For citation purposes: Tsuboy MS, Marcarini JC, de Souza AO, de Paula NA, Luiz RC, Dorta DJ, Mantovani MS, Ribeiro LR. Effects of physiological levels of daidzein on cell proliferation of tumoral and non-tumoral breast cells lines. OA Cancer 2014 Mar 10;2(1):5.

Research study

Basic Cancer Biology

Effects of physiological levels of daidzein on cell proliferation of tumoral and non-tumoral breast cells lines.

M Tsuboy1*, J Marcarini1, A de Souza 2, N de Paula 3, R Luiz 4, D Dorta2, M Mantovani5, L Ribeiro1

Authors affiliations

(1) Biosciences Institute, UNESP, São Paulo State University, Rio Claro, SP, Brazil.

(2) Faculty of Philosophy, Sciences and Letters, Chemistry Department, USP, University of São Paulo, Ribeirão Preto, SP, Brazil.

(3) Hospital of the Faculty of Medicine; Department of Clinical Medicine, USP, University of São Paulo, Ribeirão Preto, SP, Brazil.

(4) Laboratory of Molecular Pathology; Pathology Department, UEL; State University of Londrina, Londrina, PR, Brazil.

(5) Biology Department, UEL, State University of Londrina, Londrina, PR, Brazil.

* Corresponding author Email:



Soy isoflavones are associated with the lower breast cancer risk found in Asiatic populations. They can interfere in various stages of the carcinogenesis process, preventing the development of this pathology and, for this reason, its consumption became popular. Instead of the beneficial effects found in literature, most chemopreventive in vitro studies use non-physiological levels of daidzein (> 30 µM). In our study, the effects of maximal physiological levels of daidzein was investigated in two human breast cell lines: HB4a (non-tumoral) and MCF-7 (tumoral). Experiments to verify cytotoxicity, induction of cell death by apoptosis and, additionally, cell cycle arrest were performed.

Materials and methods

Cytotoxic effect of daidzein (0.1 - 100 µM) was assessed using resazurin-based assay. Flow cytometry tests were performed to investigate apoptosis induction (annexin V-FITC/PI) and cell cycle arrest (propidium iodide staining of DNA content) by daidzein at 10 and 25 µM. Changes in expression of apoptosis related genes after treatment of cells with daidzein (10 and 25 µM) were investigated using quantitative real time PCR.


Daidzein ranging from 0.1 to 100 µM (for 24 and 48h) were not cytotoxic to both cell lines. Concentrations of 10 and 25 µM did not induce apoptosis in these cells, instead of statistical significant changes in gene expression of some of the target genes (CASP-3, CASP-7, BAX, BCL-xL, COX-2). Both concentrations of daidzein arrested cell cycle in G0/G1 phase only in MCF-7.


Daidzein at maximal achievable physiologic serum levels selectively arrested cell cycle only in the tumoral MCF-7, and did not induce cell death by apoptosis, one of the major effects described in literature. We hope that our study helps in understanding the chemopreventive effects of low levels of soy isoflavones in breast carcinogenesis.


It has been more than 30 years since Doll and Peto called attention for the association between lifestyle and cancer[1] and now it is becoming clearer as research continues to show nutrition plays a major role in cancer. Epidemiological studies also point out this association as many of these show that the incidence and mortality of some kinds of cancer are higher in Western countries with meat-based diets than in Asian countries with plant-based diets[2,3]. One of the best examples of this situation is the association between the high intake of soy-based food in Asian populations and the reduced risk of breast, ovarian, prostate and other types of cancer and chronic diseases[4]. Because of these beneficial health effects, soy-based products have gained worldwide popularity and are especially consumed by women with menopause to alleviate its symptoms[5,6]. However there have been concerns that isoflavones (the main compounds found in soybean and soy products), may increase the risk of recurrence or stimulate the growth of existing tumours[7]. Despite significant research in the area, the role of isoflavones in breast and hormone-related cancers remains controversial[8,9].

The main compounds found in soy that are responsible for the human health benefits are the isoflavones daidzein (7,4´dihydroxyisoflavone) and genistein (4′ 5, 7-trihydroxyisoflavone). Before ingestion, daidzein and genistein are present as β-D-glycosides namely daidzin and genistin, which according to Setchell[10], are biologically inactive. Only after being hydrolysed by intestinal bacteria, isoflavone glycosides become bioactive aglycones daidzein and genistein and are absorbed by the intestinal tract[11].

Daidzein and genistein, also known as phytoestrogens, are able to bind to oestrogen receptors, inducing cell transcription and eliciting biological effects similar to those of other natural oestrogens[12] and, for this reason, interest has focused primarily on their effects on hormone-dependent conditions, such as breast, ovarian and prostate cancers.

Cancer chemoprevention properties of daidzein include induction of apoptosis and cell cycle arrest13,14,15. However, in most studies, high concentrations of daidzein (> 30 µM) were tested, and they are difficultly found in human plasma after ingestion of soy-based foods or supplements. According to Moiseeva and Manson[16], such studies hinder predictions of efficacy. Another limitation to investigate the preventive effects of isoflavones is the majority use of tumoral cell lines, although very well established in most areas[14].

Considering these observations, we have evaluated the cytotoxicity, induction of apoptosis, cell cycle arrest and expression of pro- and anti-apoptotic genes focusing on concentrations of daidzein considered the maximal physiologically (i.e., that have been already found in human plasma) achievable through diet or through pharmacological administration, using two human mammary cell lines: a non-tumoral lineage (HB4a) and a tumoral one (MCF-7).

Materials and methods

The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.


Daidzein (DAID.) (CAS 446-72-0), camptothecin (CAS 7689-03-4) and resazurin (CAS 62758-13-8) were acquired from Acros Organics (New Jersey, USA). Doxorubicin (CAS 29042-30-6; Adriblastina[®]) is manufactured by Actavis Italy (Milan, Italy) and distributed by Pfizer. Reagents used to maintain cell cultures and used to perform qRT-PCR were from Invitrogen Corporation (NY, USA): DMEM (Dulbecco’s Modified Eagle Medium; cat n. 12800-058), foetal bovine serum, antibiotics (cat n. 15240-062), insulin and trypsin; RNAse A, Trizol LS, DNAse I, dNTP mix, RNAse Out[TM], M-MLV reverse transcriptase, DEPC water and Platinum[®] SYBR[®] Green qPCR SuperMix-UDG kit. Hydrocortisone was from Pharma Nostra (Rio de Janeiro, Brazil). Oligo sequences were purchased from Prodimol Biotecnologia (Belo Horizonte, MG, Brazil). Propidium iodide was from Sigma-Aldrich (St. Louis, MO, USA) and annexin V-FITC from BD Pharmigen[TM] (San Diego, CA, USA).

Daidzein concentrations

Daidzein was dissolved in dimethylsulfoxide (DMSO) and used in culture with DMSO maximum concentration of 1%. In cytotoxicity assay, we evaluated five concentrations of daidzein. In the other tests, we have focused on concentrations of 10 and 25 µM of daidzein, representing, respectively, maximal dietary and pharmacological concentrations found in human plasma according to literature[8,17,18,19].

Cell lines and culture conditions

MCF-7 cells (tumoral) were kindly provided by Professor João Ernesto de Carvalho (UNICAMP, São Paulo, Brazil) and HB4a cell line (non-tumoral) was kindly provided by Professor Silvia R. Rogatto from Hospital do Câncer A. C. Camargo (São Paulo, Brazil). MCF-7 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and antibiotics. HB4a cells were cultivated in the same medium supplemented with 5 µg/mL of insulin and hydrocortisone. Cells cultures were maintained at 37 ºC, 5% CO2 and 95% relative humidity. Cell density used in each experiment was determined by pilot-tests and it aimed to avoid detaching of cells resultant from total confluence in the last day of each assay.

Cytotoxicity evaluation – resazurin-based assay

The cytotoxicity assay was standardized based on the protocols described by Nakayama et al., O’Brien et al. and McMillian et al.[20,21,22]. Approximately 4.5 x 10[4] cells were seeded in each well of a 24 wells-microplate and treated with daidzein for 24 or 48h (0.1; 1; 10; 50 and 100 µM). Control treatment was medium with 1% DMSO and control of cytotoxicity induction was doxorubicin at final concentration of 10 µg/mL. At the end of this period, cells were incubated with resazurin 60 µM for 3h. Fluorescence was measured with VICTOR 3 (Perkin Elmer) at 530-560 nm of excitation and 580-600 nm of emission ranges. Experiments were performed in biological triplicate with three wells/ each treatment (n=9).

Analysis of apoptosis induction by flow cytometry (annexin V-FITC/PI)

Approximately 10[5] cells HB4a or MCF-7 were seeded in each well of a 6 wells-microplate. Cells were treated with camptothecin (4 µg/mL) and daidzein 10 or 25 µM for 24 hours. At the end of the treatment, cells were trypsinized and centrifuged (1000 rpm, 5 min, 4ºC). Supernadant was discarded and cell pellet was resuspended in cold PBS followed by another centrifugation (1000 rpm, 10 min, 4ºC). Cells were then labelled with annexin V-FITC and propidium iodide (5 µg/mL), and protected from the light. Ten thousand events were analysed in a BD FACS CANTO[TM] flow cytometer. Experiments were performed in biological duplicate with three wells/ each treatment (n = 6).

Cell cycle analysis by flow cytometry (propidium iodide staining of DNA content)

The same way as in the previous experiment (annexin V-FITC/PI tests), cells (10[5]/well) were seeded in 6 wells-microplate and treated with camptothecin (4 µg/mL) and daidzein 10 or 25 µM for 24 hours. After trypsinization of the cells and centrifugation (1000 rpm, 5 min, 4ºC), it was added to PBS and ice cold ethanol 70% to the pellet. Then, it was resuspended and left at -20ºC for 24 hours. At the end of this period, cells were centrifuged (1000 rpm, 5 min, 4ºC), supernadant was discarded and it was added to cold PBS and RNAse A (10 mg/mL) to each tube. Samples were incubated for 30 minutes at 37ºC, followed by staining with hypothonic fluorochrome solution (propidium iodide, 5 µg/mL; sodium citrate 0.1% and Triton X-100 0.1%) one hour before flow cytometry analysis (10.000 events; BD FACS CANTO [TM]). Experiments were performed in biological duplicate with three wells/ each treatment (n = 6).

Quantitative Real-time PCR (qRT-PCR)

For analysis of gene expression, 10[6] cells were seeded in 25 cm[2] culture flasks and treated with 10 µM or 25 µM of daidzein for 12 h. Experiments were performed in biological duplicate and technical triplicate (n = 6).

Total RNA was prepared using Trizol LS reagent followed by DNAse I treatment, both according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized subsequent to verification of RNA quality in agarose gel (28S and 18S rRNA pattern of bands) and by A260/A280 ratio (Biophotometer – Eppendorf). Real time PCR experiments were performed in the Cromo 4[TM] Engine Opticon detection system (Bio-Rad) using the Platinum® SYBR® Green qPCR SuperMix-UDG kit in a total of 20 µL of reaction mix (2 µL cDNA – diluted 1:5; 10 µL SYBR® Green; 0.5 µL of each specific gene primer (10 µM), and 7 µl of H2O). Target cDNAs were amplified in separate tubes: 3 min at 95°C, then 35 cycles of denaturation (95°C for 20 s), annealing (at 60°C for 30 s) and extension (72ºC for 20 s) per cycle. Melting curves (50 to 95ºC ready every 0.5ºC) for each PCR reaction were generated to ensure the purity of the amplification product. Primers of CASP-3, CASP-7, BAX and BCL-xL were used, as well as GAPDH as reference gene[23] and COX-224. Primer sequences were designed with the IDT tool[48] CASP-3 (NM_032991.2) 5’- GTGCTACAATGCCCCTGGAT-3’ and 5’-GCCCATTCATTTATTGCTTTCC-3’ (199 bp); CASP-7 (NM_001227.3) 5’-TCACCATGCGATCCATCAAGACCA-3’ and 5’-TTTGTCTGTTCCGTTTCGAACGCC-3’ (149bp); BAX (NM_004324.3) 5’-TTTCTGACGGCAACTTCAACTGGG-3’ and 5’-TGTCCAGCCCATGATGGTTCTGAT-3’ (122bp); BCL-xL (NM_138578.1) 5’- TGGGCTCACTCTTCAGTCGGAAAT-3’ and 5’-ATGTAGTGGTTCTCCTGGTGGCAA-3’ (121bp).

Data analysis

Data obtained in cytotoxicity and in flow cytometry tests were submitted to ANOVA followed by Dunnett’t test compared to the control group (α = 0.05; GraphPad Prism5 software). Relative quantification method of analysis was employed in qRT-PCR using GAPDH as reference gene according to Pfaffl with REST© software (Relative Expression Software Tool)[25,26,49].


Cytotoxicity evaluation – resazurin-based assay

Data obtained with the resazurin-based assay showed that none of the tested concentrations of daidzein were cytotoxic, neither for non-tumoral cells (HB4a) nor for the tumoral lineage MCF-7 (Figure 1 and Figure 2).

Fluorescence intensity (mean ± SD) measured after treatment of HB4a with different concentrations of daidzein (Daid) in resazurin-based assay (24, 48 h). Daid [0.1] = cells treated with daidzein 0.1 µM; Daid [1] = cells treated with daidzein 1 µM; Daid [10] = cells treated with daidzein 10 µM; Daid [50] = cells treated with daidzein 50 µM; Daid [100] = cells treated with daidzein 100 µM; Doxorubicin = cells treated with doxorubicin 10 µg/mL. * indicates cytotoxic effect (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=9).

Fluorescence intensity (mean ± SD) measured after treatment of MCF-7 with different concentrations of daidzein (Daid) in resazurin-based assay (24, 48h). Daid [0.1] = cells treated with daidzein 0.1 µM; Daid [1] = cells treated with daidzein 1 µM; Daid [10] = cells treated with daidzein 10 µM; Daid [50] = cells treated with daidzein 50 µM; Daid [100] = cells treated with daidzein 100 µM; Doxorubicin = cells treated with doxorubicin 10 µg/mL. * indicates cytotoxic effect (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=9).

Analysis of apoptosis induction by flow cytometry

Figure 3 and Figure 4 show results obtained in analysis of apoptosis induction by flow cytometry. Both concentrations of daidzein (10 or 25 µM) were not able to induce apoptosis in HB4a or in MCF-7 cells; it was observed only when cells were treated with camptothecin.

Annexin V-FITC/PI analysis of apoptosis (flow cytometry) induction in HB4a cells treated with daidzein for 24 hours. *statistical significant (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=6).

Annexin V-FITC/PI analysis (flow cytometry) of apoptosis induction in MCF-7 cells treated with daidzein for 24 hours. *statistical significant (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=6).

Cell cycle analysis by flow cytometry

In this test, we observed that the concentrations of daidzein used (10 or 25 µM) were not able to arrest cell cycle of non-tumoral cells HB4a (Figure 5). For MCF-7 (Figure 6) we can observe a significant increase in number of cells at the G0/G1 phase after treatment of both concentrations of daidzein (10 or 25 µM) for 24 hours. As a consequence of this arrest, the percentage of cells at S phase had decreased.

Effects of daidzein (24 h) on cell cycle distribution of HB4a (propidium iodide staining of DNA content – flow cytometry). CPT = cells treated with camptothecin 4 µg/mL; Daid [10] = cells treated with daidzein 10 µM; Daid [25] = cells treated with daidzein 25 µM. *statistical significant (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=6).

Effects of daidzein (24 h) on cell cycle distribution of MCF-7 (propidium iodide staining of DNA content – flow cytometry). CPT = cells treated with camptothecin 4 µg/mL; Daid [10] = cells treated with daidzein 10 µM; Daid [25] = cells treated with daidzein 25 µM. *statistical significant (p <0.05; ANOVA followed by Dunnett’s test compared to control group; n=6).

Quantitative Real-time PCR (qRT-PCR)

Table 1 and Table 2 show the values of R (ratio) obtained in the REST© software for gene expression of the selected primers.

For HB4a cells (Table 1), we can observe that dietary concentration of daidzein (10 µM) did not modify mRNA expression of the analysed genes significantly; however, the gene expression ratios for CASP-3, BAX, BCL-xL and COX-2 became statistically significant when pharmacological concentration of daidzein (25 µM) was used to treat these lineage. In MCF-7 cells (Table 2), daidzein 10 µM modified expression of BCL-xL. When cells were treated with 25 µM of daidzein, CASP-3 and CASP-7 expression was altered. It is important to report that COX-2 expression in mammary tumoral MCF-7 cells was null.

Table 1

Relative gene expression (R value) of CASP-3, CASP-7, BAX, BCL-xL and COX-2 after treatment of 10 or 25 µM of daidzein in HB4a cells. R value ± SD and p value (qRT-PCR).

Table 2

Relative gene expression (R value) of CASP-3, CASP-7, BAX and BCL-xL after treatment of 10 or 25 µM of daidzein in MCF-7 cells. R value ± SD and p value (qRT-PCR).


Soy based-food and supplements are among the most worldwide consumed because of their various beneficial health effects. They are very popular among women at climacteric, which have been used as soy isoflavones to manage menopausal symptoms[9].

Although there are a significant number of studies on soy isoflavones, their preventive effects on breast cancer are still under consideration because of the lack of consistent human data from long-term dietary intervention trials and because of the tumour stimulating effects found in in vitro and in vivo studies. Classical examples are the proliferating effect of tumoral MCF-7 cells in low daidzein concentrations, increase in breast tumour growth in mice fed with daidzein (xenograft mouse model) or even the absence of protective effect of the animals fed with isoflavones[27,28,29,30,31]. Besides, there are much more studies and data on molecular mechanisms of the effects of genistein than of daidzein.

In our study, daidzein at low (0.1-10 µM) and at high (> 10 µM) concentrations were not cytotoxic to tumoral MCF-7 cells or to non-tumoral HB4a cells, in both experimental points analysed (24 and 48 h). In the study performed by Röhrdanz et al.[32] with H4IIE rat hepatoma cells, cytotoxicity was apparent only with concentrations of daidzein above 200 µM (MTT assay). Daidzein at concentrations of 0.05, 0.5, 5, 50 μM also did not show cytotoxicity to porcine granulosa cells harvested from large, preovulatory follicles, in the Alamar Blue[TM] assay[33]. Blay et al.[34] found a range of non-cytotoxic concentration of 0–25 µM for daidzein in RAW 264.7 cells. Lee et al.[35] also did not find cytotoxicity in their study with HCT-116 human colon cancer cells with high concentrations of daidzein (25, 50 and 100 µM). Moreover, Wang and Kurzer[36] found that daidzein 0.01-100 µM was not cytotoxic and stimulated DNA synthesis (proliferation) in MCF-7. Our results are also in accordance with the data obtained by Park et al.[37]: high concentration of daidzein (200 µM) also did not induce cytotoxicity to SK-HEP-1 (hepatocellular carcinoma cells) or Chang cells (normal hepatocytes).

Contrary, some authors have found cytotoxic activity of daidzein in different cells types, including breast cancer cells lines. Choi and Kim[38] demonstrated that daidzein significantly decreased the proliferation of both MCF-7 and MDA-MB-453 cells in a dose- and time-dependent manner, with significant decreases at concentrations of 10 µM in MCF-7 and 1 µM in MDA-MB-453 after 24 hours of treatment. Jin et al.[39] also observed cytotoxic effect of daidzein in MCF-7 cells, but they used only high concentrations of 25, 50 and 100 µM. These contradictory results are in accordance with the well documented biphasic effect of soy isoflavones, i.e., they can promote or inhibit cell proliferation[17,36].

Daidzein, like other isoflavones, can exert a wide range of effects through activation/inhibition of several cellular pathways[14,15]. Soy isoflavones activities on apoptotic and cell cycle cascades are one of the most investigated by researches in searching to obtain a cancer chemopreventive effect. Another desirable characteristic of a chemopreventive agent is to affect only tumoral cells. In our study, at maximal achievable physiologic serum levels (10 or 25 µM), daidzein was not able to induce apoptosis in the breast cell lines used (HB4a and MCF-7). Together with qRT-PCR results, we can conclude that daidzein are able to act in an apoptotic cascade, but it was not sufficient to produce a chemopreventive response in the studied cells through this classical pathway of cell death.

Anti-apoptotic effect of daidzein has been found by many authors, and despite this it might not be desirable in cancer chemoprevention, it might be useful in the prevention/treatment of neurodegenerative diseases. Adams et al.[40], for example, showed that addition of daidzein attenuated the upregulation of active caspase-3 expression in neurons cultures. Wu et al.[41] demonstrated that daidzein (1 or 5 µM) decreased the rate of apoptosis in MCF-7 cells through downregulation of bax protein. Moreover, daidzein improved antiapoptotic effect in an E2 (17β-Estradiol) dose-dependent manner, suggesting that daidzein may enhance the deleterious effect of endogenous E2 in hormone-dependent breast cancer[41].

Pro-apoptotic activities of daidzein have also been demonstrating in different cell lines, however with supraphysiological concentrations being investigated in most of the studies, like in the one performed by Park et al.[37] These authors concluded that daidzein-induced apoptosis (200, 400 and 600 µM) in SK-HEP-1 cells was a response of down-regulation of Bcl-2 and Bcl-xL up-regulates Bak, triggering the release of cyt c, which may act as an important factor in the regulation and activation of the caspase cascade[37]. In another work, Jin et al.[39] found that the generation of reactive oxygen species (ROS) by daidzein was responsible for the apoptosis observed in their study with MCF-7 cells (25, 50 and 100 µM). There was a disruption of mitochondrial transmembrane potential, downregulation of bcl-2 and upregulation of bax, which led to the release of cytochrome c, resulting in caspases 9 and 7 activation, and ultimately in cell death[39]. In the study of Choi and Kim[38], caspase 9 activity (early biomarker of apoptosis) was increased by daidzein 100 µM treatment for 72 hours in both MCF-7 and MDA-MB-453, but it only caused the accumulation of cells in sub-G0 phase (apoptotic peak) in MDA-MB-453.

As it can be observed with our results, daidzein arrested cell cycle in a selective manner: both tested concentrations (10 and 25 µM) increased percentage of cells at G0/G1 phase only for MCF-7. This effect on cell cycle was observed by Casagrande and Darbon[42] in OCM-1 melanoma cells, however with high daidzein concentration of 150 µM for 72 hours. Consistently with this result, they also verified CDK2 inhibition (cyclin-dependent kinase protein that controls G1/S transition) and increased expression of p21[CIP1] (CDK inhibitor)[42]. Daidzein (50 and 100 µM) can also arrest cells at the G2/M phase, as demonstrated by Choi and Kim[38] in the MCF-7 and in MDA-MB-453, with more pronounced results in the latter. Upregulation of p21[CIP1] (and G2/M arrest) was also observed in human tumoral prostate cells (PC-3) with soy isoflavone concentrate, which according to the authors reflects the natural composition found in soybeans[43].

Lack of COX-2 mRNA expression in our results with MCF-7 cells is consistent with data found in literature[44,45,46]. In our experiments with HB4a cells, daidzein caused dose-dependent increase in COX-2 transcripts. This observation, together with the fact of selective cell cycle arrest, lead us to infer that absence of COX-2 expression sensitizes MCF-7 cells to daidzein treatment, thus resulting in cell cycle arrest. It is well known that various drugs are COX-2 inhibitors, and because of this property, they can arrest cell cycle and induce apoptosis in different tumoral cell lines. On the other hand, overexpression of COX-2 has been associated with many aspects of tumourigenesis[47], including mechanisms that confer chemoresistence to cancerous cells.


Taken together, our results and data from literature confirm that daidzein at maximal achievable physiological levels can still be an effective cancer chemopreventive agent, arresting cell cycle only in the tumoral breast cell line (MCF-7) and maintaining the normal balance between survival and cell death signs in non malignant breast cells (HB4a). Further studies to comprehend the molecular mechanisms of low levels of daidzein on cancer chemoprevention are necessary, mainly because in recent years, new molecular targets of isoflavones are being discovered, like microRNAs.

We hope that our study alert scientific community and general population to pay attention to the physiological concentrations of soy isoflavones and their real effects on the carcinogenesis of breast tumours. It is also important to understand if their ingestion is appropriated to all kinds of public (healthy people, cancer patients, women at menopause, pregnant, children, etc), so that people can have better counselling on the consumption of soybean and its derivatives.


Authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; 471938/2008-4) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support.

Conflict of interests

None declared.

Competing interests

None declared


1. Doll R, Peto R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst. 1981; 66: 1191–308.

2. Adlercreutz CH, Goldin BR, Gorbach SL, Hockerstedt KA, et al. Soybean phytoestrogen intake and cancer risk. J Nutr. 1995; 125: 757S-770S.

3. Anand P, Kunnumakara AB, Sundaram C, Harikumar KB, et al. Cancer is a preventable disease that requires major lifestyle changes. Pharm Res. 2008; 25: 2097-2116.

4. Adlercreutz H. Phytoestrogens and cancer. Lancet Oncol. 2002; 3: 364-373.

5. Powles T. Isoflavones and women’s health. Breast Cancer Res. 2004; 6: 140-142.

6. Williamson-Hughes PS, Flickinger BD, Messina MJ, Empie MW. Isoflavone supplements containing predominantly genistein reduce hot flash symptoms: a critical review of published studies. Menopause. 2006; 13: 831–839.

7. Messina M, Mccaskill-Stevens W, Lampe JW. Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings. J Natl Cancer Inst. 2006; 98: 1275–1284.

8. Duffy C, Perez K, Partridge A. Implications of phytoestrogen intake for breast cancer. CA Cancer J Clin. 2007; 57: 260-277.

9. NAMS (North American Menopause Society). The role of soy isoflavones in menopausal health: report of The North American Menopause Society/Wulf H. Utian Translational Science Symposium in Chicago, IL (October 2010). Menopause. 2011; 18: 732-753.

10. Setchell KDR. Phytoestrogens: the biochemistry, physiology and implications for human health of soy isoflavones. Am J Clin Nutr. 1998; 68: 1333S-1346S.

11. Cederroth CR, Nef S. Soy, phytoestrogens and metabolism: a review. Mol Cell Endocrinol. 2009; 340: 30-42.

12. Setchell KD, Brown NM, Lydeking-Olsen E. The clinical importance of the metabolite equol - a clue to the effectiveness of soy and its isoflavones. J Nutr. 2002; 132: 3577-3584.

13. Ramos S. Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention. J Nutr Biochem. 2007; 18: 427-442.

14. Steiner C, Arnould S, Scalbert A, Manach C. Isoflavones and the prevention of breast cancer: new perspectives opened by nutrigenomics. Br J Nutr. 2008; 99: ES78-ES108.

15. Reiter E, Beck V, Medjakovic S, Jungbauer A. Isoflavones are safe compounds for therapeutical applications – Evaluation of in vitro data. Gynecol Endocrinol. 2009; 25: 554–580.

16. Moiseeva EP, Manson MM. Dietary chemopreventive phytochemicals: Too little or too much? Cancer Prev Res. 2009; 2: 611-616.

17. De Lemos ML. Effects of soy phytoestrogens genistein and daidzein on breast cancer growth. Ann Pharmacother. 2001; 35: 1118–1121.

18. Bloedon LT, Jeffcoat AR, Lopaczynski W, Schell MJ, et al. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women. Am J Clin Nutr. 2002; 76: 1126–1137.

19. Miltyk W, Craciunescu CN, Fischer L, Jeffcoat RA, et al. Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein and glycitein) in 20 patients with prostate cancer. Am J Clin Nutr. 2003; 77: 875-882.

20. Nakayama GR, Caton MC, Nova MP, Parandoosh Z. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J Immunological Methods. 1997; 204: 205-208.

21. O' Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000; 267: 5421-5426.

22. Mcmillian MK, Li L, Parker JB, Patel L, et al. An improved resazurin-based cytotoxicity assay for hepatic cells. Cell Biol Toxicol. 2002;18: 157-173.

23. Sugaya S, Nakanishi H, Tanzawa H. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells. Mutat Res. 2005; 578: 327–332.

24. Dussalt AA, Pouliot M. Rapid and simple comparison of messenger RNA levels using real-time PCR. Biol Proced Online. 2006; 8: 1-10.

25. Pfaffl MW. A new mathematical model for relative quantification in real time RT-PCR. Nucleic Acids Res. 2001; 29: 2003-2007.

26. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002; 30: 1-10.

27. Allred CD, Allred KF, Ju YH, Virant SM, et al. Soy diets containing varying amounts of genistein stimulate growth of estrogen-dependent (MCF-7) tumors in a dose dependent manner. Cancer Res. 2001; 61: 5045–5050.

28. Sathyamoorthy N, Wang TTY. Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells. Eur J Cancer. 1997; 33: 2384-2389.

29. Ju YH, Fultz J, Allred KF, Doerge DR, et al. Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice. Carcinogenesis. 2006; 27: 856–863.

30. Wuttke W, Jarry H, Seidlová-Wuttke D. Isoflavones – safe food additives or dangerous drugs? Ageing Res Rev. 2007; 6: 150-188.

31. Cederroth CR, Zimmermann C, Nef S. Soy, phytoestrogens and their impact on reproductive health. Mol Cell Endocrinol. 2012; 355: 192–200.

32. Röhrdanz E, Ohler S, Tran-Thi Q-H, Kahl R. The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells. J Nutr. 2002; 132: 370–375.

33. Nynca A, Jablonska O, Slomczynska M, Petroff BK, et al. Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles. J Physiol Pharmacol. 2009; 60: 95-105.

34. Blay M, Espinel AE, Delgado MA, Baiges I, et al. Isoflavone effect on gene expression profile and biomarkers of inflammation. J Pharm Biomed Anal. 2010; 51 382–390.

35. Lee DE, Kiwon L, Keun S, Lee EJ, et al. 6,7,4-Trihydroxyisoflavone inhibits HCT-116 human colon cancer cell proliferation by targeting CDK1 and CDK2. Carcinogenesis. 2011; 32: 629–635.

36. Wang C, Kurzer MS. Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells. Nutr Cancer. 1997; 28: 236-247.

37. Park HJ, Jeon YK, You DH, Nam MJ. Daidzein causes cytochrome c-mediated apoptosis via the Bcl-2 family in human hepatic cancer cells. Food Chem Toxicol. 2013; 60: 542–549.

38. Choi EJ, Kim G-H. Daidzein causes cell cycle arrest at G1 and G2/M phases in human breast cancer MCF-7 and MDA-MB-435. Phytomedicine. 2008; 15: 683-690.

39. Jin S, Zhang QY, Kang XM, Wang JX, et al. Daidzein induces MCF-7 breast cancer cells apoptosis via the mitochondrial pathway. Ann Oncol. 2010; 21: 263–268.

40. Adams SM, Aksenova MV, Aksenov MY, Mactutus CF, et al. Soy isoflavones genistein and daidzein exert anti-apoptotic actions via a selective ER-mediated mechanism in neurons following HIV-1 Tat1–86 exposure. Plos One. 2012; 7: e37540.

41. Wu X-F, Wang Y-J, Xia G-L, Zhang M-J. Dietary daidzein enhances antiapoptotic effect of 17β-estradiol (E2) on breast cancer MCF-7 cells. Chin J Cancer Res. 2010; 22: 10-16.

42. Casagrande F, Darbon J-M. Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1. Biochem Pharmacol. 2001; 61: 1205–1215.

43. Handayani R, Rice L, Cui Y, Medrano TA, et al. Soy isoflavones alter expression of genes associated with cancer progression, including interleukin-8, in androgen-independent PC-3 human prostate cancer cells. J Nutr. 2006; 136: 75–82.

44. Liu X, Rose D. Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res. 1996; 56: 5125–5127.

45. Half E, Tang XM, Gwyn K, Sahin A, et al. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res. 2002; 62: 1676-1681.

46. Zhao X, Goswami M, Pokhriyal N, Ma H, et al. Cyclooxygenase-2 expression during immortalization and breast cancer progression. Cancer Res. 2008; 68: 467–475.

47. Trifan OC, Hla T. Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis. J Cell Mol Med. 2003; 7: 207-222.

48. IDT. Integrated DNA Tecnologies. [homepage on the Internet]. Tool for primer design. Available from:

49. REST© software (Relative Expression Software Tool) for gene expression analysis. [homepage on the Internet]. Available from:

    Licensee to OAPL (UK) 2014. Creative Commons Attribution License (CC-BY)

    Relative gene expression (R value) of CASP-3, CASP-7, BAX, BCL-xL and COX-2 after treatment of 10 or 25 µM of daidzein in HB4a cells. R value ± SD and p value (qRT-PCR).


    Daidzein 10µM

    Daidzein 25 µM


    1.003± 0.232

    p = 0.987

    1.208± 0.185

    p = 0.031


    1.114± 0.193

    p = 0.522

    1.191± 0.149

    p = 0.204

    BAX(BCL2-associated X protein)

    1.124± 0.156

    p = 0.461

    1.524± 0.176

    p = 0.014

    BCL-xL(BCL2-like 1)

    -1.562± 0.176

    p = 0.058

    1.335± 0.168

    p = 0.010


    1.477± 0.477

    p = 0.11


    p = 0.001


    Data were submitted to software REST© (PFAFFL et al., 2002). Negative R value means downregulation of the gene.

    Relative gene expression (R value) of CASP-3, CASP-7, BAX and BCL-xL after treatment of 10 or 25 µM of daidzein in MCF-7 cells. R value ± SD and p value (qRT-PCR).


    Daidzein 10 µM

    Daidzein 25 µM

    CASP-3 (Caspase-3)

    1.253± 0.254

    p = 0.251

    2.773± 0.489

    p = 0.045

    CASP-7 (Caspase-7)

    1.38± 0.297

    p = 0.171

    2.059± 0.371

    p = 0.004

    BAX(BCL2-associated X protein)

    1.34± 0.238

    p =0.151

    -1.107± 0.213

    p = 0.418

    BCL-xL (BCL2-like 1)

    1.467± 0.259

    p = 0.037

    1.209± 0.218

    p = 0.368


    Data were submitted to software REST© (PFAFFL et al., 2002). Negative R value means downregulation of the gene. COX-2 expression in mammary tumoral MCF-7 cells was null.