The NSCLC A549 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). 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; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 0.1 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA). All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO₂. When cells entered into the logarithmic growth phase, they were washed three times with PBS and digested with 0.25% trypsin (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China). When the cells detached, DMEM was re-added to terminate digestion and resuspended the cells into single cell suspension, then the cells (100 µl, 1×10⁵) were seeded into a 6-well plate for subsequent experiments.

A 100 µl suspension of A549 cells was seeded into 96-well plate with the standard 1,000 cells/well. In this assay, 5 groups were used, which were control (DMEM only), vehicle [0.1% dimethyl sulfoxide (DMSO); Ameresco, Inc., Framingham, MA, USA)] and 0.2, 2 and 20 µM telmisartan (MedChem Express, Princeton, NJ, USA). Following this, cells were cultured in a CO₂ incubator and the cell viability was detected at 24, 48, 72 h. A total of 10 µl CCK-8 (Beijing Solarbio Science & Technology, Co., Ltd.) reagent was added into each well and the plates were incubated at 37°C for 1.5 h. The optical density value was measured at a wavelength of 450 nm using a microplate reader and a proliferation curve was plotted. The assay was performed in triplicate. In the following experiments, the negative control (NC) group was treated with 0.1% DMSO, and the treated group with 20 µM telmisartan.

A total of 100 µl Matrigel (diluted 1:6 in serum-free DMEM; BD Biosciences, Franklin Lakes, NJ, USA), was melted overnight and then added to the upper chamber of a 24-well transwell plate (EMD Millipore, Billerica, MA, USA). Following shaking, the Matrigel was placed into 37°C CO₂ incubator cultivating for 4–6 h until the gel set, and then the culture medium was dried. The cells, which had been treated with 20 µM telmisartan for 24 h, were prepared using serum-free DMEM in a cell suspension. A total of 100 µl (1×10⁵) cell suspension were added to the upper chamber of the transwell. Following this, 500 µl complete DMEM (containing 10% FBS) was mixed into the bottom chamber of the transwell. Cells were incubated for 24 h, then the transwell was taken out and the remaining cells in the upper chamber were removed with a cotton swab. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with 0.1% crystal violet for 20 min at room temperature. Following washing with PBS, five clear fields of vision were selected random using a light microscope (magnification, ×200), and images were captured to observe the cell count.

The migration experiment procedure was similar to the invasion assay; however, the Matrigel was not applied and the 5,000 cells were seeded in the upper chambers.

Apoptotic A549 cells were evaluated using an Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI) Apoptosis Detection kit (4A Biotech Co. Ltd., Beijing, China) according to the manufacturer's protocol. The NC group was treated with 0.1% DMSO. Following the treatment of A549 cells with 20 µM telmisartan and digested with 0.25% trypsin (without EDTA), collected in a centrifuge tube, centrifuged at 800 × g at 4°C for 5 min, resuspended in 4°C pre-cooled PBS and finally centrifuged once again at 800 × g at 4°C for 5 min. The supernatant was aspirated and the cells were resuspended in 1× Binding Buffer (Biomiga Inc., San Diego, CA, USA). Cell density was regulated to ~1×10⁶/ml. A total of 100 µl cell suspension was added into a 5-ml flow tube and stained with 5 µl Annexin V-FITC (Apoptosis Detection kit) for 5 min at room temperature in the dark. Next, cells were stained with 10 µl propidium iodide (PI) and incubated at room temperature in the dark for 5 min, and 400 µl PBS was added to detect cells. The cells were then assessed using a flow cytometer and results were analyzed using FlowJo software (version 7.6.3; FlowJo, LLC, Ashland, OR, USA). The assay was performed in triplicate.

When the cell confluence was ~80%, the cells were treated with 20 µM telmisartan or 0.1% DMSO. After 24 h, the 6-well plate was placed on ice. Next, Radioimmunoprecipitation Assay lysis buffer (including protease inhibitor; Beijing ComWin Biotech Co., Ltd., Beijing, China) was added to extract protein. The protein concentration was measured using the BCA Protein Assay kit (Beijing ComWin Biotech Co., Ltd.). Next, the protein was heated at 95°C for 5 min. A total of ~20 µg protein per lane was separated using 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% skimmed milk for 1 h at room temperature. Next, the membrane was incubated with the primary antibodies listed below at 4°C overnight. The membrane was then washed three times for 5 min with 0.1% Tween 20-TBS, then incubated with the appropriate secondary antibodies, listed below, for 1 h at room temperature. Following washing of the membrane (as aforementioned), bands were visualized using an enhanced chemiluminescence reagent (PerkinElmer, Inc., Waltham, MA, USA). Quantity One software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to perform densitometry analysis.

The following antibodies were used. Primary antibodies: AKT (dilution, 1:1,000; cat. no. 4691; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-AKT (dilution, 1:1,000; cat. no. 4060; Cell Signaling Technology, Inc.), mechanistic target of rapamycin (mTOR) (dilution, 1:1,000; cat. no. 2983; Cell Signaling Technology, Inc.), p-mTOR (dilution, 1:1,000; cat. no. 5536; Cell Signaling Technology, Inc.), Bcl-2 (dilution, 1:1,000; cat. no. 12789-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), Bax (dilution 1:1,000; cat. no. 23931-1-AP; ProteinTech Group), caspase-3 (dilution, 1:1,000; cat. no. 25546-1-AP; ProteinTech Group), cyclin D1 (dilution, 1:1,000; cat. no. 60186-1-Ig; ProteinTech Group), p70 S6 kinase (p70S6K) (dilution, 1:1,000; cat. no. 14485-1-AP; ProteinTech Group), Tubulin (dilution, 1:5,000; cat. no. 10759-1-AP; ProteinTech Group); Secondary antibodies: horseradish peroxidase-labeled goat anti-rabbit (dilution 1:5,000; cat. no. SA00001-2; ProteinTech Group). The relative expression of each protein was normalized to tubulin levels in each sample.

Statistical analysis of experimental data was performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. Student's t-test was used to analyze the difference between the telmisartan-treatment and NC groups. Analysis of variance with post hoc Fisher's least significant difference test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of telmisartan on NSCLC, the NSCLC A549 cell line was treated with 0.2, 2 and 20 µM telmisartan to detect cell proliferation rates. In this experiment, the cells were measured at 24, 48, 72 h. The results of the CCK-8 proliferation assay indicated that only the 20 µM telmisartan treatment group exhibited a significant reduction in the proliferation of A549 cells following 72 h (P<0.05; Fig. 1). These results demonstrated that telmisartan could effectively inhibit A549 proliferation in a dose- and time-dependent manner; therefore, 20 µM telmisartan was used in the following experiments.

A transwell assay was used to analyze the effects of telmisartan on the invasive and migratory ability of A549. After crystal violet staining the A549 cells were reduced via the telmisartan treatment in the invasion experiment. Additionally, the cells were also reduced in the migration experiment. The number of invaded cells in the telmisartan-expressing group was less than control group (8±2 vs. 46±4 cells invaded, respectively) and indicated a significant difference (P<0.05; Fig. 2A). The migratory capacity of cells was also inhibited in the telmisartan-expressing group compared with the control group (29±3 vs. 74±5 cells migrated, respectively), indicating the negative effect of telmisartan on the migration ability of A549 cells (P<0.05; Fig. 2B), compared with the control group. These results indicated that telmisartan could significantly inhibit the invasion and migration of A549.

An annexin V-FITC/PI double-staining assay was used to determine the effect of telmisartan on A549 cell apoptosis. Following the induction of serum-free apoptosis, the apoptosis rate in the telmisartan-treated group was significantly higher than that in the control group (21.85 vs. 5.81%, respectively; P<0.05). In addition, it was determined that telmisartan had a greater effect on early apoptosis (17.8%) compared with late apoptosis (4.05%) (Fig. 3A). Furthermore, western blot analysis was used to analyze the protein expression of apoptosis regulators, including the anti-apoptotic protein Bcl-2, and the pro-apoptotic proteins caspase-3 and Bax (Fig. 3B). The expression of the pro-apoptotic proteins caspase-3 and Bax increased in the telmisartan-treated group compared with the control group, whereas the expression of the anti-apoptotic protein Bcl-2 decreased (P<0.05; Fig. 3C-E). These results indicated that telmisartan could promote A549 cell apoptosis.

In tumors the PI3K/AKT pathway is extremely important. Following telmisartan treatment, mTOR, p70S6K and cyclin D1 proteins were selected as indicators for the evaluation of the activity of the PI3K/AKT signaling pathway. The results of western blot analysis indicated that the phosphorylation level of mTOR decreased significantly in telmisartan-treated A549 cells (P<0.05). Similarly, the expression levels of p70S6K and cyclin D1 were decreased following telmisartan treatment (P<0.05; Fig. 4). These results indicated that telmisartan-induced inhibition of A549 cell growth might be regulated via the PI3/AKT pathway.