Human MCF-7 breast adenocarcinoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). For the creation of an anchorage-dependent culture, MCF-7 cells (5×10⁵) were seeded in a Falcon™ Standard Tissue Culture Dish (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere.

MCF-7 cells (5×10⁵) were seeded in DMEM supplemented with 10% FBS. Following 24 h of culture, cells were washed twice with PBS, and fresh medium was added. Various concentrations (1, 10, 100 nM and 1 µM) of dexamethasone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and/or the glucocorticoid inhibitor RU486 (1 µM; Sigma-Aldrich; Merck KGaA) were added to the cells. The numbers of viable cells were estimated at a number of time points (following 24, 48 and 72 h of culture) using trypan blue staining.

Following incubation for 72 h, cells (1×10⁵) were fixed in 70% ethanol for 1 h at 4°C, washed with PBS and treated with 100 µg/ml RNase A (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C. Cells were then stained with 25 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. For Annexin V-fluorescein isothiocyanate (FITC) staining, cells were washed with PBS, treated with diluted trypsin-EDTA solution, centrifuged at 25°C for 3 min at 150 × g, washed twice with cold PBS, and resuspended in binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂). A 100 µl aliquot of the suspension (~1×10⁵ cells) was incubated with 5 µl Annexin V-FITC and 5 µl PI for 15 min in the dark at room temperature. Binding buffer (400 µl) was added to each mixture, and the samples were analyzed by flow cytometry within 1 h. Flow cytometry was performed using the FACSCalibur™ system (BD Biosciences, San Jose, CA, USA) and data were analyzed using the BD FACStation™ software version 6.0 (BD Biosciences). Experiments were performed in triplicate.

MCF-7 cells (5×10⁵) were seeded in DMEM supplemented with 10% FBS. Following 24 h incubation, cells were washed twice with PBS, and fresh media were added. The cells were then treated with ethanol (CTL), dexamethasone (100 nM), RU486 (1 µM), or RU486 (1 µM) and dexamethasone (100 nM) for 72 h. SP analysis was performed as previously described (11). To detach cells, cultures were trypsinized for 3 min and detachment was monitored under a phase-contrast microscope. The number of viable cells was estimated using trypan blue staining. Cells were centrifuged at 25°C for 3 min at 150 × g and resuspended in 5 ml PBS. To detect SP cells, cells at a density of 1×10⁶ cells/ml were incubated with Hoechst 33342 dye (5 µg/ml) in DMEM supplemented with 10% FBS for 90 min at 37°C, with vortexing every 10 min. At the end of the incubation, cells were centrifuged at 4°C for 3 min at 150 × g and transferred to microcentrifuge tubes. PI (1 µg/ml) was added to the tube for 15 min at 37°C prior to fluorescence-activated cell sorting for the identification and exclusion of dead cells. Samples were analyzed using the BD FACSAria™ system and the FACSDiva™ software version 6.1 (BD Biosciences). In order to confirm that cells belonging to the SP were expressing the ABCG2 transporter, Hoechst 33342 staining was also performed in cells additionally treated for 90 min with the ABCG2 inhibitor verapamil (50 µM; Merck KGaA).

Cells were detached using trypsin as aforementioned, and 5 ml DMEM supplemented with 10% FBS were added to the culture for trypsin inactivation. Cells were collected by centrifugation for 3 min at 150 × g at 25°C, resuspended in 5 ml PBS and centrifuged again. Cell pellets were resuspended in 500 µl total volume of PBS containing FITC-conjugated anti-ABCG2 antibody (cat. no. 332014; 1:50; BioLegend, Inc., San Diego, CA, USA). Following incubation for 25 min at room temperature, cells were rinsed three times with PBS and flow cytometric analyses were performed in triplicate using the FACSCalibur™ system (BD Biosciences).

The statistical significance of the differences between groups was assessed using Student's t-test using Microsoft Excel software version 2016 (Microsoft Corporation, Redmond, WA, USA). Data are expressed as the mean ± standard error of the mean of at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

The present study evaluated the effects of dexamethasone on the viability of human MCF-7 breast adenocarcinoma cells using trypan blue staining. Following treatment with various concentrations of dexamethasone for 72 h, MCF-7 cell viability was reduced to 92, 81, 54 and 45% of the control levels by 1, 10, 100 nM and 1 µM dexamethasone, respectively (Fig. 1A). Following 72 h of treatment, 100 nM dexamethasone significantly reduced the numbers of viable MCF-7 cells compared with control (Fig. 1B). Therefore, a dose of 100 nM dexamethasone was used in all subsequent experiments. To confirm that the observed reduction in MCF-7 cell viability was due to dexamethasone, the glucocorticoid inhibitor RU486 was employed. The inhibitory effects of dexamethasone on MCF-7 proliferation were abolished following treatment with 1 µM RU486 (Fig. 1C).

To determine whether the observed decrease in MCF-7 cell viability was the result of cell cycle arrest or apoptosis, the DNA contents of the surviving cells were assessed using PI staining followed by flow cytometry. The fraction of MCF-7 cells in the G₀/G₁ phase demonstrated an upward trend (~8% increase) following treatment with 100 nM dexamethasone compared with untreated control cells; however, no statistical significance was detected (Fig. 2A and B). In detail, the G₀/G₁ fraction was 71.00±9.45% in untreated cells, 73.67±6.19% in cells treated with 100 nM dexamethasone, 72.00±9.33% in cells treated with 1 µM RU486 and 72.33±8.28% in cells treated with RU486 and dexamethasone. The results of the apoptosis assay demonstrated that dexamethasone did not produce any statistically significant effects on MCF-7 cell apoptosis (Fig. 3A and B).

To investigate the effects of dexamethasone treatment on the SP fraction of MCF-7 cells, cells were treated with 100 nM dexamethasone and 1 µM RU486 as aforementioned. SP cells were detected using Hoechst 33342 staining followed by flow cytometry. The size of the SP fraction was 1.6±0.1% in untreated cells, 0.6±0.1% in cells treated with 100 nM dexamethasone, 1.6±0.1% in cells treated with 1 µM RU486 and 1.1±0.2% in cells treated with RU486 and dexamethasone (Fig. 4A and B). The SP fraction was significantly decreased following dexamethasone treatment (P<0.01) compared with untreated cells, whereas co-administration of RU486 attenuated the effects of dexamethasone (P<0.05 compared with dexamethasone-treated cells). Following treatment with verapamil, the SP fraction was not detected.

To investigate whether the effects of dexamethasone on the SP of MCF-7 cells may be associated with the ABCG2 transporter, the expression of ABCG2 was assessed using flow cytometry. The present results demonstrated that ABCG2 expression was significantly downregulated following treatment with dexamethasone compared with untreated cells (P<0.01; Fig. 5). Furthermore, RU486 co-administration appeared to restore the dexamethasone-induced ABCG2 downregulation in MCF-7 cells (P<0.01 compared with dexamethasone-treated cells; Fig. 5). ABCG2-positive cells were detected as 6.8±0.06% in untreated cells, 6.7±0.17% in the presence of 1 µM RU486, 3.7±0.32% in the presence of 100 nM dexamethasone, and 5.5±0.24% in the presence of both 1 µM RU486 and 100 nM dexamethasone.