Xiao-fei Wu, Yi-jing Wang, Guo-liang Xia, Mei-jia Zhang

State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E2) on Breast Cancer MCF-7 Cells

Xiao-fei Wu, Yi-jing Wang, Guo-liang Xia, Mei-jia Zhang*

State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Objective:To investigate whether dietary daidzein interact with endogenous 17β-Estradiol (E2) to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells.

Methods:Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein (1, 5 μmol/L) and E2(0.1?10 nmol/L) for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ) and ERβ-estrogen response element (ERE) dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptoticp53,baxand antiapoptoticbcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot.

Results:Daidzein enhanced the antiapoptotic effect in an E2dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression ofp53andbax, but had no effect on E2-upregulated expression ofbcl-2.

Conclusion:Daidzein enhances the antiapoptotic effect of E2on breast cancer cells by inhibiting E2-mediatedp53-baxproapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2in hormone-dependent breast cancer.

Daidzein; E2; Breast cancer; MCF-7 cells; Antiapoptotic effect; Estrogen receptor (ER)

INTRODUCTION

Daidzein, the second-most prominent insoflavone in soy products, has attracted attention because intake of it redounds to the protection against breast cancer[1,2]. However, the anticancer effect of daidzein is only observed in vitro experiments at high concentrations (＞10 μmol/L)[3]. In contrast, the dietary concentration of daidzein only reaches 1?5 μmol/L[4], which stimulates the 11111111growth of estrogen receptor (ER)-positive breast cancer cells in vitro, exhibiting estrogenic properties[5]. Estrogens are the most important risk factors for breast cancer, and endogenous E2present in breast tissue may achieve 0.1 to 10 nmol/L[6,7]. The role of dietary daidzein in an endogenous estrogen environment still remains obscure.

Activation of ERα is known to promote cellular growth, but activation of ERβ has been proposed to inhibit proliferation and induce apoptosis in breast tumors[8?10]. ERβ mRNA was dose-dependently upregulated by high concentrations of E2(≥10 nmol/L) in breast cancer T47D cells[11]. E2may stimulate cellular growth via ERα, and synchronously induce cellular apoptosis via ERβ above 10 nmol/L in T47D cells. For example, theproliferative rate rose from 0.1 to 10 nmol/L and dropped from 10 to 100 nmol/L[12]. Proapoptotic effect involves thep53-baxpathway. Proapoptotic proteinbaxcan induce the mitochondrial pathway of apoptosis and tumor suppressor proteinp53can inducebaxexpression[13].bcl-2 is an antiapoptotic protein and its overexpression has been shown to inhibit apoptosis[14]. Thebcl-2/baxprotein ratio modulates cellular apoptosis[15].

Therefore, we examined whether dietary daidzein affected E2-mediated proliferation and apoptosis of breast cancer cells, by altering E2-modulated expression of ERα, ERβ,p53,baxandbcl-2.

MATERIALS AND METHODS

Reagents and Cell Culture

Daidzein, 17β-Estradiol (E2) and Propidium iodide (PI) were purchased from Sigma and MPP dihydrochloride (highly selective ERα antagonist) from Tocris. Human breast cancer MCF-7 cells were obtained from ATCC. Cells were routinely maintained in DMEM medium (Gibco), supplemented with 10% FBS (Hyclone) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Gibco) at 37°C in a humidified atmosphere containing 5% CO2. Prior to each experiment, Media were changed into phenol red-free DMEM (Gibco) supplemented with 10% charcoal dextran-treated FBS (CDT-FBS, Hyclone) and cells were incubated for at least one week to deplete steroid hormones. Then, cells were incubated for five days in medium supplemented with 5% CDT-FBS containing various concentrations of daidzein or/and E2.

Cell Cycle Analysis and Apoptosis Measurement

After treatment for 5 d, cells from two dishes in each group were put into a conical tube, and washed twice with PBS by centrifugating at 3000g for 5 min at room temperature. Cell cycle distribution and the rate of apoptosis were measured using flow cytometry as described before[16].

Values were expressed asof two representative experiments, each performed in 4?6 replicates.

RNA Extraction and RT-PCR

After treatment for 72 h, Total RNA was isolated from cells by extraction using TRIZOL reagent. Lysates were extracted with chloroform and total RNA was precipitated with isopropanol. Verification of changed expression was done by semi-quantitative RT-PCR for ERα,p53,baxandbcl-2. Details of the primers for target genes and reaction conditions are listed in Table 1. GAPDH severed as the internal standard. The PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized by ethidium bromide staining. Relative intensities were quantified using Gel-pro Analyzer 4.0. The ratio between the intensity of the bands was reported as the. For that the level of ERβ mRNA was low in MCF-7[17], real-time quantitative RT-PCR was performed to investigate the expression of ERβ as previously described[18]. Data represent the average of replicates with their standard errors.

Luciferase Induction Assays

The estrogen response element (ERE) reporter construct (pGL3-ERE-luciferase) and expression vectors for ERβ (psG5-hERβ) were kindly provided by Professor Dalong Ma (Chinese National Human Genome Center). Cells were transferred into 24-well plates (5×104cells/well) with 500 μl phenol red-free DMEM (without antibiotics or fungicides) containing 3% CDT-FBS/well the day before transfection. One day later, 0.1 ml medium containing plasmid DNA (0.2 μg ERE reporter plasmid, 0.05 μg PRL-TK, and 0.5 μg ERβ expression vector) and FuGENE HD Transfection reagent (Roche) at a charge ratio of 1:3 was added, then cells were incubated for 14 h. The cells were incubated in medium containing 3% CDT-FBS plus test compounds for another 24 h. MPP dihydrochloride (100 nmol/L), an ERα-specific antagonist, was used with test compounds together to avoid the effect of ERα-ERE transactivation.

When ERβ is activated by the test ligands, ERβ forms a dimer that binds ERE, activating the transcription of luciferase reporter gene. Cell extracts were prepared for luciferase reporter assay (Dual-Luciferase Reporter Assay System, Promega). Transcriptional activity is represented as relative light units (RLUs) calculated as percentage of the maximal induction by E2(100 nmol/L) and standardized to the internal transfection control provided byrenillaluciferase activity.

Western Blot Analysis

Cells were lysed in ice-cold lysis buffer containing 50 mmol/L Tris-HCL, pH 7.5, 150mmol/L NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mmol/L PMSF, 1 mmol/L NaF, 1% SDS (v/v), 2 mmol/L sodium orthovanadate, 0.2 mmol/L DTT, and complete TM protease inhibitor cocktail. The cellular debris was cleared by centrifugation (12,000×g, 10 min, 4°C). Protein content of the samples was determined by BCA procedure. Equal amounts of protein (80 μg/well) were separated by SDS-PAGE (15%) and electrotransferred onto nitrocellulose membranes (Bio-Rad, Hercules; Amersham Biosciences). The membranes were incubated overnight at 4°C with primary antibodies (Santa Cruz, CA, USA). Then, the membrane was incubated for 1 h with horseradish peroxidaseconjugated antibody at room temperature. Finally, ECL Plus reagent (Pierce Biotechnology, IL, USA) was used to detect the peroxidase activity and the signal was visualized by autoradiography.

Statistical Analysis

All experiments were repeated at least three times and the values are given as. All frequencies were subjected to arcsine transformation and analyzed by ANOVA followed by Duncan’s multiple range tests.P＜0.05 was considered statistically significant and groups with a common letter are not significantly different.

Table 1. Primer sequences and cycling conditions used for RT-PCR for ERα, ERβ, p53, bax and bcl-2 mRNA in MCF-7 cells

RESULTS

Effects of Daidzein and E2on Cell Cycle Progression and Apoptosis in MCF-7 Cells

The concentrations of the compounds, the percentages of cells in G0/G1, S, G2/M phases and sub-G0/G1peak were presented in Table 2. The proliferation was indicated by the percentage of cells in the S phase of the cell cycle. Compared with control, Daidzein or E2significantly induced cellular proliferation (P＜0.05, Figure 1). Neither additive nor antagonistic effects on proliferation could be observed with any of the daidzein/E2combinations (Figure 1). The rate of apoptosis was indicated by the percentage of sub-G0/G1nuclei of all nuclei measured. In comparison with control, Daidzein significantly decreased the rate of apoptosis (P＜0.05, Figure 1). However, E2produced a biphasic effect on cellular apoptosis (Figure 1), namely inhibiting apoptosis at low concentration (0.1 nmol/L,P＜0.05) and inducing apoptosis at high concentration (10 nmol/L,P＜0.05). E2exhibited (1 nmol/L) no significant effect on apoptosis. Compared with daidzein or E2alone treatment, the rate of apoptosis significantly decreased when cells were co-treated with daidzein and E2(P＜0.05, Figure 1). In addition, daidzein enhanced the antiapoptotic effect in an E2dose-dependent manner.

Effects of Daidzein and E2on Gene Expression of ERα and ERβ

We first investigated whether daidzein could interfere with E2-modulated expression of ERα and ERβ mRNA by RT-PCR after cells were treated for 72 h with various doses of daidzein or/and E2. As shown in Figure 2A and B, E2dose-dependently decreased ERα mRNA and increased ERβ mRNA compared with the control. Daidzein could inhibit E2-increased ERβ mRNA, but had no effect on ERα mRNA expression when cells were co-treated with daidzein and E2.

Table 2. Effects of daidzein and E2on cell cycle and apoptosis in MCF-7 cells,, n≥ 3

Table 2. Effects of daidzein and E2on cell cycle and apoptosis in MCF-7 cells,, n≥ 3

Compounds G0/G1S G2/M Sub-G0/G1Control 77.6%±1.6 15.8%±2.2 6.6%±3.1 27.8%±2.1 Cell cycle Apoptosis 1 μmol/L daidzein 71.2%±2.2 21.2%±3.6 7.6%±3.5 20.8%±5.3 5 μmol/L daidzein 69.8%±4.4 22.8%±2.0 7.4%±2.6 22.3%±2.6 0.1 nmol/L E270.9%±2.2 20.9%±4.4 8.2%±3.1 21.1%±3.8 1 nmol/L E268.1%±2.5 25.6%±3.5 6.3%±2.8 25.6%±5.6 10 nmol/L E265.9%±4.1 29.8%±4.5 4.3%±3.3 33.6%±4.4 1 μmol/L daidzein +0.1 nmol/L E271.4%±2.1 21.5%±5.4 7.1%±3.2 15.2%±4.0 1 μmol/L daidzein +1 nmol/L E270.6%±3.7 23.1%±3.4 6.3%±4.2 13.8%±3.5 1μmol/L daidzein +10 nmol/L E268.1%±1.9 26.8%±4.2 5.1%±2.8 11.2%±5.5 5 μmol/L daidzein +0.1 nmol/L E272.3%±3.2 22.9%±2.5 4.8%±2.0 17.1%±4.8 5 μmol/L daidzein +1 nmol/L E267.8%±4.1 25.3%±4.5 6.9%±2.1 16.1%±3.2 5 μmol/L daidzein +10 nmol/L E265.7%±4.5 28.6%±5.3 5.7%±3.7 12.5%±4.1

Figure 1. Effects of daidzein and E2on cell cycle progression and apoptosis in MCF-7 cells.

Luciferase Induction Assays

In order to investigate whether daidzein could inhibit E2-mediated transcription via ERβ, we further performed luciferase induction assays in MCF-7 cells. Transcriptional activity is represented as relative light units (RLUs) calculated as percentage of the maximal induction by 100 nmol/L E2. E2induced transactivation of the ERE reporter gene via ERβ in a concentrationdependent manner (Figure 3). Although daidzein induced ERβ-ERE transactivation, it could attenuate E2-mediated ERE transactivation via ERβ when daidzein was used with 1 or 10 nmol/L E2together.

Figure 2. Effects of daidzein and E2on gene expression of ERα and ERβ. A: Representative gel showing ERα mRNA expression; B: ERβ mRNA expression values were expressed as fold changes compared with control (defined as 1).

Effects of Daidzein and E2on the Expression ofp53,baxandbcl-2 at both mRNA and Protein Levels

After cells were treated for 72 h, the effects of daidzein on E2-modulated the expression ofp53,baxandbcl-2 at both mRNA and protein levels were investigated by RT-PCR and Western blot. In comparison with the control, 1 or 5 μmol/L daidzein significantly decreasesedbaxmRNA (P＜0.05), and E2dose-dependently increasedp53,baxandbcl-2 mRNA (Figure 4A, B). When cells were co-treated with daidzein and E2, daidzein only antagonized E2-upregulated mRNA expression ofp53andbax(Figure 4A, B). Additional Western blot assays were also examined and the results were shown in Figure 4C. Daidzein did not alter the protein levels ofp53andbcl-2, but markedly decreased the level ofbaxprotein. However, E2dose-dependently increased the protein levels ofp53andbax, and upregulatedbcl-2 protein expression to the same level (Figure 4C). When cells were co-treated with daidzein and E2, daidzein antagonized E2-induced expression ofp53andbaxproteins, but had no effect on E2-upregulated expression ofbcl-2 protein andbcl-2/baxprotein ratio was higher than daidzein or E2treatment alone (Figure 4C). Hence, daidzein antagonized E2-induced expression ofp53andbaxat both mRNA and protein levels.

Figure 3. Daidzein attenuated E2-mediated transactivation via ERβ.

DISCUSSION

Figure 4. Effects of daidzein and E2on expression ofp53,baxandbcl-2 at both mRNA and protein levels were investigated by RT-PCR and Western blot. A: Representative gel showing gene expression ofp53,baxandbcl-2; B: Summary of the relative densities ofp53,baxandbcl-2 genes; C: Representative blot showing protein expression ofp53,baxandbcl-2.

In the present study, we investigated the combinatory effect of dietary daidzein and endogenous E2(0.1?10 nmol/L) on proliferation and apoptosis of breast cancer cells. Daidzein enhanced the antiapoptotic effect in an E2dose-dependent manner, but had no effect on E2-induced proliferation when MCF-7 cells were cotreated with daidzein and E2. The result is similar to previous report that anticancer effect of genisteinwas abolished by cotreatment with higher concentration of E2(25 nmol/L or 50 nmol/L)[19]. Our results suggest that dietary daidzein may increase the risk of estrogen-dependent breast cancer especially for postmenopausal women, in whose breast tissue E2is transnormal.

In addition, we further examined the mechanism by which daidzein enhances the antiapoptotic effect of E2on breast cancer cells. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transactivation, but had no effect on E2-modulated expression of ERα andbcl-2 mRNA. Daidzein antagonized E2-upregulated expression ofp53andbaxat both mRNA and protein levels, inducing a marked increase inbcl-2/baxprotein ratio when cells were co-treated with daidzein and E2. Thus, the molecular mechanism is that daidzein do not interfere with the antiapoptotic effect by E2, but inhibits E2-induced proapoptotic effect throughp53-baxpathway.

Daidzein had no antagonistic effect on E2-induced cellular proliferation in our and other study[20]. It has been reported that genistein is more effective than daidzein in competing with E2for binding to ERα[20]. Its strong affinity for ERα generally correlates with the antagonistic effect of genistein on E2-induced proliferation, possibly by downregulating ERα expression[8,20]. Daidzein had no effect on E2-downregulated expression of ERα mRNA in our study. All these studies show that daidzein has no effect on E2-induced proliferation due to its weak affinity for ERα.

Activation of ERβ may inhibit cellular growth in breast cancer cells[9,10]. For example, Pyranocoumarin compound induced apoptosis, accompanied by an increased expression of ERβ[21]. The antiproliferative effect of apigenin was effectively abrogated by ERβ siRNA to downregulate ERβ[22]. Our results showed that daidzein could antagonize E2-upregulated ERβ mRNA expression and attenuate E2-mediated transactivation via ERβ. Generally, the ability for binding of a phytoestrogen to a specific ER subtype correlates with its ability to transactivate gene expression through that receptor. So, daidzein attenuates E2-mediated transactivation via ERβ by competing with E2for binding to ERβ. This is likely associated with that daidzein has a stronger affinity for ERβ than ERα, and E2binds to ERα and ERβ with equal affinity[23,24]. Our result is inconsistent with previous investigation that 1 μmol/L daidzein could not interfere with 0.5 nmol/L E2-mediated transactivation via ERβ[24]. The differences result from that endogenous ERα might interfere with the activity of the exogenously introduced ERβ in their system, however ERα-specific antagonist was used to avoid the effect of ERα-ERE transactivation in ours.

Previous results have demonstrated that pharmacological concentration of daidzein induced cellular apoptosis of MCF-7 cells[3], followed by upregulation of the expression of ERβ,p53andbaxmRNA[25]. In contrast, the expression ofp53andbaxmRNA were downregulated by daidzein in MCF10a devoid of ERβ[25]. Proapoptotic effect may be relevant to the activation of ERβ-p53-baxpathway[25,26]. Thus, daidzein antagonized E2-activatedp53-baxpathway possibly through attenuating E2-upregulated ERβ mRNA expression and ERβ-ERE dependent transactivation. Proapototicp53protein induces apoptosis by transcriptional activation of proapoptotic genebax[13]. However,bcl-2 protein can dimerize withbaxto silence its apoptotic functions[14]. The ratio ofbcl-2/baxprotein modulates apoptosis[15]. In this study, E2dose-dependently increased the levels of proapoptoticp53andbaxproteins in MCF-7 cells. These increases coincided with that E2induces cellular apoptosis at high concentration. Daidzein only antagonized E2-upregulated expression ofp53andbaxproteins. E2can inducebcl-2 expression via ERα-ERE in MCF-7 cells[27]. As mentioned above, daidzein hardly competes with E2for ERα binding and interferes with its transactivation via ERα[20,24]. That daidzein has no effect on E2-upregulated expression ofbcl-2 may result from these. So,bcl-2/baxprotein ratio markedly increased when cells were co-treated with daidzein and E2. It can be seen that daidzein attenuates the proapoptotic effect of E2, and do not inhibit the antiapoptotic effect of E2.

In summary, dietary daidzein enhances the antiapoptotic effect in an E2dose-dependent manner by inhibiting E2-induced proapoptoticp53-baxpathway. Thus, we suggest that dietary daidzein may potentiate deleterious effect of endogenous estrogen in hormone-dependent breast cancer.

Acknowledgements

We thank Professor Da-long Ma (Chinese National Human Genome Center, P.R. China) for donating the plasmids that were used in this study.

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R737.9 Document code: A Article ID: 1000-9604(2010)01-0010-07

10.1007/s11670-010-0010-2

2009?08?12; Accepted 2009?10?23

This work was supported by the National Natural Science Foundation of China (No.30671508) and by State Key Laboratory for Agrobiotechnology of China (No.2009SKLAB07-5).

*Corresponding author.

E-mail: zmeijia@cau.edu.cn

? Chinese Anti-Cancer Association and Springer-Verlag Berlin Heidelberg 2010