ELE (98% purity) was purchased from Dalian Yuanda Pharmaceuticals (Liaoning, China). The primary antibodies against E-cadherin (ab1416), ERα (ab2746), metastasis-associated protein 3 (MTA3) (ab87275), Snail (ab53519) and β-actin were from Abcam (Cambridge, UK). The secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse-IgG and anti-rabbit-IgG antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The MCF-7 human breast cancer cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and propagated in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA). MCF-7 cells were cultured at 37°C in a humidified incubator (Heraeus, Germany) with 5% CO₂ and seeded at 2.5×10⁵ cells/ml in 6-well plates (Corning, Inc., Corning, NY, USA). The cells were divided into three treatment groups (0, 50 and 100 μg/ml ELE), observed and examined after 24 h.

For E-cadherin staining, cells were plated on glass coverslips in 6-well plates and treated with ELE for 24 h. The cells were washed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde solution containing 0.1% Triton X-100 for 20 min at room temperature (RT), incubated in 5% bovine serum albumin for 30 min at RT, and then treated with the anti-E-cadherin monoclonal antibody for 16 h at 4°C. After being washed three times in PBS, cells were further incubated with CY3 conjugated goat anti-mouse IgG (Beyotime Biotechnology, Haimeng, China) for 1 h at RT. After three washes with PBS, the cells were analyzed using a fluorescence inverted microscope (IX71; Olympus, Japan). The cells were counterstained with Hoechst 33258 (Beyotime Biotechnology) to label the cell nuclei.

Cells were washed twice with ice-cold PBS and lysed in 1% Triton buffer [1% Triton X-100, 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na₃VO₄ (Sigma, St. Louis, MO, USA), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 μg/ml aprotinin] on ice. Total proteins were quantified using the Lowry method. Proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, USA). The membranes were blocked with 5% bovine serum albumin (Sigma) at RT for 1 h and incubated overnight at 4°C with the following primary antibodies: E-cadherin (1:1,000), ERα (1:1,000), MTA3 (1:2,000) and β-actin (1:500). After washing with Tris-buffered saline with Tween-20 (TBST) buffer, the membranes were probed with HRP-conjugated secondary antibodies and developed with enhanced chemiluminescence reagent (Beyotime Biotechnology). To detect Snail, nuclear proteins were extracted with a nuclear extraction kit (Beyotime Biotechnology) and probed with the anti-Snail antibody (1:2,000). The images were analyzed by NIH ImageJ software.

Total RNA was extracted by homogenization in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform re-extraction and isopropanol precipitation. RT-PCR was performed in a final volume of 20 μl containing 1.6 ml of cDNA template, 1 ml of primer (10 mM) and 10 ml of SYBR-Green Master Mix (Takara, Dalian, China). Primers used were 5′-TCCCATCAGCT GCCCAGAAA-3′ (sense) and 5′-TGACTCCTGTGTTCCTG TTA-3′ (antisense) for E-cadherin; 5′-GCACCGTCAAGG CTGAGAAC-3′ (sense) and 5′-TGGTGAAGACGCCAGT GGA-3′ (antisense) for human GAPDH. GAPDH was used as an endogenous housekeeping gene.

Cell invasion was measured using a Transwell chamber. In brief, 2×10⁵ cells were added per Transwell invasion chamber coated with 1–2 mg/ml Matrigel (reconstituted basement membrane; BD Biosciences, Mississauga, ON, Canada). MCF-7 cells were treated with ELE. Twenty-four hours later, the cells in the upper chamber were removed with a cotton swab. The remaining cells on the membrane were fixed for 10 min in methanol, stained with 1% crystal violet solution and washed with PBS. The number of invaded cells was counted for 5 fields/field of view at ×200 magnification.

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded at 5×10⁴ cells/well in 96-well plates, incubated overnight and then exposed to the indicated concentrations of ELE for the indicated times. Next, 20 μl of MTT (Sigma, St. Louis, MO, USA) solution (5 mg/ml) was added to each well, and the cells were incubated for another 4 h at 37°C. After removal of the culture medium, the cells were lysed in 200 μl of dimethyl sulfoxide (DMSO), and the optical density (OD) was measured at 570 nm with a microplate reader (Model 550; Bio-Rad Laboratories, USA). The following formula was used: Cell viability = (OD of the experimental sample/OD of the control group) × 100%.

The experiments were repeated at least three times. Data are expressed as the means ± standard deviation. Differences in the results between two groups were evaluated by the Student’s t-test. P<0.05 was considered to indicate a statistically significant result.

To examine the effect of ELE on the cell viability of MCF-7 cells, we treated the cells with 50 or 100 μg/ml ELE for 0, 12, 24, 36, 48, 60 and 72 h and determined the rate of cell survival with an MTT assay. MCF-7 cell proliferation was not inhibited at 50 or 100 μg/ml ELE for 24 h (P>0.05), whereas treatment of cells with both doses of ELE for 36 h significantly inhibited cell survival (Fig. 1) (P<0.05). Therefore, for our subsequent experiments, we treated cells with 50 and 100 μg/ml ELE for 24 h, respectively.

We used a microscope to approximately evaluate the cell motility capacity with the change in cell morphology of MCF-7 cells treated with 50 and 100 μg/ml ELE for 24 h, respectively. ELE-treated cells demonstrated a loosely aggregated cell phenotype that switched gradually to a tight adherence, indicating increased cell-cell interactions and reduced capacity for motility (Fig. 2).

To support the above-mentioned alteration in cell morphology, we next determined the E-cadherin protein levels in the MCF-7 cells by an immunofluorescence assay and found that E-cadherin protein was upregulated by ELE (Fig. 3A). Further western blot analysis showed that 50 and 100 μg/ml ELE increased E-cadherin in a concentration-dependent manner (Fig. 3B and C) (P<0.05 and <0.01, respectively). Thus, ELE upregulates E-cadherin protein levels.

To investigate whether ELE enhances the gene transcription of E-cadherin, we determined the E-cadherin mRNA levels in MCF-7 cells using an RT-PCR assay. ELE at 50 and 100 μg/ml significantly increased the mRNA levels of E-cadherin in a concentration-dependent manner (Fig. 4) (P<0.05 and 0.01, respectively), which was consistent with the upregulation of E-cadherin protein (Fig. 3). These results showed that ELE increased E-cadherin gene transcription.

To determine the underlying mechanism by which ELE increases the expression of E-cadherin, we determined the protein levels of ERα, MTA3 and Snail. ELE at 50 and 100 μg/ml increased both ERα and MTA3, whereas it decreased Snail (Fig. 5A and B) (P<0.05 and <0.01, respectively). In addition, increasing the concentration of ELE apparently enhanced these effects. Thus, ELE regulates E-cadherin expression via the ERα/MTA3/Snail signaling pathway.

An increase in E-cadherin may explain the increased cell-cell interactions and decreased capacity for motility of the MCF-7 cells following ELE treatment (Fig. 2). To further support this conclusion, we measured the cell migration and invasion of MCF-7 cells treated with ELE with a Transwell assay and found that cells successfully penetrated the basement membrane-coated chambers; however, the number of cells that penetrated the membrane was reduced significantly in the ELE-treated cells (Fig. 6) (P<0.05 and <0.01, respectively, for the 50 and 100 μg/ml treatment groups). These data demonstrated that ELE inhibits both cell migration and invasion.