The A549 cells were provided by the Drug Engineering Research Center of Chongqing Medical University (Chongqing, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator containing 5% CO₂ in air. The medium was changed every 2 days. A549 cells in the logarithmic growth phase treated with DEX (Tianjin Jinyao Amino Acid Co., Ltd., Tianjin, China) and SB431542 (Med Chem Express Co., Monmouth Junction, NJ, USA) were used for the following experiments. All the cells were harvested using pancreatin (Thermo Fisher Scientific, Inc.), washed with PBS and collected following centrifugation at 200 × g for 5 min at room temperature.

The MTS cell proliferation assay was used to quantify viable A549 cells. In brief, A549 cells were seeded onto 96-well microplates (5×10⁴ cells/well) in 100 µl culture medium (DMEM with 10% FBS) and cultured until the cells reached 70% confluency. Following this, cells were further cultured for 0, 12, 24 or 48 h, with medium containing DEX at a range of concentrations (0, 0.1, 1.0 and 10 mmol/l). The cell proliferation assay was performed using the MTS reagent kit (MTS; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. In brief, 10 µl MTS was added, and cells were incubated at 37°C in a humidified incubator for 1–2 h. The absorbance values for each well were detected at 490 nM using a micro plate reader (Omega Bio-Tek Inc., Norcross, GA, USA).

The DEX- or SB431542-treated A549 cells were fixed in 0.5 ml formalin (4%) at room temperature for 10 min, washed twice in PBS, and stained with 0.5 ml Hoechst 33342 at room temperature for 5 min. Cells were washed twice with PBS, and cell nuclei in five random fields were observed using a fluorescence microscope (Olympus Corporation, Tokyo, Japan; magnification, ×200). The apoptosis rate of A549 cells was measured using flow cytometry with Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection kit, which was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). In brief, all cells were washed with PBS to remove the medium. A minimum of 1×10⁵ cells were resuspended in 100 µl binding buffer containing Annexin V-FITC and PI (Annexin V-FITC/PI Apoptosis Detection kit; Beyotime Institute of Biotechnology). A FACScan flow cytometer was used to quantify Annexin V-FITC and PI binding using channels FL-1 (Annexin V-FITC) and FL-3 (PI). Quadrant analysis was performed using the Cellquest Pro software (version 5.1; BD Biosciences, Franklin Lanes, NJ, USA).

The pre-cooled cells from each treatment group were harvested for total protein extraction and treated with a lysis buffer containing 20 mmol/l Tris (PH 7.5), 150 mmol/l NaCl, 1% Triton X-100 and inhibitors of protease and phosphates on ice for 30 min. The cell lysis products were centrifuged for 10 min at 2,000 × g in a 4°C refrigerated centrifuge and the supernatants were collected. The final protein concentration was measured using a BCA protein kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and supernatants were boiled for 5 min. An aliquot of 40 µg of cellular protein was electrophoresed on an 8% gel using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was then blocked for 2 h with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature, and incubated overnight with primary antibodies against TGF-β1 (cat. no. ab92486), phosphorylated (p-)Smad2 (cat. no. ab53100), cleaved caspase-3 (cat. no. ab136812) and β-actin (cat. no. ab8226; 1:1,000; Abcam, Cambridge, MA, USA) at 4°C. Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 and incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G secondary antibody (cat. no. ab97035) at room temperature (1:1,000; Abcam). The immunoreactivity of each protein was visualized using the Millipore western blot chemiluminescence horseradish peroxidase substrate ECL Chemiluminescence reagent kit (EMD Millipore, Billerica, MA, USA). The results were analyzed using Quantity One software (version 4.4.02; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

All the experiments were performed in triplicate and data were expressed as the mean ± standard deviation. Statistical significance was analyzed using a one-way analysis of variance followed by Dunnett's post hoc test to analyze the difference between DEX groups. A student's t-test was performed to compare the differences among medicine groups (GraphPad Prism, version 5.01; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate whether DEX decreases the proliferation of A549 cells in a dose-time-dependent manner, cells were treated with DEX at concentrations of 0.1, 1.0 and 10.0 mmol for 0, 12, 24 and 48 h (Fig. 1A). A significant time- and dose-dependent decrease of proliferation in A549 cells was observed from 1.0–10.0 mmol DEX, following 24 and 48 h of culture. Hoechst 33342 staining was performed to observe the nuclei change in A549 cells. The nuclei of the DEX-treated group emitted white blue fluorescence, whereas the control group emitted blue fluorescence, which indicated a dose-dependent increase in apoptosis in the DEX-treated group (Fig. 1B). Flow cytometry was conducted to test the apoptotic rate of cells. The results demonstrated that the rate of early apoptotic death in DEX-treated groups was significantly higher compared with that of the control. In addition, DEX induced apoptosis in a time and dose-dependent manner (Fig. 1C-D).

To investigate whether DEX induced the expression of TGF-β1 and Smad2 in A549 cells, cells were treated with 0.1–10.0 mmol/l DEX for 48 h. The results demonstrated that DEX significantly increased the expression of TGF-β1 and Smad2 when compared with the control (Fig. 2A and B). Cleaved caspase-3, an indicator of apoptosis, was also measured. The results from the present study revealed that DEX significantly increased the expression of cleaved caspase-3 (Fig. 2C and D).

To explore whether TGF-β1/Smad2 signaling was involved in DEX-induced apoptosis of A549 cells, the TGF-β1 receptor was blocked with SB431542 at a concentration of 10 mmol/l, which was previously verified in a preliminary experiment (data not shown). The results of flow cytometry demonstrated that DEX increased the apoptosis rate of A549 cells, and in response to SB431542, the apoptosis of A549 cells induced by DEX was significantly inhibited (Fig. 3A and B; P<0.05). Hoechst staining revealed that SB431542 treatment also protected A549 cells from DEX induced apoptosis (Fig. 3C).

The present study demonstrated that SB431542 inhibited apoptosis of A549 cells, however the mechanism involved remains unknown. Therefore, in the present study, the TGF-β1 receptor was blocked with SB431542, and the expression of TGF-β1, Smad2 and caspase-3 were analyzed by western blot. The expression of TGF-β1, Smad2 and caspase-3 were significantly decreased in the SB431542 group when compared with the DEX group alone (Fig. 4A and B).