The human NSCLC A549 and human bronchial epithelial 16HBE cell lines were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in 5% CO₂ at 37°C. BNN was obtained from Chengdu Herbpurify Co., Ltd. DMSO was purchased from Beijing Solarbio Science & Technology Co., Ltd. The PPARγ antagonists GW9662 and T0070907 were obtained from Beyotime Institute of Biotechnology.

Both A549 and 16HBE cells were seeded on 96-well culture plates (2×10³ cells/well) and the cell culture medium was removed 24 h later. Medium containing different concentrations of BNN (0, 25, 50, 100 and 150 µmol/l) was added to the cells. Following treatment for 24 h at 37°C, 10 µl CCK-8 solution was added to each well and incubated for 1 h at 37°C with 5% CO₂. The absorbance value at 450 nm was then measured using a SpectraMax iD3 microplate reader (Molecular Devices, LLC). The IC₅₀ was calculated by comparing the cell viability with the BNN concentration.

A549 cells were seeded on 96-well culture plates (2×10³ cells/well) and treated with different concentrations of BNN (10, 20, 40 or 80 µM) or in the presence of PPARγ antagonists GW9662 (20 µM) and T0070907 (10 µM) for 24 h. Cell culture medium (100 µl) was collected for LDH determination using an LDH cytotoxicity assay kit according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Foll owing the reaction, the absorbance was read at a wavelength of 490 nm using the SpectraMax iD3 microplate reader.

An ROS assay kit (Beyotime Institute of Biotechnology) was used to measure intracellular ROS accumulation. A549 cells were seeded on 6-well culture plates (8×10³ cells/well) and treated with different concentrations of BNN (10, 20, 40 µM), with or without PPARγ antagonists GW9662 (20 µM) and T0070907 (10 µM). The cells were incubated with 10 µM DCFH-DA for 20 min at 37°C, and images were captured using a fluorescence microscope (Olympus Corporation). Cells were then collected and measured using a SpectraMax iD3 microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Cells seeded in 48-well plates (3×10³ cells/well)were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.3% Triton X-100 (Beyotime Institute of Biotechnology) at room temperature for 30 min. The cells were then blocked at 37°C for 1 h with Immunol Staining Blocking Buffer (Beyotime Institute of Biotechnology), followed by incubation with an anti-PPARγ antibody (16643-1-AP, 1:200; ProteinTech Group, Inc.) at room temperature for 1 h. Next, cells were incubated with an Alexa-488-conjugated secondary antibody (SA00013-2, 1:500; ProteinTech Group, Inc.) at 37°C for 50 min. Nuclei were stained with DAPI (Beyotime Institute of Biotechnology). PPARγ was stained green and the nuclei were stained blue. Fluorescent imaging was performed using a laser scanning confocal microscope (Olympus Corporation).

Cells were lysed with RIPA buffer containing 1% protease inhibitor and centrifuged at 14,000 × g for 20 min at 4°C. The supernatants were collected and the protein concentrations were measured by bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). The extraction of nuclear and cytoplasmic protein was performed according to the manufacturer's instructions using nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology). Protein (20 µg) was applied to 10% SDS-PAGE, and then transferred on to 0.45 µm PVDF membranes. The membranes were incubated in blocking solution (5% non-fat milk) for 1 h at room temperature and then incubated with the following primary antibodies: Bax (50599-2-Ig; 1:2,000; Proteintech Group, Inc.), Bcl-2 (12789-1-AP; 1:1,000, Proteintech Group, Inc.), PPARγ (16643-1-AP; 1:2,000; ProteinTech Group, Inc), Caspase-3 (AC030; 1:2,000; Beyotime Institute of Biotechnology), Caspase-9 (10380-1-AP; 1:2,000; ProteinTech Group, Inc), GAPDH (60004-1-Ig; 1:3,000; Proteintech Group, Inc.) and Histone-H3 (17168-1-AP; 1:2,000; ProteinTech Group, Inc) overnight at 4°C. Then the membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG (A0208; 1:2,000; Beyotime Institute of Biotechnology) for 1 h at room temperature. Western blotting bands were visualized by enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) and quantified using ImageJ software (version 1.51j8; National Institutes of Health) and standardized against GAPDH or Histone-H3 (nuclear protein quantification).

DARTS was performed as described in our previous study (10). Cells were lysed with M-PER lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with 1% phosphatase and protease inhibitors and centrifuged at 16,000 × g for 20 min at 4°C. Next, 10X TNC buffer (50 mM Tris·Cl, 50 mM NaCl, 10 mM CaCl₂) was added to the supernatant at room temperature for 10 min. The lysates were incubated with DMSO or BNN (1, 10 or 100 µM) at room temperature for 1 h, followed by incubation with 0.03 mg/ml pronase (Roche Diagnostics GmbH) at room temperature for 30 min. The proteolysis was stopped using SDS loading buffer. All samples were analyzed by western blotting.

Cells treated with BNN (10 µM) or DMSO at 37°C for 24 h were collected, and the cell suspension was distributed into 0.2 ml PCR tubes, with 200 µl cell suspension in each tube. The PCR tubes were heated at the designated temperature (42, 45, 48, 51 and 54°C) for 3 min. They were then removed and incubated at 4°C immediately following heating. Cells were then lysed using cell lysis buffer for western (Beyotime Institute of Biotechnology) and analyzed by western blotting as described in the western blotting methods above.

Statistical analysis of the data was performed using GraphPad Prism v.6.0 (GraphPad Software, Inc.). Data are presented as the mean ± standard deviation. Significance was determined by one-way ANOVA, and Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

In order to detect the toxicity of BNN, A549 cell morphology following BNN exposure was first examined. When human A549 cells were treated with BNN for 24 h, the cell number decreased while the cell size increased. When the BNN concentration was increased to 25 µM, the cell density was decreased. When the BNN concentration was increased to 50 µM, the cell density was markedly decreased, and a great number of dead cells were suspended in the culture medium (Fig. 1B). Next, a CCK-8 assay was performed to detect the cell viability using the A549 and 16HBE cell lines following treatment with different concentration of BNN (0, 25, 50, 100 or 150 µmol/l) for 24 h. As shown in Fig. 1C, the proliferation of A549 cells was significantly suppressed by BNN in a dose-dependent manner; BNN exerted less toxicity on 16HBE than on A549 cells. To further evaluate the toxicity of BNN, the LDH release from A549 cells treated with BNN was measured in order to determine whether necrosis was involved in the BNN-induced decrease in cell viability. LDH release is a typical property of cell necrosis (11). As shown in Fig. 1D, BNN dose-dependently increased the release of LDH, which reflected the levels of necrotic cell death. In addition, BNN increased the expression of apoptosis-related factors caspase (CAS)-3, CAS9 and Bax, but decreased the expression of Bcl-2 (Fig. 2A). These proteins act as pro- or anti-apoptotic regulators in cellular activities (12).

During carbohydrate and lipid metabolism, BNN exhibits pan-PPAR activity (8). We therefore speculated that BNN may exert a growth inhibition effect on human A549 cells by activating PPARγ, which may serve as a promising therapeutic target in lung cancer. As shown in Fig. 2A, BNN significantly (P<0.05) increased PPARγ protein expression. PPARγ is a ligand-activated nuclear transcription factor that belongs to the nuclear hormone receptor superfamily (13). The results of the immunofluorescence experiments showed that PPARγ protein levels in both the cytoplasm and the nucleus were markedly higher following treatment with BNN (Fig. 2B). To further confirm this result, cytoplasmic and nuclear proteins were separated from cultured A549 cells following treatment with BNN. The results of western blotting were consistent with those of immunofluorescence (Fig. 2C).

Since BNN affected PPARγ expression at the protein level, BNN may directly bind to PPARγ and act as a PPARγ agonist to exert antitumor effects. To identify whether PPARγ directly binds to BNN, DARTS and CETSA were employed to validate the affinity between PPARγ and BNN. DARTS is a label-free strategy to identify the small molecule targets that are stabilized by binding to small molecules and can therefore be protected from proteolysis (14,15). Following incubation with 0.03 mg/ml pronase for 30 min at room temperature, significant (P<0.01) protection from the proteolysis of PPARγ was observed in the A549 whole cell lysate in the presence of 10 and 100 µM BNN (Fig. 3A). The physical interaction of BNN with PPARγ was further investigated by CETSA, which is based on the physical phenomenon of small molecule-induced thermal stabilization of target proteins (16). The A549 cells were incubated with 10 µM BNN for 24 h and the collected cells were heated. Compared with the DMSO-treated cell lysate, BNN markedly changed the thermal stability of PPARγ at 42, 45, 48, 51 and 54°C (Fig. 3B).

To clarify whether the effect of BNN on A549 was dependent on PPARγ, cells were co-treated with BNN and PPARγ antagonists. The BNN-induced alterations in the expression of the apoptosis-related factors Bcl-2 and Bax were inhibited by the PPARγ antagonists T0070907 and GW9662 (Fig. 4A). The upregulation of PPARγ and the BNN-induced translocation of PPARγ to the nucleus were also restrained by T0070907 and GW9662 (Fig. 4A and B). Additionally, the present study investigated the impact of T0070907 and GW9662 alone on A549, which had no significant effect on A549 cell viability. In order to measure the effect of PPARγ antagonists on cell toxicity, CCK-8 and LDH release assays were performed. The cell viability of cells co-treated with PPARγ antagonists and BNN was higher than that observed in the BNN only group, while LDH release was decresed, when compared with the BNN only group (Fig. 4C and D).

ROS exerts a complex role in tumor survival and proliferation; excessive ROS leads to DNA damage, which results in cell necrosis and apoptosis (17). The present study therefore investigated whether ROS participated in the BNN-induced anti-cancer effect. ROS was detected using a fluorescenct microscope, and the fluorescence intensity was then measured using a microplate reader. BNN promoted ROS generation in a dose-dependent manner (Fig. 5A and B). In order to clarify whether the generation of ROS was dependent on PPARγ, PPARγ antagonists were applied for co-treatments with BNN. The upregulation of ROS induced by BNN could be inhibited by PPARγ antagonists (Fig. 5C and D). These results indicated that the generation of ROS in A549 cells was dependent on the expression of PPARγ.