The A549 and NCI-H1975 human lung cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, L-glutamine (5 mmol/l), non-essential amino acids (5 mmol/l), penicillin (100 U/ml), and streptomycin (100 U/ml) (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ atmosphere. The mitogen-activated or extracellular signal-regulated protein kinases 1 and 2 (MEK1/2) inhibitor, U0126, was obtained from Cell Signaling Technology (Beverly, MA, USA). Sorafenib was purchased from Bayer Corporation (West Haven, CT, USA) and dissolved in dimethyl sulfoxide (DMSO) with cell medium to the given concentration, with a final DMSO concentration of 0.1%.

Stock solutions of sorafenib and U0126 were prepared at appropriate concentrations in DMSO and stored at −20°C. For treatment, inhibitor solutions were diluted 1:1,000 to appropriate working concentrations (5 or 20 μmol/l cyclopamine and 3 μmol/l U0126) in serum-free medium. Control cultures received medium containing the solvent DMSO at a concentration of 0.1%.

Cell proliferation was determined using an MTT viability assay, the most commonly used assay for determining cell growth and death. The MTT survival assay has been described in detail in previous studies (9). Exponentially growing cells were recultured (5,000 cells/well) overnight in 96-well tissue culture plates. Up to 20 μl MTT (Sigma-Aldrich, St. Louis, MO, USA) was directly added to the media in each well, with a final concentration of 2 mg/ml. After 4 h incubation, the medium containing MTT was discarded, and 120 μl DMSO was added for 10 min. The absorbance was measured using an enzyme-linked immunosorbent assay reader (Bio-Tek, Inc., Winooskie, VT, USA) at 570 nm, with the absorbance at 630 nm as the background correction. The cell viability is expressed as the percentage of untreated controls. All experiments were performed at least three times.

Apoptosis analysis by a flow cytometer Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, Sparks, MD, USA) was used to measure the percentage of apoptosis induced by sorafenib. Cells were removed with trypsin and collected into centrifuge tubes together with the culture medium. A total of 5 μl Annexin V-FITC solution and 10 μl PI (1 μg/ml) were added to these cells for 30 min in the dark. Flow cytometry and Annexin V-FITC apoptosis analysis were performed as previously described (10). Apoptotic rates were calculated from 10,000 cells using ModFit LT software with FACSCalibur (both Becton Dickinson, San Jose, CA, USA).

The cells were removed with trypsin and collected in centrifuge tubes, together with the culture medium. The contents were centrifuged for 5 min at 1,800 × g. The supernatant was poured out, washed once with 1X phosphate-buffered saline (PBS), and centrifuged for a subsequent 5 min. The cells were then fixed with 5 ml pre-cooled 70% ethanol for at least 4 h. The fixed cells were centrifuged and washed with 1X PBS. Following centrifugation, the cell pellets were resuspended in 500 μl PI (10 μg/ml) containing 300 μg/ml RNase (Sigma-Aldrich). The cells were incubated on ice for 30 min, and filtered with a 53 μm nylon mesh. The cell cycle distribution was calculated from 10,000 cells using ModFit LT software (Becton Dickinson) using FACSCalibur.

Cell lysates were prepared and western blot analysis was performed as previously described (10). Equal aliquots of total cell protein (50 μg/lane) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, transferred onto polyvinylidene fluoride membranes, and blotted using the primary antibodies: β-actin (C-4), cyclin D1 (A-12), cyclin-dependent kinase (CDK)2 (M2), nuclear factor (NF)-κB (P65A), matrix metallopeptidase (MMP)-2 (2C1), MMP-9 (6-6B), BAX (P-19) and Bcl-2 (C-2; all 1:1,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by the secondary antibodies horseradish peroxidase-labeled goat anti-mouse (GAM-007) and goat anti-rabbit (SC-2004) IgG, and phospho-ERK1/2 (T202/Y204) and ERK1/2 (1:1,000 dilution; Cell Signaling Technology, Beverly, MA, USA). The protein bands were visualized using an enhanced chemiluminescence system (Union Bioscience Corporation, Hangzhou, China) with prestained markers as molecular size standards. At least three independent experiments were performed for each cell type studied.

Data are presented as the mean ± standard deviation. Experimental results of the treated and control groups were compared using two-tailed Student’s t-test. All statistical tests were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

MTT viability assays were conducted to elucidate the potential biological effects of sorafenib in different lung cancer cells. As shown in Fig. 1A, the NCI-H1975 cells treated with cyclopamine exhibited significant reduction in proliferation rates compared with the control cells at the two concentrations (5 and 20 μM), and sorafenib also markedly inhibited A549 cells at a concentration of 20 μM. However, at a concentration of 5 μM and 6 h time point, the proliferation rate was increased compared with the control cells (Fig. 1B).

To investigate the different proliferation results in sorafenib-treated lung cancer cells, cell cycle proliferation and apoptosis was examined by flow cytometry. The results showed that sorafenib decreased the proportion of A549 cells in the G1 phase of the cell cycle and increased that of cells in the S phase (Fig. 2A), and the cell cycle redistribution was not observed in NCI-H1975 cells at this time point (Fig. 2B). The apoptosis assay revealed that sorafenib decreased the proportion of apoptotic cells in A549 cells at the 6 h time point, which was not observed in NCI-H1975 cells (Fig. 2C and D). These data indicate that cell cycle distribution and apoptosis were involved in the proliferation of sorafenib-treated lung cancer cells.

The alterations in the cell cycle, invasion- and apoptosis-associated proteins in sorafenib-treated cells were evaluated and compared with the control cells using western blot analysis. As shown in Fig. 3A, the expression levels of cyclin D1, CDK2, NF-κB, MMP2, MMP9 and Bcl 2 decreased significantly in sorafenib-treated NCI H1975 cells. In A549 cells, sorafenib significantly enhanced the expression of cyclinD1 and CDK2, and suppressed BAX expression at the 6 h time point (Fig. 3B). With regard to invasion-associated proteins, there was no difference between the two cell lines (Fig. 3C and D). Sorafenib regulates the proliferation and apoptosis of A549 cells by activating the ERK1/2 signaling pathway. To examine the effects of sorafenib on the ERK1/2 signaling pathway in A549 cells, immunoblot analysis was performed with antibodies specific for phosphorylated ERK1/2. The results of this study reveal that sorafenib markedly enhanced ERK phosphorylation in A549 cells at the 5 μM concentration and 6 h time point, but had no marked effect on the expression of this protein (Fig. 4). Phosphorylation of ERK1/2 is a key initial response to cell cycle distribution and apoptosis. With regard to downstream proteins, sorafenib markedly upregulated the expression of cyclin D1, and inhibited the expression of BAX at the same concentration and time point. The specific signaling cascade involved in this response was investigated using the MEK1/2 inhibitor (U0126) specific to the MAPK/ERK pathway. The increased phospho-ERK1/2 following sorafenib-treatment was inhibited by U0126, as well as the downstream protein cyclin D1, Bcl-2 and BAX (Fig. 4), which suggests that sorafenib mediates cell cycle distribution and apoptosis by modulating the ERK signaling pathway.