1. Introduction {#sec1-molecules-23-01557}
===============

Epidemiological evidence suggests that an increased consumption of soybean food is associated with a decreased cancer risk \[[@B1-molecules-23-01557]\]. Soybean food has been recommended in cancer prevention for a number of anticarcinogens exist in it. Soybean isoflavones, a family of nonnutritive compounds, were demonstrated to be associated with modulation of a variety of biological processes in carcinogenesis \[[@B2-molecules-23-01557]\]. Equol, derived from isoflavones by the action of gut microflora, was reported to inhibit cancer cell growth and invasion with higher bioavailability \[[@B3-molecules-23-01557]\]. Phytosterols are plant sterols chemically similar to cholesterol but exclusively in plants, particularly legumes. Awad et al. \[[@B4-molecules-23-01557]\] found that it could induce apoptosis in breast cancer cells. In addition, a previous study indicated that soybean saponins could suppress the growth of colon tumor cells in vitro \[[@B5-molecules-23-01557]\]. Except these phytochemicals, soybean also provides a source of bioactive proteins and polypeptides which are now being intensively studied as cancer chemopreventive agents, such as trypsin inhibitor, lectin and a recently discovered peptide lunasin \[[@B6-molecules-23-01557],[@B7-molecules-23-01557],[@B8-molecules-23-01557]\].

Breast cancer is the most frequently diagnosed cancer and one of the major causes of mortality in woman \[[@B9-molecules-23-01557]\]. The conventional treatment methods of breast cancer would bring irreversible side effects to patients. Although a lot of work on prevention and intensive treatment of breast cancer has been done, effective strategies with minor side effects are still lacking \[[@B10-molecules-23-01557]\]. Breast cancer is a heterogeneous disease, classified into different subtypes according to the gene expression profile or immunohisto chemical markers such as estrogen receptor (ER) \[[@B11-molecules-23-01557]\]. MCF-7 and MDA-MB-231 human cancer cell lines are ER+ and ER− cells, which are well-established in vitro model for evaluation of estrogen responsive or estrogen independent antineoplastic drugs \[[@B12-molecules-23-01557]\]. Recently, increasing evidences suggested that the abundant anticarcinogens in soybean have obvious ability to prevent and treat breast cancer. However, it now remains to identify the most bioactive anticarcinogens from soybean on different subtypes of breast cancer cells.

The low concentration and low bioavailability of active components in natural plants limited their clinical application \[[@B13-molecules-23-01557]\]. It is reported that chemotherapeutic combination approaches could reach a greater effect than with one active compounds single at equal concentrations \[[@B10-molecules-23-01557]\], which is called a synergistic effect. Synergistic anticancer effect has been observed with different compounds or bioactive components derived from natural plants such as arctigenin and quercetin \[[@B14-molecules-23-01557]\], and phytochemicals enriched apple extracts and quercetin 3-β-[d]{.smallcaps}-glucoside \[[@B10-molecules-23-01557]\]. However, limited knowledge is available regarding the synergistic effect of anticarcinogens in soybean on inhibition of proliferation of breast cancer cells.

In this study, the anti-proliferative activity of 12 reported anticarcinogens individually from soybean in MDA-MB-231 and MCF-7 human breast cancer cells was firstly evaluated by single treatment. Then, anticarcinogens showing stronger activity were taken forward to investigate whether there is a synergistic effect on human breast cancer cell proliferation by two-way combination treatment.

2. Results and Discussion {#sec2-molecules-23-01557}
=========================

2.1. Anti-Proliferative Effects of 12 Treatment Solutions on Human Breast Cancer Cells by Single Treatment and Combination Treatment {#sec2dot1-molecules-23-01557}
------------------------------------------------------------------------------------------------------------------------------------

To realize the aims of this study, 12 reported anticarcinogens from soybean were collected and screened for their anti-proliferative activity against the MCF-7 and MDA-MB-231 human breast cancer cells. As shown in [Figure 1](#molecules-23-01557-f001){ref-type="fig"}a,b, within the concentrations determined (0--200 μM), BBI and lunasin have no significant anti-proliferative effect on MCF-7 cells. Genistein, daidzein, glycitein, genistin, daidzin, glycitin, equol, soyasaponin, β-sitosteroland lectin inhibited the MCF-7 cell proliferation in a dose-dependent manner. Among these 10 bioactive anticarcinogens, genistein, daidzein, glycitein, genistin and daidzin were demonstrated to have stronger anti-proliferative activity, with the EC~50~ values ([Figure 2](#molecules-23-01557-f002){ref-type="fig"}a) of 66.98 ± 4.87 μM, 130.14 ± 2.10 μM, 190.67 ± 5.65 μM, 72.82 ± 2.66 μM and 179.21 ± 6.37 μM, respectively. MCF-7 human breast cancer cells are estrogen-responsive breast cancer cells. The anti-proliferative activity of soybean isoflavones has been previously and widely demonstrated in estrogen-responsive breast cancer cells in vitro \[[@B15-molecules-23-01557]\]. Recently, considerable interest has focused on the triple negative breast cancer which is an aggressive subtype of breast cancer and more frequent in younger and pre-menopausal women \[[@B16-molecules-23-01557]\]. MDA-MB-231 human breast cancer cells are the estrogen-independent, triple negative breast cancer cells. Results of the present study showed that single treatments with daidzin, BBI, equol, lectin and lunasin at concentrations less or equal than 200 μM showed no significant inhibition effects on MDA-MB-231 cell proliferation. However, genistein, glycitein, genistin and β-sitosterol exhibited stronger anti-proliferative effects against MDA-MB-231 cells in a dose-dependent manner, with the EC~50~ values ([Figure 2](#molecules-23-01557-f002){ref-type="fig"}b) of 93.75 ± 5.15 μM, 142.67 ± 5.88 μM, 127.82 ± 4.70 μM and 196.28 ± 4.45 μM, respectively. These results suggested that MCF-7 cells were more sensitive to anticarcinogens from soybean than MDA-MB-231 cells.

Due to obvious anti-proliferative activity of compounds from natural plants, the dietary modification is thought to be an alternative strategy to prevent and reduce the risk of breast cancer \[[@B10-molecules-23-01557]\]. However, the effective doses of these compounds can barely be achieved by oral consumption. Nowadays, the synergistic effect generated from drug combination has attracted great attention due to the advantage of increased anti-cancer effect, lesser drug dose, reduced side effects. To further investigate whether there is a synergistic effect of anticarcinogens from soybean, samples showing stronger anti-proliferative activity by single treatment were selected and two-way combination treatments were conducted respectively in MCF-7 and MDA-MB-231 cells. Ten combination treatments including genistein plus daidzein, genistein plus glycitein, genistein plus genistin, genistein plus daidzin, daidzein plus glycitein, daidzein plus genistin, daidzein plus daidzin, glycitein plus genistin, glycitein plus daidzin and genistin plus daidzin were performed on MCF-7 cells ([Figure 2](#molecules-23-01557-f002){ref-type="fig"}a). The CI values were calculated for all the ten combination treatment at 50% inhibition of MCF-7 proliferation. Results ([Table 1](#molecules-23-01557-t001){ref-type="table"}) showed that only the CI~50~ value of the combination of genistin plus daidzin was less than 1 (0.89 ± 0.12), indicating that there was a synergistic effect by the combined treatment of genistin plus daidzin in MCF-7 cells. The EC~50~ value of genistin and daidzin were reduced to 26.21 ± 3.72 μM and 64.50 ± 3.88 μM, respectively, due to the synergistic effect. Six combination treatments including genistein plus glycitein, genistein plus genistin, genistein plus β-sitosterol, glycitein plus genistin, glycitein plus β-sitosterol and β-sitosterol plus genistin were conductedtoward MDA-MB-231 cells and results are presented in [Figure 2](#molecules-23-01557-f002){ref-type="fig"}b. The CI~50~ values of two-way combination treatments of genistein plus genistin, genistein plus β-sitosterol and β-sitosterol plus genistin for MDA-MB-231 cell proliferation were 0.56 ± 0.13, 0.54 ± 0.20 and 0.45 ± 0.12, respectively ([Table 1](#molecules-23-01557-t001){ref-type="table"})These results suggested that there were significant synergistic effects of these three combination treatments. The EC~50~ values of genistein, genistin and β-sitosterol in these two-way combination treatments were significantly lower than in single treatments. The EC~50~ values of β-Sitosterol and genistin toward MDA-MB-231 reduced to 37.71 μM and 24.55 μM, less than 50 μM. It is reported that soybean isoflavone concentrations in prostatic fluid can reach up to 50 μM in persons with a long-term soybean-rich dietary habits \[[@B17-molecules-23-01557]\]. The synergistic effects of these combination treatments increase the possible application of natural anticarcinogens in humans.

2.2. Cytotocicity of Bioactive Anticarcinogens by Single or Two-Way Combination Treatment {#sec2dot2-molecules-23-01557}
-----------------------------------------------------------------------------------------

At the concentration of the EC~50~ values, the cytotoxicity of genistein, daidzein, glycitein, genistin, daidzin and genistin plus daidzin toward MCF-7 cells, and the cytotoxicity of genistein, glycitein, genistin, β-sitosterol, genistein plus genistin, genistein plus β-sitosterol and β-sitosterol plus genistin toward MDA-MB-231 cells are shown in [Figure 3](#molecules-23-01557-f003){ref-type="fig"}a,b. No cytotoxicity was observed in genistein, daidzein, genistin and genistin plus daidzin toward MCF-7 cells. In addition, there was no cytotoxicity in genistein, genistin, genistein plus genistin, genistein plus β-sitosterol and β-sitosterol plus genistin toward MDA-MB-231 cells.

2.3. Inhibition of Cell Invasion and Migration {#sec2dot3-molecules-23-01557}
----------------------------------------------

The invasive and migratory ability is the important characteristic of metastasis of tumor cells \[[@B18-molecules-23-01557]\]. To further evaluate the pharmacological activity of these anticarcinogens against cancer metastasis, wound-healing assay and transwell chamber assay were performed to evaluate the inhibition of cell invasion and migration. As shown in [Figure 4](#molecules-23-01557-f004){ref-type="fig"}a,b, treatment of MCF-7 cells with genistein, daidzein, glycitein, genistin or daidzin single led to decreased wound closure compared to control cells by 49.49%, 46.22%, 47.24%, 34.98% and 38.49%, respectively, suggesting that these samples reduced the motility of MCF-7 cells. The mixture of genistin plus daidzin inhibited the wound closure by 59.48%, significantly more (*p* \< 0.05) than treatment with genistin or daidzin single. Genistein, glycitein, genistin and β-sitosterol were able to inhibit the wound closure in MDA-MB-231 ([Figure 4](#molecules-23-01557-f004){ref-type="fig"}c,d). They decreased wound closure compared to control cells by 57.27%, 24.93%, 5.19% and 15.67%. Genistein plus genistin, Genistein plus β-sitosterol, and β-sitosterol plus genistin decreased wound closure by 55.56%, 27.49% and 45.51%. However, among these combination treatments, only the mixture of β-sitosterol and genistin showed significantly stronger (*p* \< 0.05) inhibitory effect than single treatment in MDA-MB-231 cells.

Results of the transwell assay showed that gensitein, daidzein, glycitein, genistin and daidzin have a positive effect on MCF-7 cell migration ([Figure 4](#molecules-23-01557-f004){ref-type="fig"}e). The migration of MCF-7 cells was respectively inhibited by 65.43%, 57.04%, 42.04%, 38.19% and 62.03% with gensitein, daidzein, glycitein, genistin and daidzin treatment compared to control. The combination treatment of genistin plus daidzin significantly enhanced (*p* \< 0.05) the inhibition compared to genistin and daidzin alone. The inhibitory effects of genistein, glycitein, genistin and β-sitosterol on MDA-MB-231cell migration were also examined ([Figure 4](#molecules-23-01557-f004){ref-type="fig"}f). Similar to results of the wound-healing assay, genistein, glycitein, genistin and β-sitosterol led to a significant decrease (*p* \< 0.05) in MDA-MB-231 cell migration, however, genistin had no significant effect by single treatment. Three combination treatments also showed significant inhibitory effects on MDA-MB-231 cell migration, however, only combination of genistin plus β-sitosterol enhanced the effects compared to genistin and β-sitosterol alone.

Overall, all the data indicate that the bioactive anticarcinogens, except genistin, from soybean effectively decreased the invasive and migratory ability of MCF-7 cells and MDA-MB-231 cells. The combination treatment could enhance the inhibitory effect. The inhibitory effect on the invasion and migration of cancer cells is not due to the cytotoxicity.

2.4. Modulations of Protein Expression and Signaling Pathways {#sec2dot4-molecules-23-01557}
-------------------------------------------------------------

The anti-proliferative effect and inhibitory effect on cell invasion and migration of anticarcinogens was associated with changes in multiple signaling pathways \[[@B14-molecules-23-01557]\]. In order to explore the involved signaling pathways of single treatments and combination treatments in MCF-7 and MD-MB-231 cells, the protein expression levels of 18 intracellular signaling molecules ([Figure 5](#molecules-23-01557-f005){ref-type="fig"}a) were examined.

In MCF-7 cells ([Figure 5](#molecules-23-01557-f005){ref-type="fig"}b,c), results showed that genistein significantly (*p* \< 0.05) decreased phosphorylation of stat3, Akt, and PRAS40; Daidzein significantly (*p* \< 0.05) decreased phosphorylation of stat3 and PRAS40; Glycitein significantly (*p* \< 0.05) decreased phosphorylation of stat3, and increased phosphorylation of 5′AMP-activated protein kinase (AMPK) α; Daidzin significantly (*p* \< 0.05) increased phosphorylation of AMPKα. By combination treatment of genistin plus daidzin, phosphorylation of stat3, mammalian target of rapamycin (mTOR) and PRAS40 were significantly (*p* \< 0.05) decreased, and phosphorylation of AMPKα was significantly (*p* \< 0.05) increased. In MDA-MB-231 cells, genisitein significantly (*p* \< 0.05) downregulated phosphorylation of Akt, mTOR, p38, PRAS40 and GSK-3β, and upregulated phosphorylation of BAD and p53; Glycitein significantly (*p* \< 0.05) downregulated phosphorylation of Akt, mTOR, p38, PRAS40 and GSK-3β, and upregulated phosphorylation of AMPKα; Genistin significantly (*p* \< 0.05) downregulated phosphorylation of Akt, mTOR and GSK-3β, and upregulated phosphorylation of BAD and p53; β-Sitosterol significantly downregulated phosphorylation of Akt, BAD, p53, p38 PRAS40 and GSK-3β, and upregulated AMPKα. Three combination treatments showed similar pattern with significantly (*p* \< 0.05) upregulation of phosphorylated AMPKα, BAD and p53, but not significantly (*p* \> 0.05) downregulation of phosphorylated Akt and mTOR. These results suggested that these anticarcinogens exerted anticancer effect in MCF-7 and MDA-MB-231 cells via different signaling pathways.

From these results, almost all treatments upregulated phosphorylation of AMPKα in varied degrees. AMPK is a serine/threonine protein kinase which responsible for cellular energy homeostasis \[[@B18-molecules-23-01557]\]. Published studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumour cells. These actions of AMPK appear to be mediated through multiple mechanisms including regulation of the cell cycle and inhibition of protein synthesis, de novo fatty acid synthesis, specifically the generation of mevalonate as well as other products downstream of mevalonate in the cholesterol synthesis pathway. It is reported that AMPK can regulate cell proliferation, cell growth and autophagy. It has been proposed to be a potential target for cancer chemotherapy \[[@B19-molecules-23-01557],[@B20-molecules-23-01557]\].

It reported that PI3K/Akt/mTOR signaling pathway activation is heavily implicated in cancer pathogenesis \[[@B21-molecules-23-01557]\]. Inhibited phosphorylation of Akt and mTOR in this study suggested that inactivation of PI3K/Akt/mTOR pathway seems to be the main mechanism involved in anticancer activity of bioactive anticarcinogens from soybean both in MCF-7 and MDA-MB-231 and cells. To further confirm this hypothesis, the protein expression level of PI3K p85 was determined using Western blot assay ([Figure 6](#molecules-23-01557-f006){ref-type="fig"}a--d). As shown, a reduction in the expression level of PI3K p85 was observed in these tested samples, which supported our hypothesis. In addition, change in the phosphorylation of molecular proteins including stat3 (a key target in cancer cell growth), Bad (a pro-apoptoic protein from Akt pathway), p53 (a pro-poptotic protein well known tumor suppressor), p38 (a member of MAPK), PRAS40 (a protein which can inhibit Akt pathway) and GSK-3β (a key regulator in tumor development) was observed in this study \[[@B22-molecules-23-01557],[@B23-molecules-23-01557],[@B24-molecules-23-01557],[@B25-molecules-23-01557],[@B26-molecules-23-01557]\].

3. Materials and Methods {#sec3-molecules-23-01557}
========================

3.1. Chemicals {#sec3dot1-molecules-23-01557}
--------------

Genistein, daidzein, glycitein, genistin, daidzin, glycitin, equol, soyasaponin, β-sitosterol, Bowman-birk trypsin inhibitor (BBI), lectin and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lunasin was synthesized by the American Peptide Company (Sunnyvale, CA, USA). 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulphonic acid (HEPES), Hank's balancedsalt solution (HBSS), fetal bovine serum (FBS), penicillin-streptomycin, phosphate-buffered saline (PBS) and α-minimum essential medium (α-MEM) were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). Methylene blue was purchased from BBL (Cockeysville, MD, USA). All reagents are of analytical grade.

3.2. Preparation of Treatment Solution {#sec3dot2-molecules-23-01557}
--------------------------------------

Genistein, daidzein, glycitein, genistin, daidzin, glycitin, equol, soyasaponin and β-sitosterol screened were prepared in 100% dimethyl sulfoxide (DMSO) at 10,000 μM. BBI, lunasin and lectin were prepared in growth media at 5000 μM. These solutions were stored at −80 °C and used as stock solution. Working solutions of all treatments were made through supplement with growth media until the final concentrations of 0--200 μM.

3.3. Cell Culture {#sec3dot3-molecules-23-01557}
-----------------

The MDA-MB-231 and MCF-7 human breast cancer cell lines were obtained from American Tissue Type Culture Collection (ATCC, Bethesda, MD, USA). MDA-MB-231 cells were maintained in α-MEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1mM sodium pyruvate. MCF-7 cells were maintained in α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate and 10 μg/mL insulin. All cells were grown in a humidified atmosphere at 37 °C, with 5% CO~2~ in air.

3.4. Cell Proliferation Assay {#sec3dot4-molecules-23-01557}
-----------------------------

The cell proliferation was determined using the methylene blue (98% HBSS, 0.67% glutaraldehyde, 0.6% methylene blue) assay as previously reported by Felice et al. \[[@B27-molecules-23-01557]\]. MDA-MB-231 and MCF-7 human breast cancer cells were respectively seeded into a 96-well plate at a density of 2.5 × 10^5^ cells/well. After 16 h for MDA-MB-231 cells or 8 h for MCF-7 cells, the growth media containing various concentrations (0--200 μM) of different treatment solutions were added to MDA-MB-231 cells. After 72 h of incubation, cell proliferation was determined by measuring the absorbance at 570 nm using a microplate reader (Bio-Rad, Hercules, MA, USA). An inhibition curve of cell proliferation was achieved for each treatment solution and the effective median dose (EC~50~) was determined.

3.5. Combination Study {#sec3dot5-molecules-23-01557}
----------------------

The samples that showed stronger anti-proliferative effects (EC~50~ value \< 200 μM) towards human breast cancer cells were selected for the combination study. According to the EC~50~ value of each treatment solution, two-way combination treatments were conducted. In detail, MCF-7 and MDA-MB-231 cells were respectively seeded into a 96-well plate at a density of 2.5 × 10^5^ cells/well. For MCF-7 cells, the growth media was changed to media containing a series of concentrations at different ratios of EC~50~ (0.125 × EC~50~, 0.25 × EC~50~, 0.50 × EC~50~, 0.75 × EC~50~, 1.00 × EC~50~, and 1.25 × EC~50~) value of two selected samples, which include genistein plus daidzein, enistein plus daidzein, genistein plus glycitein, genistein plus genistin, genistein plus daidzin, daidzein plus glycitein, daidzeinplus genistin, daidzein plus daidzin, glycitein plus genistin, glycitein plus daidzin or genistin plus daidzin, after 16 h incubation. For MDA-MB-231 cells, the growth media was changed to media containing a series of concentrations at different ratios of EC~50~ (0.125 × EC~50~, 0.25 × EC~50~, 0.50 × EC~50~, 0.75 × EC~50~, 1.00 × EC~50~, and 1.25 × EC~50~) value of genistein plus glycitein, genistein plus genistin, genistein plus β-sitosterol, glycitein plus genistin, glycitein plus β-sitosterol, β-sitosterol plus genistin, after 8 h incubation. After 72 h of incubation, cell proliferation was determined and the new EC~50~ value for each sample in combination study was calculated. Finally, a combination index (CI) was calculated for each combination treatment at inhibition of 50% according to the method of Yang et al. \[[@B10-molecules-23-01557]\]. The CI~50~ value of a combination treatment less than 1 indicates a synergistic effect.

3.6. Cytotoxicity Assay {#sec3dot6-molecules-23-01557}
-----------------------

Cell cytotoxicity of bioactive anticarcinogens by single or two-way combination treatment, at the concentration of their EC~50~ values, was assessed using the methylene blue assay \[[@B10-molecules-23-01557],[@B26-molecules-23-01557]\] with some modification. MDA-MB-231 and MCF-7 human breast cancer cells were respectively seeded into a 96-well plate at density of 5 × 10^5^ cells/well and incubated for 24 h. For MCF-7 cells, cells were then treated with genistein at 66.98 μM, daidzein at 130.14 μM, glycitein at 190.67 μM, genistin at 72.82 μM, daidzin at 179.21 μM and genistin plus daidzin at 26.21 μM plus 64.50 μM, or control for 24 h, respectively. For MDA-MB-231 cells, cells were treated with genistein at 93.75 μM, glycitein at 142.67 μM, genistin at 127.82 μM, β-sitosterol at 196.28 μM, genistein plus genistin at 33.61 μM plus 52.21 μM, genistein plus β-sitosterol at 24.08 μM plus 50.42 μM and β-sitosterol plus genistin at 37.71 μM plus 24.55 μM, or control for 24 h, respectively. The viability of cells was expressed as % of control.

3.7. Wound Healing Assay {#sec3dot7-molecules-23-01557}
------------------------

Samples which showed stronger anti-proliferative effects by single and combination treatments having a synergistic effect were taken forwards to further investigate their capacity to inhibit breast cancer cells invasion and migration.

Firstly, a wound-healing assay was conducted as reported by Kozlova et al. \[[@B28-molecules-23-01557]\]. Briefly, MCF-7 and MDA-MB-231 cells were respectively seeded into 6-well plate at 5 × 10^5^/well and grown in growth media until 90--100% confluency. After starvation in serum-free media overnight, a cell-free area (wound) was constructed using 200 μL pipette tip in each well and washed gently with ice-cold PBS for three times. Then serum-free media containing different samples were added to cells, or control. The concentration of each sample used was the same with that described in cytotoxicity assay. Healing of the wound was observed after 24 h by a light microscopy (CX-2, Olympus, Tokyo, Japan) and analyzed using Image J software (NIH, Bethesda, MD, USA).

3.8. Transwell Chamber Assay {#sec3dot8-molecules-23-01557}
----------------------------

A transwell chamber assay was conducted using an extracellular matrix (ECM) invasion assay kit (ECMatrix Cell Invasion Assay (Millipore, Billerica, MA, USA) according to the manufacturer's instruction. Briefly, MCF-7 and MDA-MB-231 cells were respectively suspended at 5 × 10^5^ cells in 300 μL of serum-free media and plated on the top of an ECM-coated membrane insert. The assay was terminated after 48 h with different treatment solutions or control. The concentration of each sample used was the same with that described in the cytotoxicity assay. Then the cells on the upper side of the insert were wiped off with a cotton swab and the cells on the lower side were stained with the solution included in the invasion assay. The number of invasive cells was determined as described earlier for the matrigel invasion assays in co-culture \[[@B29-molecules-23-01557]\].

3.9. Intracellular Signaling Array {#sec3dot9-molecules-23-01557}
----------------------------------

MCF-7 and MDA-MB-231 cells were respectively seeded into a 6-well plate at a density of 2.5 × 10^5^ cells/well and treated with the different treatment solutions, or control, as described in the anti-proliferative activity assay. The concentration of each sample used was the same with that described in the cytotoxicity assay. Whole cell lysates were collected using ice-cold lysis RIPA buffer (Fluorescent Readout, Cell Signaling Technology, Danvers, MA, USA) supplemented with 1% PMSF and stored at −20 °C until analysis. The PathScan Intracellular Signaling array kit (Fluorescent Readout, Cell Signaling Technology, Danvers, MA, USA) was used according to the manufacturer's instructions, to simultaneously detect 18 phosphorylated or cleaved signaling molecules. The images were captured and quantified using a LiCor Odyssey imaging system (Li-Cor Biosciences, Lincoln, NE, USA).

3.10. Western Blot Assay {#sec3dot10-molecules-23-01557}
------------------------

The whole cell lysates collected in the last section were used to the Western blot assay was performed according to previously described methods \[[@B30-molecules-23-01557]\]. Total cellular protein was solubilized in ice-cold lysis RIPA buffer. Anti-PI3K p85 HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. GAPDH protein was used as control. Target protein were detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

3.11. Data Analysis {#sec3dot11-molecules-23-01557}
-------------------

Data in this study were analyzed using one way ANOVA and Tukey's test performed by SPSS (Statistics for Social Science) version 17.0 (IBM, New York, NY, USA). All the assays were conducted in triplicate (n = 3). Significant differences were reported at *p* \< 0.05.

4. Conclusions {#sec4-molecules-23-01557}
==============

In conclusion, the present study provides insights into the anti-proliferative activity of 12 anticarcinogens from soybean against MCF-7 and MDA-MB-231 human breast cancer cell lines by single or two-way combination treatment. Genistein, daidzein, gycitein, genistin and daidzin were demonstrated to have stronger anti-proliferative activity against MCF-7cells with EC50 values less than 200 uM. There is a synergistic effect of the combination treatment of genistin plus daidzinin MCF-7 cells. Gensitein, glycitein, genistin and β-sitosterol showed stronger anti-proliferative activity (EC~50~ values \< 200 μM) against MDA-MB-231 cells and a synergistic effect was observed in the combination of genistein plus genistin, genistein plus β-sitosterol and β-sitosterol plus genistin. In addition, these bioactive anticarcinogens were also able to inhibit invasion and migration of breast cancer cells and combination treatment enhanced the inhibitory effect. Regulation of PI3K/Akt/mTOR pathway seems to be the main mechanisms involved in the anticancer activity. Our results may partly contribute to the understanding of anticancer effect of dietary components in soybean. However, further studies are needed in clinical trials.

This work was supported by the Hong Kong, Macao, and Taiwan Science and Technology Cooperation Program of China (2013DFH30050), the Special Fund for Agro-scientific Research in the Public Interest (201403063), the Agricultural Science and Technology Program for Innovation Team on Identification and excavation of Elite Crop Germplasm, CAAS. We thank the University of Liège-Gembloux Agro-Bio Tech and more specifically the research platform AgricultureIsLife for the funding of the scientific stay in Belgium that made this paper possible.

**Sample Availability:** Samples of the compounds are available from the authors.

Conceptualization, Y.Z. and Y.Y.; Methodology, N.E.; Software, Z.S.; Validation, Y.Z., Y.Y., Z.S. and G.R.; Formal Analysis, Y.Z.; Investigation, Y.Z.; Resources, Y.Y.; Data Curation, Y.Y.; Writing-Original Draft Preparation, Y.Z.; Writing-Review & Editing, N.E.; Visualization, N.E.; Supervision, G.R.; Project Administration, G.R. and N.E.; Funding Acquisition, G.R.

This research was funded by Technical System Special of Modern Agricultural Industry \[CARS-08-G20\] and Team Project on Nutrition and Function Innovation in Scientific and Technological Innovation Project of Chinese Academy of Agricultural Sciences.

The authors declare no conflict of interest.

![Percent inhibition of MCF-7 (**a**) and MDA-MB-231 (**b**) human cancer cells proliferation by 12 anticarcinogens from soybean. Cells were respectively treated with different concentrations (0--200 μM) of 12 samples. Data are presented as mean ± SD. \* indicates a significant difference compared to the control in cell proliferation assay at concentration of 200 μM (*p* \< 0.05).](molecules-23-01557-g001){#molecules-23-01557-f001}

###### 

EC~50~ values of bioactive anticarcinogens by single or two-way combination treatment in MCF-7 (**a**) and MDA-MB-231 (**b**) human cancer cells. Data are presented as mean ± SD. \* Indicates a significant difference compared to the EC~50~ value of single treatment (*p* \< 0.05).

![](molecules-23-01557-g002a)

![](molecules-23-01557-g002b)

![Cytotoxicity of bioactive anticarcinogens by alone or two-way combination treatment in MCF-7 (**a**) and MDA-MB-231 (**b**) human cancer cells. Data are presented as mean ± SD. \* Indicates a significant difference compared to the control (*p* \< 0.05).](molecules-23-01557-g003){#molecules-23-01557-f003}

###### 

Inhibition of cell invasion and migration measured by wound healing assay and transwell chamber assay. For wound-healing assay, wounds were made when cells were 90--100% confluent. Overnight, cells were treated with samples, or control. The closure of wounds in MCF-7 cells and MDA-MB-231 cells were imaged (**a**,**c**) and quantitatively measured (**b**,**d**) at 0 h and 24 h. For transwell chamber assay, MCF-7 (**e**) and MDA-MB-231 (**f**) Cells were treated with samples, or control for 48 h. Cells suspended in serum-free medium were seeded on the upper membrane of transwell chamber and incubated for 48 h. Complete growth medium was added on the bottom. Cells on the lower membrane of chambers were counted. Data are presented as mean ± SD. \* indicates a significant difference compared to the control (*p* \< 0.05). \# indicates a significant difference compared to treatment with samples by single (*p* \< 0.05).

![](molecules-23-01557-g004a)

![](molecules-23-01557-g004b)

![](molecules-23-01557-g004c)

![](molecules-23-01557-g004d)

###### 

Modulations of intracellular signaling pathways. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis. A slide-based antibody array was used for simultaneous detection of 18 signaling molecules (**a**) when phosphorylated or cleaved using a PathScan Intracellular Signaling Array kit. Image (**b**) and quantitative data (**c**) for MCF-7 cells as well as image (**d**) and quantitative (**e**) data for MDA-MB-23 cells were obtained using a LiCor Odyssey imaging system. Data are presented as mean ± SD. \* indicates a significant difference compared to the control (*p* \< 0.05). \# indicates a significant difference compared to treatment with samples by single (*p* \< 0.05).

![](molecules-23-01557-g005a)

![](molecules-23-01557-g005b)

![](molecules-23-01557-g005c)

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The protein expression level of PI3K p85. Cells were treated with samples, or control for 72 h. Total protein was extracted for the analysis using Western blot. The signal of target protein in MCF-7 cells (**a**,**b**) and MDA-MB-231 cells (**c**,**d**) was detected and quantified using SuperSignal ELISA Pico Chemiluminescent Substrate. Data are presented as mean ± SD. \* indicates a significant difference compared to the control (*p* \< 0.05).

![](molecules-23-01557-g006a)

![](molecules-23-01557-g006b)

molecules-23-01557-t001_Table 1

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CI~50~ values ^a^ of bioactive anticarcinogens by single or two-way combination treatment in MCF-7 and MDA-MB-231 human cancer cells.

                             Combined Treatments     CI Value at Inhibition of 50%
  -------------------------- ----------------------- -------------------------------
  MCF-7                      Genistein + Daidzein    2.02 ± 0.30
  Genistein + Glycitein      1.86 ± 0.22             
  Genistein + Genistin       1.53 ± 0.33             
  Genistein + Daidzin        1.52 ± 0.18             
  Daidzein + Glycitein       1.68 ± 0.44             
  Daidzein + Genistin        1.69 ± 0.31             
  Daidzein + Daidzin         1.89 ± 0.23             
  Glycitein + Genistin       1.92 ± 0.58             
  Glycitein + Daidzin        1.01 ± 0.10             
  Genistin + Daidzin         0.89 ± 0.12             
  MBA-MD-231                 Genistein + Glycitein   1.30 ± 0.08
  Genistein + Genistin       0.56 ± 0.13             
  Genistein + β-Sitosterol   0.54 ± 0.20             
  Glycitein + Genistin       1.05 ± 0.10             
  Glycitein + β-Sitosterol   1.29 ± 0.07             
  β-Sitosterol + Genistin    0.45 ± 0.12             

^a^ The combination index at inhibition of 50%.

[^1]: These authors contributed equally to this work.
