Quercetin-3-methyl ether, human insulin-like growth factor 1 (IGF-1) and DAPT were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Gibco; Thermo Fisher Scientific, Inc. Antibodies against Cyclin B1 (cat. no. 4138), Cyclin-dependent kinase 1 (CDK1; cat. no. 2546), B-cell lymphoma (Bcl)-2 (cat. no. 2870), Bcl-extra large (Bcl-xl; cat. no. 2764), PI3K (cat. no. 3358), phosphorylated PI3K (cat. no. 3821), total Akt (cat. no. 4691S), phosphorylated Akt (Ser473; cat. no. 4060S), mTOR (cat. no. 2983), phosphorylated mTOR (cat. no. 5536), phosphorylated Glycogen synthase kinase β (GSK3β; cat. no. 5558), Notch1 (cat. no. 4380), EZH2 (cat. no. 5246S), tri-methyl-histone H3 (Lys27; cat. no. 9733), E-cadherin (cat. no. 3195), Vimentin (cat. no. 5741), Matrix metalloproteinase 2 (MMP2; cat. no. 4022), SRY-box 2 (SOX2; cat. no. 3579), Nanog (cat. no. 4903) and GAPDH (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Human breast cancer cells (MCF-7, T47D and MDA-MB-231) and human breast non-tumorigenic MCF-10A epithelial cells were obtained from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MCF-7 and MDA-MB-231 cells originate from human invasive adenocarcinoma, and T47D cells originate from human ductal carcinoma. MCF-7 and T47D cells are known to express estrogen and progesterone receptors, belong to the luminal A group, and have marginal metastatic potential. MDA-MB-231 cells belong to the triple-negative basal-like group and are highly metastatic (31). The MCF-7, T47D and MDA-MB-231 cells were cultured at 37°C in a 5% CO₂ incubator in DMEM containing 10% FBS and 1% penicillin/streptomycin. The MCF-10A cells were cultured in high-glucose DMEM medium with 10% FBS and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator.

To examine the effect of quercetin-3-methyl ether on breast cancer cell growth, the cells were seeded (3×10³ cells/well) in 96-well plates with 10% FBS/DMEM and cultured at 37°C in a 5% CO₂ incubator. After 24 h, the cells were replenished with fresh medium and treated with quercetin-3-methyl ether (0–20 µM) and/or DAPT (10 µM). Following culturing for the indicated times (0–72 h), 10 µl of CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well and the wells were incubated for 1 h at 37°C in a 5% CO₂ incubator. Finally, the absorbance was measured at 450 nm.

The breast cancer cells were seeded (2×10⁵ cells/well) into six-well plates with 10% FBS/DMEM and were incubated overnight at 37°C in a 5% CO₂ incubator. The cells were then starved in serum-free medium for 24 h, treated with quercetin-3-methyl ether (0–20 µM) for 48 h, stained with propidium iodide and subjected to cell cycle analysis according to a previously described method (29,30).

The cancer cells (1×10⁶) were cultured overnight in a 10-cm dish, and starved in serum-free medium for 24 h. The starved cells were then treated with quercetin-3-methyl ether (0–20 µM) or DAPT (10 µM) for 48 h in culture medium containing 10% FBS. For the induction of phosphorlated PI3K and Akt, the cells were pretreated with quercetin-3-methyl ether for 2 h, and then exposed to 100 ng/ml IGF-1 for 20 min. The harvested cells were lysed with lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) on ice for 30 min. Following centrifugation at 12,000 × g for 15 min at 4°C, the supernatant was collected for further analysis. The total protein concentration of the cell lysates was determined with a dye-binding protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. The whole cell lysate proteins were subjected to western blot analysis according to previous protocols (29,30). Following denaturation, the proteins (50–100 µg the amount of total proteins varied depending on different targeted proteins) were separated via 8–15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer (5% non fat milk) for 1 h at room temperature and then incubated with dilutions of primary antibodies (1:1,000) in blocking buffer overnight at 4°C Subsequently, the membranes were incubated with secondary goat anti-rabbit or goat-anti-mouse antibodies conjugated with horseradish peroxidase (1:2,000) in blocking buffer for 1 h at room temperature. Protein bands were visulized using an ECL detection kit (Bio-Rad Laboratories, Inc). Quantity One 1-D Anylisis software (Bio-Rad Laboratories, Inc.) was used for scanning and densitometric analysis.

The breast cancer cells were seeded (2×10⁵ cells/well) into six-well plates with 10% FBS/DMEM and incubated overnight at 37°C in a 5% CO₂ incubator. The cells were then starved in serum-free medium for 24 h, followed by treatment with quercetin-3-methyl ether (0–20 µM) for 48 h. Annexin V/propidium iodide staining was used to analyze apoptotic cells according to the manufacturer's protocol (AD10-10; Dojindo Molecular Technologies, Inc.). The fluorescence intensity of the Annexin V/propidium iodide staining was detected by flow cytometry.

A total of 4×10⁵ cells were seeded into 12-well plates and cultured for 24 h, when an artificial wound was made with a P20 pipette tip in each well. Following washing of the cells three times with culture medium, fresh medium supplemented with querectin-3-methyl ether (0–20 µM) was added. Images were captured under an inverted microscope equipped with a camera at different time points (24–72 h). The wound gap distance was quantitatively determined with Image J software (National Institutes of Health, Bethesda, MD, USA) and closure was determined as follows: Closure (%)=migrated cell surface area/total surface area ×100.

Migration and invasion assays were performed using a Transwell chamber (8-µm pore size; Corning Incorporated, Corning, NY, USA). For the migration assays, 5×10⁴ cells in serum-free medium were placed into the upper chamber. For the invasion assays, the same number of cells in serum-free medium were placed into the upper chamber with an insert coated in Matrigel (BD Biosciences, San Jose, CA, USA). The lower compartments were filled with 10% FBS-medium, and querectin-3-methyl ether (0–20 µM) was added to the upper and lower compartments at the same concentration. Following culture for 48 h, the cells in the upper chamber were removed, and the cells in the lower chamber were stained with crystal violet and counted in four views for each well using a light microscope (TS100, Nikon Corporation, Tokyo, Japan).

Single-cell suspensions were cultured at a density of 2,000 cells per well in six-well Ultra-Low Attachment Plates (Corning Incorporated) with DMEM/F-12 medium containing 2% B27, 20 ng/ml EGF, 20 ng/ml bFGF, 10 ng/ml heparin, 0.4% bovine serum albumin (EMD Millipore) and 1% penicillin/streptomycin. After 3 days, the resulting spheroids were treated with querectin-3-methyl ether (0–20 µM) for another 7 days, following which the mammospheres were counted under a light microscope (TS100, Nikon Corporation).

The cells were seeded at a density of 500 cells per well in 60-mm dishes with 10% FBS/DMEM and were incubated overnight at 37°C in a 5% CO₂ incubator. The cells were then supplied with fresh medium and treated with vehicle control (DMSO), quercetin-3-methyl ether (10 µM) and/or DAPT (10 µM). The medium, with or without drug, was replaced every 3 days. Following 8 days of incubation, the colonies were fixed with 10% formaldehyde for 15 min and stained with 1.0% crystal violet solution for 20 min. Finally, images were captured and colonies were counted.

As appropriate, data are expressed as the means ± standard deviation, and significant differences were evaluated using Student's t-test or one-way analysis of variance, with the post hoc test following the latter being Tukey's test. GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to process the data. P<0.05 was considered to indicate a statistically significant difference.

First, the effect of querectin-3-methyl ether on the growth of MCF-10A, MCF-7 and T47D cells was examined. The results revealed that treatment with this compound (0–20 µM) for 72 h had a marked inhibitory effect on the growth of breast cancer cell lines, but had no significant effect on the growth of the non-tumorigenic epithelial cells (MCF-10A; Fig. 1A). Subsequently, whether quercetin-3-methyl ether inhibited cancer cell proliferation through the regulation of cell cycle processes was determined. Flow cytometric analysis of cell cycle distribution indicated that treatment with 10 µM quercetin-3-methyl ether for 48 h significantly increased the number of cancer cells at the G₂-M phase (P<0.05; Fig. 1B). As the G₂-M phase is regulated primarily by Cyclin B1/CDK1, the protein expression of Cyclin B1 and CDK1 in quercetin-3-methyl ether-treated breast cancer cells was subsequently determined. The results revealed a marked decrease in the protein expression of these two genes in MCF-7 and T47D cells following treatment with quercetin-3-methyl ether for 48 h (Fig. 1C). To examine whether quercetin-3-methyl ether inhibited cell growth via the induction of cell death, the rate of apoptosis was detected in the two hormone-sensitive breast cancer cell lines (MCF-7 and T47D) treated with quercetin-3-methyl ether. The findings indicated that, following treatment for 48 h, 10 µM quercetin-3-methyl ether significantly induced apoptosis in the two cell lines (P<0.05; Fig. 1D). The Bcl-2 family is crucial in the regulation of apoptosis. Therefore, the effects of quercetin-3-methyl ether on the protein expression of Bcl-2 family members was also determined. The results demonstrated that the levels of the key anti-apoptotic proteins Bcl-2 and Bcl-xl were downregulated in the two cell lines following treatment for 48 h (Fig. 1E). Collectively, these data suggested that quercetin-3-methyl ether inhibited breast cancer cell growth through inducing apoptosis and G₂-M phase cell cycle accumulation.

The effects of querectin-3-methyl ether on the migration and invasion of breast cancer cells were subsequently determined. A scratch wound-healing assay demonstrated that breast cancer cells (MCF-7 and MDA-MB-231) had high mobile capacities; whereas treatment with querectin-3-methyl ether (0-20 µM) for 24-72 h significantly decreased the motility of breast cancer cells into the wound gap in a dose- and time-dependent manner (Fig. 2A). Accordingly, a Transwell migration assay demonstrated that querectin-3-methyl ether significantly inhibited breast cancer cell migration through the Transwell insert membrane (Fig. 2B). Following treatment with 10 µM of querectin-3-methyl ether for 48 h, the rates of cell migration in the MDA-MB-231 and MCF-7 cells were reduced to 38 and 26%, respectively (P<0.05; Fig. 2B). Similarly, the Matrigel invasion assay revealed that querectin-3-methyl ether treatment of the two cell lines dose-dependently reduced the number of invasive cells that migrated through the Matrigel base membrane from the upper to the lower chamber (P<0.05; Fig. 2C). Notably, the cell invasion rates were reduced to 55% (MDA-MB-231 cells) and 43% (MCF-7 cells) following treatment with 10 µM of querectin-3-methyl ether for 48 h (P<0.001). These data suggested that querectin-3-methyl ether had marked inhibitory effects on the migratory and invasive capacities of breast cancer cells. In addition, as shown in the images in Fig. 2C, the cell number in the MCF-7 0 µM group was higher than that in the MDA-MB-231 0 µM group, however, a contrasting trend of cell number is shown in the bar graph when comparing the two cell lines. The reason for this was that cell number was counted in four views of each well using a microscope (total of four views), whereas the images shown in Fig. 2C were randomly selected from one of the four views (single view). Although the images suggest the cell number in the MCF-7 0 µM group was markedly higher than that in the MDA-MB-231 0 µM group, the image represents a single view and does not show the total number. However, the cell counting results shown in the bar graph represent the average values per well in three independent experiments. The reason for the change in the cell lines was that suppression of the migration and invasion in breast cancer cells was found. Therefore, MDA-MB-231 cells were selected to examine the effects of quercetin-3-methyl ether on cell invasion, metastasis and cancer stem cell formation due to the fact that this cell line is a TNBC line with high metastatic and malignant potential.

EMT, an important program enabling epithelial cells to acquire a mesenchymal phenotype, characterized by the decreased expression of E-cadherin and the overexpression of Vimentin and cellular proteases, including MMP-2, is closely linked with the invasion and metastasis of breast cancer (1). The present study determined the effect of querectin-3-methyl ether on the expression of EMT-related genes in breast cancer cells. The data indicated that treatment with querectin-3-methyl ether led to a reduction in the expression of mesenchymal cell biomarkers, including Vimentin and MMP-2, and concomitantly, the overexpression of epithelial cell biomarker E-cadherin (Fig. 3A). As EMT may contribute to the increase of CSC-like properties, and CSCs have been implicated in cancer invasion and metastasis, the present study examined whether querectin-3-methyl ether can suppress mammosphere formation and the expression of stemness regulatory genes, including SOX2 and Nanog, in breast cancer cells. The results demonstrated that querectin-3-methyl ether effectively reduced the number of mammospheres (P<0.05; Fig. 3B) and inhibited the protein expression of SOX2 and Nanog in a dose-dependent manner (Fig. 3C). These findings suggested that querectin-3-methyl ether inhibited breast cancer invasion and metastasis, possibly by suppressing the intrinsic EMT process and CSC formation.

EZH2, the catalytic subunit of polycomb repressive complex 2, can downregulate gene transcription through histone H3 trimethylation on lysine 27 (H3K27me3). It has been reported that EZH2 causes expansion of breast cancer stem cells through activation of the Notch and PI3K/Akt signaling pathways (32). Therefore, the present study determined changes in the levels of these proteins in the hormone-sensitive and TNBC cells in response to querectin-3-methyl ether treatment. The results indicated that querectin-3-methyl ether markedly decreased the constitutive protein levels of phosphorylated PI3K, phosphorylated AKT and phosphorylated mTOR in MCF-7 and T47D cells (Fig. 4A). Furthermore, the compound suppressed the levels of IGF-1-induced phosphorylated PI3K, Akt and GSK3β in hormone-sensitive cells (Fig. 4B). As quercetin-3-methyl ether had a significant repressive effect on the expression of Notch1 (Fig. 4C), the combinational efficacy of this agent and the Notch signaling inhibitor DAPT (a γ-secretase complex inhibitor) was examined. The combination of these compounds exhibited an additive inhibitory effect on the protein expression of Notch1, PI3K, Akt and mTOR in MCF-7 and MDA-MB-231 cells (Fig. 4D). Furthermore, combining these two compounds had more marked inhibitory effects on breast cancer cell proliferation and colony formation (Fig. 4E and F). It was also found that quercetin-3-methyl ether inhibited H3K27me3 signals in a dose-dependent manner, but had no significant effect on the expression of EZH2 (Fig. 4G). These data suggested that quercetin-3-methyl ether suppressed EMT and CSC formation in the hormone-sensitive breast cancer cells and TNBC cells, possibly via the downregulation of H3K27me3 epigenetic marks and the Notch1 and PI3K/Akt pathways.