on cell proliferation. We found that genistein not only highly significantly increases the SHBG production by Hep-G2 cells, but also suppresses the proliferation of these cancer cells already at a stage when SHBG production continues to be high. We conclude that, in addition to the lignan enterolactone, the most abundant urinary isoflavonoid genistein stimulates SHBG production and inhibits Hep-G2 cancer cell proliferation.

Nutrition and Cancer.

Lee J, Ju J, Park S, et al. Nutrition and Cancer. Volume 64, Issue 1, 2012 DOI:10.1080/01635581.2012.630161

Elevated levels of insulin-like growth factor-1 (IGF-1) are associated with an increased risk of several different cancers, including prostate cancer. Inhibition of IGF-1 and the downstream signaling pathways mediated by the activation of the IGF-1 receptor (IGF-1R) may be involved in inhibiting prostate carcinogenesis. We investigated whether genistein downregulated the IGF-1/IGF-1R signaling pathway and inhibited cell growth in hormone refractory PC-3 prostate cancer cells. Genistein treatment caused a significant inhibition of IGF-1-stimulated cell growth. Flow cytometry analysis revealed that genistein significantly decreased the number of IGF-1-stimulated cells in the G0/G1 phase of the cell cycle. In IGF-1-treated cells, genistein effectively inhibited the phosphorylation of IGF-1R and the phosphorylation of its downstream targets, such as Src, Akt, and glycogen synthase kinase-3? (GSk-3? ). IGF-1 treatment decreased the levels of E-cadherin but increased the levels of ? -catenin and cyclin D1. However, genistein treatment greatly attenuated IGF-1-induced ?-catenin signaling that correlated with increasing the levels of E-cadherin and decreasing cyclin D1 levels in PC-3 cells. In addition, genistein inhibited T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity. These results showed that genistein effectively inhibited cell growth in IGF-1-stimulated PC-3 cells, possibly by inhibiting downstream of IGF-1R activation.

Anticancer Res

Shao ZM, Shen ZZ, Fontana JA, Barsky SH. Anticancer Res. 2000 Jul-Aug;20(4):2409-16.

Genistein, a natural flavone found in soy has been postulated to be responsible for lowering the rate of breast cancer in Asian women. Our previous studies have shown that genistein exerts multiple suppressive effects on both estrogen receptor positive (ER+) as well as estrogen receptor negative (ER-) human breast carcinoma lines suggesting that the mechanisms of these effects may be independent of ER pathways. In the present study however we provide evidence that in the ER+ MCF-7, T47D and 549 lines but not in the ER-MDA-MB-231 and MDA-MB-468 lines both presumed "ER-dependent" and "ER-independent" actions of genistein are mediated through ER pathways. Genistein's antiproliferative effects are estrogen dependent in these ER+ lines, being more pronounced in estrogen-containing media and in the presence of exogenous 17-beta estradiol. Genistein also inhibits the expression of ER-downstream genes including pS2 and TGF-beta in these ER+ lines and this inhibition is also dependent on the presence of estrogen. Genistein inhibits estrogen-induced protein tyrosine kinase (PTK) activity. Genistein is only a weak transcriptional activator and actually decreases ERE-CAT levels induced by 17-beta estradiol in the ER+ lines. Genistein also decreases steady state ER mRNA only in the presence of estrogen in the ER+ lines thereby manifesting another suppression of and through the ER pathway. Our observations resurrect the hypothesis that genistein functions as a "good estrogen" in ER+ breast carcinomas. Since chemopreventive effects of genistein would be targeted to normal ER-positive ductal-lobular cells of the breast, this "good estrogen" action of genistein is most relevant to our understanding of chemoprevention.

Mol Med Rep

Choi EJ, Kim GH. Mol Med Rep. 2013 Mar;7(3):781-4. doi: 10.3892/mmr.2013.1283. Epub 2013 Jan 21.

In this study, we investigated the antiproliferative activity of the isoflavones daidzein and genistein in three breast cancer cell lines with different patterns of estrogen receptor (ER) and c-erbB-2 protein expression (ERα-positive MCF-7 cells, c-erbB-2-positive SK-BR-3 cells and ERα/c-erbB-2-positive ZR-75-1). After treatment at various concentrations (1-200 µM for 72 h), the effect of daidzein and genistein on the proliferation of different cell types varied; these effects were found to be associated with ER? and c-erbB-2 expression. Daidzein and genistein exhibited biphasic effects (stimulatory or inhibitory) on proliferation and ER? expression in MCF-7 cells. Although 1 µM daidzein significantly stimulated cell growth, ER? expression was unaffected. However, genistein showed marked increases in proliferation and ER? expression after exposure to <10 µM genistein. Notably, the inhibition of cell proliferation by 200 µM genistein was greater compared to that by daidzein at the same concentration. Daidzein and genistein significantly inhibited proliferation of SK-BR-3 and ZR-75-1 cells in a dose-dependent manner. In addition, ER? and c-erbB-2 expression was reduced by daidzein and genistein in both SK-BR-3 and ZR-75-1 cells in a dose-dependent manner. However, the effect of genistein was greater compared to that of daidzein. In conclusion, the isoflavones daidzein and genistein showed anti-breast cancer activity, which was associated with expression of the ER? and c-erbB-2 receptors.

Mol Cell Endocrinol

Pugeat M, Nader N, Hogeveen K, et al. Mol Cell Endocrinol. 2010 Mar 5;316(1):53-9. doi: 10.1016/j.mce.2009.09.020. Epub 2009 Sep 26.

Sex hormone-binding globulin (SHBG) is the main transport binding protein for sex steroid hormones in plasma and regulates their accessibility to target cells. Plasma SHBG is secreted by the liver under the control of hormones and nutritional factors. In the human hepatoma cell line (HepG2), thyroid and estrogenic hormones, and a variety of drugs including the antioestrogen tamoxifen, the phytoestrogen, genistein and mitotane (Op'DDD) increase SHBG production and SHBG gene promoter activity. In contrast, monosaccharides (glucose or fructose) effectively decrease SHBG expression by inducing lipogenesis, which reduces hepatic HNF-4alpha levels, a transcription factor that play a critical role in controlling the SHBG promoter. Interestingly, diminishing hepatic lipogenesis and free fatty acid liver biosynthesis also appear to be associated with the positive effects of thyroid hormones and PPARgamma antagonists on SHBG expression. This mechanism provides a biological explanation for why SHBG is a sensitive biomarker of insulin resistance and the metabolic syndrome, and why low plasma SHBG levels are a risk factor for developing hyperglycemia and type 2 diabetes, especially in women. These important advances in our knowledge of the regulation of SHBG expression in the liver open new approaches for identifying and preventing metabolic disorder-associated diseases early in life.

Cancer Epidemiol Biomarkers Prev

Low YL, Taylor JI, Grace PB, Dowsett M, Scollen S,et al. Cancer Epidemiol Biomarkers Prev. 2005 Jan;14(1):213-20.

Cross-sectional studies investigating the relationship between phytoestrogens in diet, urine, or blood with plasma estradiol and sex hormone binding globulin (SHBG) have been inconclusive. We investigated the relationship among phytoestrogen exposure, polymorphisms in the ESR1, COMT, CYP19, and SHBG genes, and plasma estradiol and SHBG levels in 125 free-living postmenopausal women taking part in a cohort study (European Prospective Investigation of Cancer and Nutrition-Norfolk) using three different markers: dietary, urinary, and serum phytoestrogens. Phytoestrogen levels (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) in spot urine and serum were analyzed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, respectively. Plasma estradiol and SHBG were measured by immunoassays. Adjusting for age and body mass index, urinary daidzein, genistein, glycitein, and serum daidzein and glycitein were negatively correlated with plasma estradiol (R = -0.199 to -0.277, P <0.03), with particularly strong associations found in the 18 women with CC genotype for ESR1 PvuII polymorphism (R = -0.597 to -0.834, P < 0.03). The negative correlations observed between isoflavones and estradiol in women as a whole became no longer significant when we excluded women with ESR1 PvuII CC genotype, indicating that the correlations observed were due mainly to this group of women. There was no relationship between dietary isoflavones and plasma estradiol and no association was found between any of the dietary, urinary, and serum phytoestrogen and plasma SHBG or between these factors and polymorphisms in CYP19, SHBG, and COMT. We conclude that higher isoflavone exposure is associated with lower plasma estradiol in postmenopausal women and that this preliminary study is suggestive of the involvement of diet-gene interactions.

Mol Nutr Food Res

Rietjens IM, Sotoca AM, Vervoort J, Louisse J. Mol Nutr Food Res. 2013 Jan;57(1):100-13. doi: 10.1002/mnfr.201200439. Epub 2012 Nov 23.

Isoflavones are phytoestrogens that have been linked to both beneficial as well as adverse effects in relation to cell proliferation and cancer risks. The present article presents an overview of these seemingly contradicting health effects and of mechanisms that could be involved in this dualistic mode of action. One mechanism relates to the different ultimate cellular effects of activation of estrogen receptor (ER) ?, promoting cell proliferation, and of ER?, promoting apoptosis, with the major soy isoflavones genistein and daidzein activating especially ER?. A second mode of action includes the role of epigenetics, including effects of isoflavones on DNA methylation, histone modification and miRNA expression patterns. The overview presented reveals that we are only at the start of unraveling the complex underlying mode of action for effects of isoflavones, both beneficial or adverse, on cell proliferation and cancer risks. It is evident that whatever model system will be applied, its relevance to human tissues with respect to ER? and ER? levels, co-repressor and co-activator characteristics as well as its relevance to human exposure regimens, needs to be considered and defined.


Rahal OM, Simmen RC. Endocrinology. 2011 Sep;152(9):3409-21. doi: 10.1210/en.2011-1085. Epub 2011 Jun 28.

Mammary stromal adipocytes constitute an active site for the synthesis of the adipokine, adiponectin (APN) that may influence the mammary epithelial microenvironment. The relationship between "local," mammary tissue-derived APN and breast cancer risk is poorly understood. Here, we identify a novel mechanism of APN-mediated signaling that influences mammary epithelial cell proliferation, differentiation, and apoptosis to modify breast cancer risk. We demonstrate that early dietary exposure to soy protein isolate induced mammary tissue APN production without corresponding effects on systemic APN levels. In estrogen receptor (ER)-negative MCF-10A cells, recombinant APN promoted lobuloalveolar differentiation by inhibiting oncogenic signal transducer and activator of transcription 3 activity. In ER-positive HC11 cells, recombinant APN increased ER? expression, inhibited cell proliferation, and induced apoptosis. Using the estrogen-responsive 4X-estrogen response element promoter-reporter construct to assess ER transactivation and small interfering RNA targeting of ER? and ER?, we show that APN synergized with the soy phytoestrogen genistein to promote ER? signaling in the presence of estrogen (17?-estradiol) and ER?-specific agonist 2,3-bis(4-hydroxyphenyl)-propionitrile and to oppose ER? signaling in the presence of the ER?-specific agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol. The enhancement of ER? signaling with APN + genistein cotreatments was associated with induction of apoptosis, increased expression of proapoptotic/prodifferentiation genes (Bad, p53, and Pten), and decreased antiapoptotic (Bcl2 and survivin) transcript levels. Our results suggest that mammary-derived APN can influence adjacent epithelial function by ER-dependent and ER-independent mechanisms that are consistent with reduction of breast cancer risk and suggest local APN induction by dietary factors as a targeted approach for promotion of breast health.


Wang Sh, DeGroff VL, Clinton SK. J. Nutr. July 1, 2003 vol. 133 no. 7 2367-2376

We examined the ability of polyphenols from tomatoes and soy (genistein, quercetin, kaempferol, biochanin A, daidzein and rutin) to modulate insulin-like growth factor-I (IGF-I)–induced in vitro proliferation and apoptotic resistance in the AT6.3 rat prostate cancer cell line. IGF-I at 50 ?g/L in serum-free medium produced maximum proliferation and minimized apoptosis. Polyphenols exhibited different abilities to modulate IGF-I–induced proliferation, cell cycle progression (flow cytometry) and apoptosis (Annexin V/propidium iodide and terminal deoxynucleotidyltransferase–mediated deoxyuridine 5?-triphosphate nick end labeling). Genistein, quercetin, kaempferol and biochanin A exhibited dose-dependent inhibition of growth with a 50% inhibitory concentration (IC50) between 25 and 40 ?mol/L, whereas rutin and daidzein were less potent with an IC50 of >60 ?mol/L. Genistein and kaempferol potently induced G2/M cell cycle arrest. Genistein, quercetin, kaempferol and biochanin A, but not daidzein and rutin, counteracted the antiapoptotic effects of IGF-I. Human prostate epithelial cells grown in growth factor–supplemented medium were also sensitive to growth inhibition by polyphenols. Genistein, biochanin A, quercetin and kaempferol reduced the insulin receptor substrate-1 (IRS-1) content of AT6.3 cells and prevented the down-regulation of IGF-I receptor ? in response to IGF-I binding. IGF-I–stimulated proliferation was dependent on activation of mitogen-activated protein kinase/extracellular signal–regulated kinase (ERK) and phosphatidylinositide 3-kinase pathways. Western blotting demonstrated that ERK1/2 was constitutively phosphorylated in AT6.3 cells with no change in response to IGF-I, whereas IRS-1 and AKT were rapidly and sensitively phosphorylated after IGF-I stimulation. Several polyphenols suppressed phosphorylation of AKT and ERK1/2, and more potently inhibited IRS-1 tyrosyl phosphorylation after IGF-I exposure. In summary, polyphenols from soy and tomato products may counteract the ability of IGF-I to stimulate proliferation and prevent apoptosis via inhibition of multiple intracellular signaling pathways involving tyrosine kinase activity.

Breast Cancer Res

Fox EM, Kuba MG, Miller TW, Davies BR, Arteaga CL. Breast Cancer Res. 2013 Jul 11;15(4):R55.

Estrogen receptor alpha-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of IGF-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation. Inhibition of AKT using the catalytic inhibitor AZD5363 was examined in 4 ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the FGFR inhibitor AZD4547. Treatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3alpha/beta and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of 3 of 4 ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1, and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERalpha mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation. These data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.

Genistein & Cancer

1. 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

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.

J Steroid Biochem Mol Biol. 2009 May 3. 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. Division of Basic Medical Sciences, St George's University of London, Cranmer Terrace, London SW17 0RE, United Kingdom.

2. Effects of EGF and Genistein on Proliferation of Human Breast Cancer Cell

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.

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

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

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.

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.

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.

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.

Gilda G Hillman, Yu Wang, Mingxin Che, Julian J Raffoul, Mark Yudelev, Omer Kucuk and Fazlul H Sarkar. Progression of renal cell carcinoma is inhibited by genistein and radiation in an orthotopic model. BMC Cancer 2007, 7:4doi:10.1186/1471-2407-7-4

Genistein blocks breast cancer cells in the G2M phase of the cell cycle

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 M genistein showed a dose-dependent accumulation in the G2M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G2M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G2M arrest was coupled with a parallel depletion of the G0/G1 phase. To understand the mechanism of action underlying the block in G2M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G2M transition. As expected, p34cdc-2 expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34cdc-2 catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 M genistein, cyclin B1 (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G2M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34cdc-2/cyclin B1  complex only in all of the studied cell lines. Cappelletti, V., Fioravanti, L., Miodini, P. & Di Fronzo, G. J. Cell. Biochem. 79:594-600, 2000.

Genistein inhibition of the growth of human breast cancer cells: Independence from oestrogen receptors and the multi-drug resistance

The effect of isoflavones on the growth of the human breast carcinoma cell lines, MDA-468 (oestrogen receptor negative), and MCF-7 and MCF-7-D-40 (oestrogen receptor positive), has been examined. Genistein is a potent inhibitor of the growth of each cell line (IC50 values from 6.5 to 12.0 μg/ml), whereas biochanin A and daidzein are weaker growth inhibitors (IC50 values from 20 to 34 μg/ml). The isoflavone β-glucosides, genistin and daidzin, have little effect on growth (IC50 values > 100 μg/ml). The presence of the oestrogen receptor is not required for the isoflavones to inhibit tumour cell growth (MDA-468 vs MCF-7 cells). In addition, the effects of genistein and biochanin A are not attenuated by over expression of the multi-drug resistance gene product (MCF-7-D40 vs MCF-7 cells).
ER, oestrogen receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MDR, multi-drug resistance; TPK, tyrosine protein kinase; EGF-R, epidermal growth factor receptor; HPLC, high performance liquid chromotography; gp 170, a 170,000 Da glycoprotein, a product of the multidrug resistance gene. Peterson, G. & Stephen Barnes, S., Biochemical and Biophysical Research Communications  Volume 179, Issue 1, 30 August 1991, Pages 661-667

Genistein inhibits both oestrogen and growth factor-stimulated proliferation of human breast cancer cells

Genistein is a naturally occurring dietary protein tyrosine kinase (PTK)  inhibitor that is hypothesized to be responsible for the lower rate of  breast cancer observed in Asian women consuming soy. Although genistein is a potent in vitro PTK inhibitor, its mechanism of action in vivo is not  known. In vivo, breast cancer growth is regulated by estrogens and peptide growth factors, such as epidermal growth factor (EGF), the receptor of  which has intrinsic PTK activity. Therefore, genistein may block mammary  epithelial cell growth by interfering with signal transduction events  stimulated by oestradiol or growth factors. The effect of genistein, related isoflavones, and other tyrosine kinase inhibitors on foetal bovine serum-, oestradiol-, and EGF-stimulated cell growth and signal transduction pathways was examined in five human breast cancer cell lines. Genistein inhibited  the growth of these cells by each of the growth stimuli with IC50 values  ranging from 2.6 to over 20 micrograms/ml. Growth inhibition by genistein was cytostatic and reversible at IC50 concentrations. Related isoflavones were less potent growth inhibitors than genistein, whereas the synthetic  PTK inhibitor tyrphostin A25 was an equally potent growth inhibitor. The  mechanism of genistein growth inhibition in human breast cancer cells did not depend on the presence of functional oestrogen receptor signalling  pathways or on inhibition of EGF-receptor PTK activity. Furthermore,  genistein (< or = 20 micrograms/ml) did not decrease constitutive or  EGF-induced tyrosine phosphorylation as determined by Western blotting with antiphosphotyrosine antibodies. These data suggest that although genistein inhibits the growth of breast cancer cells in culture, it does so without gross inhibition of PTK activity.
Peterson, G. & Barnes, S.

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

There has been a variety of opinions regarding the use os Genistein in oestrogen receptor positive (ER+) breast cancer (BCa) and the discussion continues. There is ample evidence as seen in the first study to conclude that the use of Genistein is highly beneficial, however there are several factors including those discussed in the second study. Firstly dosage, as too low a dose CAN stimulate tumour growth. The dosage must be dependant on body size as well as liver and intestinal oestrogen elimination. A recent study I posted on Diadzein shows the phytoestrogen metabolism pathway in the intestine. Secondly, as the second study shows Genistein must not be used with aromatase inhibitors. This would also indicate that caution should be used when the patient is using Tamoxifen, although again at low doses (see study 3). In study 4, more support for the use of Genistein, however the final list from suggests other dietary factors are involved. I therefore suggest that all Genistein/Daidzein supplementation is combined with I3C and/or high levels of brassica vegetables.

Potent genistein derivatives as inhibitors of estrogen receptor alpha-positive breast cancer.
The estrogen receptor (ER) is a major target for the treatment of breast cancer cells. Genistein, a soy isoflavone, possesses a structure similar to estrogen and can both mimic and antagonize estrogen effects although at high concentrations it inhibits breast cancer cell proliferation. Hence, to enhance the anti-cancer activity of Genistein at lower concentrations, we have synthesized seven structurally modified derivatives of Genistein (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) based on the structural requirements for an optimal anti-cancer effect. Among those seven, three derivatives (MA-6, MA-8 and MA-19) showed high antiproliferative activity with IC(50) levels in the range of 1-2.5 μM, i.e., at much lower concentrations range than Genistein itself, in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) studied. In our analysis, we noticed that at IC(50) concentrations, the MA-6, MA-8 and MA-19 Genistein derivatives induced apoptosis, inhibited ER-α messenger RNA expression and increased the ratio of ER-β to ER-α levels in a manner comparable to the parent compound Genistein. Of note, these three modified Genistein derivatives exerted their effects at concentrations 10-15 times lower than the parent compound, decreasing the likelihood of significant ER- α pathway activation, which has been a concern for Genistein. Hence these compounds might play a useful role in breast cancer chemoprevention. Marik R, Allu M, Anchoori R, Stearns V, Umbricht CB, Khan S. Cancer Biol Ther. 2011 May 15;11(10):883-92. Epub 2011 May 15.

Genistein induces breast cancer-associated aromatase and stimulates estrogen-dependent tumor cell growth in in vitro breast cancer model
In breast cancer, the interaction between estrogen-producing breast adipose fibroblasts (BAFs) and estrogen-dependent epithelial tumor cells is pivotal. Local estrogen production is catalyzed by aromatase, which is differentially regulated in disease-free and tumorigenic breast tissue. The use of aromatase inhibitors to block local estrogen production has proven effective in treatment of estrogen-dependent breast cancer. However, a major problem during breast cancer treatment is the sudden onset of menopause and many women seek for alternative medicines, such as the soy isoflavone genistein. In this study, we show that genistein can induce estrogen-dependent MCF-7 tumor cell growth and increase breast cancer-associated aromatase expression and activity in vitro. We have previously developed an in vitro breast cancer model where the positive feedback loop between primary BAFs and estrogen-dependent MCF-7 tumor cells is operational, thereby representing a more natural in vitro model for breast cancer. In this model, genistein could negate the growth inhibitory action of the aromatase inhibitor fadrozole at physiologically relevant concentrations. These data suggest that soy-based supplements might affect the efficacy of breast cancer treatment with aromatase inhibitors. Considering the high number of breast cancer patients using soy supplements to treat menopausal symptoms, the increasing risk for adverse interactions with breast cancer treatment is of major concern and should be considered with care.
M.B.M. van Duursen MBM, Nijmeijer SM, de Morree ES, et al. Toxicology. Article in Press 2011 doi:10.1016/j.tox.2011.07.005

Study 3:
Effects of soy phytoestrogens genistein and daidzein on breast cancer growth.
To determine whether genistein and daidzein, the major phytoestrogens in soy, can stimulate breast cancer growth.
Systematic search through primary English-language literature on MEDLINE (1966-January 2001), EMBASE (1982-January 2001) and Current Contents (1998-January 2001).
Genistein and daidzein at low concentrations were found to stimulate breast tumor growth in in vitro and in vivo animal studies, and antagonize the antitumor effect of tamoxifen in vitro. At high concentrations, genistein inhibited tumor growth and enhanced the effect of tamoxifen in vitro.
Genistein and daidzein may stimulate existing breast tumor growth and antagonize the effects of tamoxifen. Women with current or past breast cancer should be aware of the risks of potential tumor growth when taking soy products.
de Lemos ML. Ann Pharmacother. 2001 Sep;35(9):1118-21.

Study 4:
Genistein's "ER-dependent and independent" actions are mediated through ER pathways in ER-positive breast carcinoma cell lines.
Genistein, a natural flavone found in soy has been postulated to be responsible for lowering the rate of breast cancer in Asian women. Our previous studies have shown that genistein exerts multiple suppressive effects on both estrogen receptor positive (ER+) as well as estrogen receptor negative (ER-) human breast carcinoma lines suggesting that the mechanisms of these effects may be independent of ER pathways. In the present study however we provide evidence that in the ER+ MCF-7, T47D and 549 lines but not in the ER-MDA-MB-231 and MDA-MB-468 lines both presumed "ER-dependent" and "ER-independent" actions of genistein are mediated through ER pathways. Genistein's antiproliferative effects are estrogen dependent in these ER+ lines, being more pronounced in estrogen-containing media and in the presence of exogenous 17-beta estradiol. Genistein also inhibits the expression of ER-downstream genes including pS2 and TGF-beta in these ER+ lines and this inhibition is also dependent on the presence of estrogen. Genistein inhibits estrogen-induced protein tyrosine kinase (PTK) activity. Genistein is only a weak transcriptional activator and actually decreases ERE-CAT levels induced by 17-beta estradiol in the ER+ lines. Genistein also decreases steady state ER mRNA only in the presence of estrogen in the ER+ lines thereby manifesting another suppression of and through the ER pathway. Our observations resurrect the hypothesis that genistein functions as a "good estrogen" in ER+ breast carcinomas. Since chemopreventive effects of genistein would be targeted to normal ER-positive ductal-lobular cells of the breast, this "good estrogen" action of genistein is most relevant to our understanding of chemoprevention.
Shao ZM, Shen ZZ, Fontana JA, Barsky SH. Anticancer Res. 2000 Jul-Aug;20(4):2409-16.

Studies of the impact of genistein, daidzein or equol on breast cancer cells have found the following:
A study designed to evaluate the effects of low-dose, long-term genistein exposure on (ER+/PR+) MCF-7 breast cancer cells was performed by culturing the cells in genistein for 10-12 weeks. It was found that the treatment significantly reduced the growth promoting effects of estradiol (E2) and resulted in a down-regulation of the PI3-K/Akt signaling pathway (a mechanism through which genistein might offer protection against breast cancer). However, it did not result in decreased expression of estrogen receptor alpha (ERα).

A study examining the mechanism by which genistein can trigger G2/M cell cycle arrest and inhibit cell growth in (ER-/PR-) MDA-MB-231 breast cancer cells found that the Ras/MAPK/AP-1 signal pathway might be involved.

A study designed to determine whether soy genistein has a genotoxic effect on normal breast cancer cells at low-dose, physiological concentrations found that after three months of exposure, the cells evinced loss of a normal chromosome 8, gain of an extra chromosome 20, and loss of a chromosomal segment of chromosome 9, leading to the homozygous deletion of the two tumor suppressor genes CDKN2A (p16INK4a) and CDKN2B (p15INK4b). The authors comment that these genotoxic effects may contribute to soy- and genistein-associated risk.

A study designed to examine mechanisms by which daidzein inhibits the growth of breast cancer cells found that daidzein significantly inhibited MCF-7 and (fibroblast growth factor receptor overexpressing) MDA-MB-453 breast cancer cell proliferation in a dose- and time-dependent manner. Further testing found that daidzein exerted its anticancer effects via cell cycle arrest at the G1 and G2/M phases.

A study designed to examine gene expression patterns in MCF-7 breast cancer cells at both physiologic (1 or 5 μM) and pharmacologic (25 μM) genistein concentrations found that genistein modified the expression of genes belonging to multiple pathways, including estrogen- and p53-mediated pathways. At physiologic concentrations, genistein induced expression suggestive of increased proliferation, while at pharmacologic concentration, genistein induced expression suggestive of increased apoptosis, decreased proliferation and decreased total cell number.

A study found that caffeine enhanced the inhibition of cell proliferation induced by genistein in the hormone-independent breast cancer cell line MDA-MB-435S. Genistein induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle which caffeine reversed. The authors comment that this reversal by caffeine of genistein-induced G2/M phase could enhance genistein-induced inhibition of cell growth.

A study focusing on the effects of equol in human breast cancer cells found that equol significantly inhibited proliferation in MDA-MB-453 cells in a dose- and time-dependent manner. However, equol stimulated proliferation in MCF-7 cells at low concentrations (<1 µM) and only inhibited proliferation at a high concentration (100 µM). During equol-induced apoptosis, equol increased the number of cells in the sub-G0 phase and enhanced the level of p53, leading the authors to conclude that equol-induced cell cycle arrest and apoptosis involves a p53-dependent pathway.

In another study, phytoestrogens extracted from soymilk were used to treat human target cells that represent a common model system for mammary tumorigenesis. The soy phytoestrogens were found to induce a genomic fingerprint indistinguishable from the transcriptional effects of 17β-estradiol. More diverging transcriptional profiles were generated when steps were taken to reconstitute the expression of estrogen receptor β (ERβ). The authors conclude that soy phytoestrogens appear to mitigate estrogenic signaling in the presence of both estrogen receptor subtypes but, in late-stage cancer cells lacking ERβ, these phytochemicals contribute to a tumor-promoting transcriptional signature.

A study designed to examine whether certain carotenoids (lycopene, phytoene, phytofluene, and β-carotene) can inhibit signaling of phytoestrogens found that treatment of MCF-7 and (ERα-positive) T47D breast cancer cells with genistein induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element, actions similar to the known effects of 17β-estradiol in promoting breast cancer. Each of the carotenoids tested reduced proliferation induced by 17β-estradiol and genistein, thereby potentially reducing their harmful effect in hormone-dependent cancers.

A study designed to evaluate the impact of the combination of genistein and indole-3-carbinol (found in broccoli and other brassica vegetables) on MCF-7 breast cancer cells found a synergistic effect of genistein and I3C in increasing apoptosis and decreasing ER-α expression.

A study found that genistein induced aromatase activity in liver cells. After menopause, when the ovaries stop producing the hormone, localized estrogen synthesis in other tissues become more significant physiologically. The study illustrated an extragonadal pathway by which genistein might increase estrogen synthesis.

Bottom line: Soy phytoestrogens have been shown both to inhibit and to promote breast cancer cell growth in the laboratory, depending on the concentrations and numerous other factors (such as whether they are administered in the presence of caffeine, certain carotenoids, or indole-3-carbinol (found in brassica vegetables)). The evidence hints that the potential for breast cancer promotion may be greater in later stages of breast cancer.

Results suggest that the isoflavone derivative genistein, inhibits the cell growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis (Yanagihara et al, 1993).

Yanagihara K et al. Antiproliferative Effects of Isoflavones on Human Cancer Cell Lines Established from the Gastrointestinal Tract. Cancer Res December 1, 1993 53; 5815
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