The human breast cancer cell line MCF-7 was obtained from the Shanghai Cell Bank (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) at 37°C in a humid environment with 5% CO₂.

Human breast cancer cells MCF-7 were inoculated in a 96-well plate with 100 µl culture medium/well at density of 5×10³ cells. The cells were treated with 0, 0.25, 0.50, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0 and 4.0 µM TAM (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h at 37°C. Alternatively, cells were pretreated with 5 mM N-acetylcysteine (NAC; Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, and then treated with 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0 and 10.0 µM TAM for 24 h at 37°C. Cell proliferation was measured using a Cell Counting kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Haimen, China). A total of 10 µl CCK-8 solution was added to each well and the cells were incubated for 2 h at 37°C. The optical density (OD) was measured at 450 nm with a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cell survival fraction was calculated as the Survival fraction=OD_Experimental/OD_Control. Three independent experiments were performed in quadruplicate.

MCF-7 cells were plated in 6-well plates at a cell density of 3×10⁵ cells and incubated until the anchoring rate was between 70 and 80%. The cells were treated with 0, 1, 2 and 4 µM TAM for 48 h at 37°C. Cells from each experimental group were collected and fixed with 70% ice-cold ethanol at 4°C overnight. Cells were centrifuged at 150 × g for 5 min at room temperature and resuspended with 500 µl propidium iodide (PI; 100 µg/ml) for 30 min in the dark at room temperature, prior to analysis. The cell cycle profiles were assayed using the FACScan ESP flow cytometer (BD Biosciences, San Jose, CA, USA) at 488 nm, and data were analyzed using MultiCycle AV software for Windows 32-bit (Phoenix Flow Systems, San Diego, CA, USA). For analysis of apoptosis, cells from each experimental group were collected and processed as described in the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit I manual (BD Biosciences) and analyzed by FACScan flow cytometry using FlowJo.7.6.2 software (BD Biosciences).

Cells were plated in 6-well plates and allowed to form a confluent monolayer for 24 h. The monolayer was scratched with the tip of a 200 µl pipette and then washed twice with PBS to remove the floating and detached cells. Fresh serum-free medium, which contained 0, 1 and 2 µM TAM, was added and images of the wound area were captured at 0, 24 and 48 h to assess cell migration using an Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan).

Cell invasion was assessed using 24-well Matrigel invasion chambers (Corning Incorporated, NY, USA). The inserts were pre-coated with 40 µl Matrigel (dilution, 1:4; BD Biosciences). Cells were suspended in serum-free DMEM, which contained 0 or 1 µM TAM. Subsequently, 1×10⁴ cells were added to the upper chambers. The lower chambers were filled with medium containing 10% FBS. Following incubation for 24 h at 37°C, the chambers were fixed with 3.7% paraformaldehyde for 10 min, and stained with 2% crystal violet for 30 min at room temperature. The penetration of cells through the membrane was quantified by counting the number of cells that penetrated the membrane in 10 microscopic fields/filter (×100).

MCF-7 cells were plated in 6-well plate and incubated until the anchoring rate reached 70 to 80%. Cells (1×10⁶) were treated with 0, 1, 2 and 4 µM TAM for 24 h and trypsinized to obtain single-cell suspensions. Subsequently, cells were incubated in 0.5 ml 1X JC-1 (Thermo Fisher Scientific, Inc.) for 15 min at 37°C and analyzed using a FACSCalibur flow cytometer with FlowJo.7.6.2 software.

MCF-7 cells were plated in 6-well plates at a cell density of 3×10⁵ cells and allowed to attach overnight. Cells were treated with 0, 1, 2 and 4 µM TAM for 24 h. Cells were harvested, resuspended in serum-free DMEM containing 10 µmol/l chloromethyl-2′,7′-dichlorofluorescein diacetate (Beyotime Institute of Biotechnology) and incubated at 37°C in the dark for 30 min. The cells were subsequently washed three times with PBS and the intensity of fluorescence was assayed using the FC500 flow cytometer at 488 nm.

MCF-7 cells were plated in 6-well plates at a cell density of 1×10⁵ and allowed to attach overnight. Cells were treated with 0, 1, 2 and 4 µM TAM for 24 h and lysed using 200 µl lysis buffer (Beyotime Institute of Biotechnology). A total of 100 µl ATP Assay kit (Beyotime Institute of Biotechnology) was added into each well of a 96-well plate and incubated at room temperature for 3 to 5 min to remove the background ATP. Subsequently, 100 µl of sample was added to each well and ATP was analyzed on a flow cytometer following mixing.

Cells were washed with PBS and lysed in cell lysis buffer containing 1X phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology, China) for 30 min. Protein concentration was determined by BCA Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (50 µg) from each group were separated by 10% SDS-PAGE, transferred onto nitrocellulose membranes and blocked with 5% skimmed milk for 1 h at room temperature. Following blocking, the membranes were incubated with antibodies against β-actin (1:1,000, #3700), apoptosis regulator Bcl-2 (1:1,000, #15071), apoptosis regulator BAX (1:1,000, #5023) or caspase-3 (1:1,000, #9662) (all from Cell Signaling Technology, Inc., Danvers, MA, USA) over night at 4°C. Blots were subsequently incubated with horseradish peroxidase-conjugated anti-mouse (1:2,000, #7076) or anti-rabbit IgG antibodies (1:2,000, #7074) (both from Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized with enhanced chemiluminescence solution (Beyotime Institute of Biotechnology).

The data are presented as the mean ± standard deviation. SPSS software (version 19; IBM SPSS, Armonk, NY, USA) was used to perform the statistical analysis. The data were analyzed using a paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated three times.

Breast cancer cells were treated with varying concentrations of TAM to investigate the effect of TAM on cell proliferation. As presented in Fig. 1, the proliferation of these cells was markedly suppressed by TAM in a dose-dependent manner.

The effects of TAM on the MCF-7 cell cycle were further investigated. Following treatment with TAM for 24 h, the number of MCF-7 cells in the G0/G1 and G2/M phases increased when compared with the untreated cells. By contrast, in the S phase the number of cells decreased. In addition, as the concentration of TAM increased, the SubG1 Peak also significantly increased (P<0.05; Fig. 2A).

The effects of TAM on cell apoptosis were determined using an Annexin V-FITC and PI staining assay. As presented in Fig. 2B, TAM induced MCF-7 cell apoptosis and the percentage of apoptotic cells significantly increased in a dose-dependent manner (P<0.05).

Cancer metastasis is a complex process that involves cell migration and invasiveness. To examine the effect of TAM on the migration of breast cancer cells, MCF-7 cells were subjected to in vitro wound healing assays. Confluent cell cultures were scraped to create a wound and were subsequently treated with three concentrations of TAM. Cell motility was determined at different time points. As presented in Fig. 3A, TAM reduced the rate of wound healing in MCF-7 cell. The effect of inhibition was enhanced with increased concentrations of TAM.

Matrigel invasion assays were performed to explore the effect of TAM on the invasiveness of breast cancer cells. Following treatment for 24 h, the migratory cells at the bottom surface of the membrane were stained with crystal violet and cell penetration through the membrane was calculated manually. As presented in Fig. 3B, TAM treatment significantly decreased the ability of MCF-7 cells to invade through the Matrigel when compared with the control cells (P<0.01).

A decrease in MMP is a sign of early cell apoptosis. Once the MMP has decreased, apoptosis is irreversible (12). The fluorescent dye JC-1 is widely-used for detecting the functionality of mitochondria. Following treatment with TAM for 24 h, the fluorescence intensity significantly decreased in MCF-7 (P<0.05; Fig. 4A), which indicates that TAM may mediate MMP dysfunction. In addition, the expression levels of anti-apoptotic factor Bcl-2 and pro-apoptotic factors Bax and caspase-3, which are involved in the mitochondrial apoptosis pathway, were detected. As presented in Fig. 4B, TAM reduced the expression of Bcl-2 and caspase-3, and increased the expression of Bax. These results indicate that the promotion of cell apoptosis by TAM may be mediated by the mitochondrial apoptosis pathway.

To investigate the variation of energy metabolism in cells following TAM-induced suppression of proliferation, the level of ATP in MCF-7 cells was measured. As presented in Fig. 4C, following treatment with 1, 2 and 4 µM TAM, the production of ATP significantly decreased in a dose-dependent manner (P<0.05). These results indicate that TAM may have reduced the production of ATP and thus, energy production was not able to be compensated due to mitochondrial dysfunction. This may be one of the important mechanisms underlying TAM-induced ER-positive breast cancer cell death.

Accumulation of intracellular ROS may activate a number of pathways that are important for the induction of apoptosis and inhibition of invasion (13,14). Therefore, the present study examined the involvement of ROS in TAM-induced ER-positive breast cancer cell death. Following treatment with 1, 2 and 4 µM TAM for 24 h, the levels of ROS in MCF-7 cells were 1.3, 2.4 and 3.1 times higher when compared with the control group, respectively (P<0.05; Fig. 5A). To further elucidate the role of ROS in TAM-induced cell death, NAC (a type of ROS inhibitor) was used in conjunction with TAM to treat MCF-7 cells, then cell proliferation was determined using a CCK-8 assay. As presented in Fig. 5B, NAC prevented TAM-induced MCF-7 cell death. These results indicate that TAM promotes the formation of ROS in ER-positive breast cancer cells, thereby inducing cell death.