The three-dimensional (3D) structure of the Kruppel-like factor 5 (KLF5) protein (PDB ID: 2EBT) was obtained using the PDB database (https://www.rcsb.org/). Thereafter, the KLF5 protein structure file was edited using the PyMOL software (version 2.2.0; Delano Scientific LLC) to remove the excess water molecules and delete small ligands so that only the protein structure remained. Since the downloaded protein structure contained ligands, the original ligands such as water molecules and other bound ligands were deleted from the structure and the original ligand positions were set as docking sites. The PubChem database (http://pubchem.ncbi.nlm.nih.gov) was used to obtain the 3D structure of Britannin, which was then imported into OpenBabel software (version 2.2.1; Open Babel development team) for hydrogenation and conversion into the ‘mol2’ file format (6). AutoDock software (version 4.2; Scripps Research Institute) was utilized to display the specific docking energy value. The position with the lowest binding energy (-6.3 kcal/mol) was selected for visualization. Finally, the results were analyzed using the Protein-Ligand Interaction Profiler (version 2.3.0; https://plip-tool.biotec.tu-dresden.de/plip-web).

Lung carcinoma cell A549 cells (cat. no. B211337) and bronchial epithelial 16HBE cells (cat. no. MZ-1420) purchased from Ningbo Mingzhou Biotechnology Co., Ltd. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂. Different concentrations of Britannin (5, 10 and 20 µM; purity, 99.90%; cat. no. HY-N3005; MedChemExpress) dissolved in DMSO were used to treat A549 or 16HBE cells for 24 h at 37˚C and untreated cells were used as negative controls.

A549 cells were seeded into 96-well plates (8x10⁴ cells/well) for 24 h at 37˚C. Different concentrations of Britannin (5, 10 and 20 µM) were then added to the cells and incubated for 24 h at 37˚C. Subsequently, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added for 3 h of incubation after the removal of the original culture medium. The optical density of the samples were measured at 450 nm using a plate reader (Infinite^® 200 PRO; Tecan Group, Ltd.).

Cell proliferation capacity was determined by 5-ethynyl-2'-deoxyuridine (EdU) staining (Beyotime Institute of Biotechnology). A549 cells were seeded into 12-well chambers, treated with the aforementioned concentrations of Britannin and incubated for 24 h at 37˚C. The cells were then incubated with 200 µl EdU (500 µM) at 37˚C for 2 h. Next, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.3% Triton X-100 for 10 min. Cells were then labeled with 2 µg/ml Hoechst 33342 (Beyotime Institute of Biotechnology) for 30 min at room temperature. EdU-positive cells were imaged using a fluorescence microscope (Leica Microsystems GmbH).

A549 cells were seeded into 6-well plates (5x10³ cells/well) before the aforementioned concentrations of Britannin were added to the cells. The culture medium was replaced every 3 days. After 14 days at 37˚C, the cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min and then stained with 0.05% crystal violet at room temperature for 20 min to visualize cell colonies under a BX53 light microscope (Olympus Corporation). Colonies (≥50 cells) were counted with Image J software v.1.52 (National Institutes of Health).

A549 cells were seeded into 6-well plates (5x10⁵ cells/well) before the aforementioned concentrations of Britannin were added to the cells. Subsequently, the samples were scratched using a 200-µl pipette tip. Following removal of the floating cells using PBS, the cells were cultured in serum-free DMEM containing the aforementioned treatments. Cells were imaged at 0 and 24 h under a light microscope. ImageJ software v.1.52 (National Institutes of Health) was used to measure the scratch areas. The recovery rate of the wound was calculated using the following equation: [(Width at 0 h-width at 24 h)/width at 0 h] x100.

A549 cells at a density of 3x10⁴/well in serum-free medium were added to the upper Transwell chamber (8-µm pore diameter; Merck KGaA) whilst the bottom chamber was filled with complete DMEM containing 7% FBS. The cells were the incubated for 24 h at 37˚C before cells on the upper side of the filter membrane were removed using a cotton swab. Cells that passed through the membrane were fixed with 70% methanol for 15 min at room temperature, stained with 0.1% crystal violet at room temperature for 30 min and imaged using a light microscope (Olympus Corporation). Cell migration rates were determined using ImageJ software (version 1.48; National Institutes of Health).

A549 cells (1x10⁵ cells/well) were seeded into 6-well plates. After the cells were treated with the aforementioned concentrations of Britannin, the proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology). The supernatant was used to determine protein concentration by bicinchoninic acid protein concentration determination kit (cat. no. RTP7102; Real-Times (Beijing) Biotechnology Co., Ltd.). Proteins (6 µg/lane) were subjected to SDS-PAGE on 10% gels and then transferred onto a PVDF membrane (0.45 µm; MilliporeSigma). Membranes were blocked with 5% BSA (Beyotime Institute of Biotechnology) for 2 h at room temperature and incubated with primary antibodies overnight at 4˚C, followed by incubation with secondary antibodies for 1 h at room temperature, specifically Goat Anti-Rabbit IgG H&L (HRP; 1:2,000; cat. no. ab6721; Abcam). Primary antibodies used were as follows: E-cadherin (1:1,000; cat. no. ab227639; Abcam); N-cadherin (1:1,000; cat. no. ab76011; Abcam); Snail (1:1,000; cat. no. ab216347; Abcam); KLF5 (1:1,000; cat. no. ab137676; Abcam); and GAPDH (1:1,000; cat. no. ab181602; Abcam). Bands were visualized using ECL Western Blotting Detection Reagent (cat. no. RPN2106; GE Healthcare) and ImageJ software (version 1.8.0; National Institutes of Health) was used for the semi-quantification of protein expression levels.

Total RNA was extracted from Britannin-treated 16HBE cells or A549 cells subjected to Britannin treatment and transfection of Oe-KLF5 or Oe-NC using the RNeasy Kit^® and DNase Set (DNAse H; Qiagen China Co., Ltd.) assay kits according to the manufacturer's protocols. RNA concentration was measured using a NanoDrop photometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). A total of 1 µg RNA was reverse-transcribed to cDNA using the iScript™ RT kit (Bio-Rad Laboratories, Inc.). The temperature protocol was 15 min at 37˚C, 5 sec at 85˚C and 30 min at 4˚C. Amplification of cDNA was the performed using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with the iQ SYBR^® Green Supermix (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. The thermal cycling conditions used were as follows: Initial denaturation at 95˚C for 5 min; followed by 40 cycles of 95˚C for 10 sec, 57-60.5˚C for 10 sec and 72˚C for 30 sec. GAPDH served as the internal control gene for normalization and the expression levels were quantified using the 2^-ΔΔCq method (22). The PCR primers used were as follows: KLF5 forward (F), 5'-AGCTACAATACGCTTGGCCT-3' and reverse (R). 5'-ATGTGTGTTACGCACGGTCT-3' and GAPDH F, 5'-AATGGGCAGCCGTTAGGAAA-3' and R, 5'-GCGCCCAATACGACCAAATC-3'.

A Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent Technologies, Inc.) was used to detect the cellular ECAR and OCR. In brief, A549 cells (2x10⁴ cells/well) subjected to Britannin treatment and transfection of Oe-KLF5 or Oe-NC were seeded into 96-well cell culture XF microplates and incubated overnight at 37˚C for further testing according to the manufacturer's instructions.

A549 cells subjected to Britannin treatment and transfection of Oe-KLF5 or Oe-NC were seeded into a 24-well plate (2x10⁵ cells/ml). Aliquots of media from each well were assessed for the concentration of lactate present using a lactate test kit (cat. no. A019-2; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. For glucose uptake assay, A549 cells were cultured under normoxic conditions in glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) for 16 h at 37˚C and were then incubated with 25 mmol/l high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) for 24 h at 37˚C. Then, the intracellular glucose levels were measured using a fluorescence-based glucose assay kit (cat. no. ab136956; BioVision, Inc.) according to the manufacturer's instructions. Values were normalized accordingly to calculate lactic acid production and glucose consumption.

Cells were transfected with pcDNA3.1 containing KLF5 (Oe-KLF5; GeneChem, Inc.) and negative control plasmid (Oe-NC; GeneChem, Inc.) using the Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at final concentration of 50 nM according to the manufacturer's instructions. After 48 h at 37˚C, RT-qPCR and western blotting were used to evaluate the transfection efficiency. Further experiments were performed after 48 h.

All data were expressed as mean ± standard deviation. One-way analysis of variance with Tukey's post hoc test was performed for multiple comparisons using GraphPad Prism software (version 5; Dotmatics). A two-tailed unpaired Student's t-test was applied for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference. Each experiment was conducted at least three times.

The chemical formula for Britannin was shown in Fig. 1A. Different concentrations of Britannin were used to treat 16HBE and A549 cells for 24 h, then a CCK-8 assay was used to detect whether Britannin had an effect on cell viability. The results demonstrated that Britannin was not toxic to 16HBE cells, but the viability of A549 cells was significantly decreased with the increase of Britannin concentrations compared with that in the control group (Fig. 1B). Considering that Britannin exerted no effect on the viability of 16HBE cells, A549 cells whose viability was greatly diminished by Britannin were selected for the ensuing experiments to further work out the role of Britannin in lung cancer. A549 cells were then treated with different concentrations of Britannin for 24, 48 and 72 h before cell proliferation was analyzed using CCK-8 assay. The results demonstrated that with the increase of Britannin treatment time, the inhibitory ability of Britannin on the viability of A549 cells was significantly enhanced compared with that in the untreated cells (Fig. 1C). EdU staining and colony formation assay were next performed to measure the extent of cell proliferation, where it was demonstrated that the proliferation of A549 cells treated with Britannin was markedly decreased in a dose-dependent manner compared with that in the control group (Fig. 1D and E).

The migratory ability of A549 cells was subsequently detected by wound healing and Transwell assays. It was demonstrated that the migratory ability of cells was significantly decreased with increasing concentrations of Britannin treatment (Fig. 2A and B). The expression of epithelial-mesenchymal transition (EMT)-related proteins E-cadherin, N-cadherin and Snail was then detected by western blotting. Compared with those in the control group, the protein expression levels of E-cadherin were increased significantly in Britannin-treated cells, whilst those of N-cadherin and Snail were decreased significantly (Fig. 2C).

The XF96 extracellular flux analyzer was used to detect ECAR and OCR in Britannin-treated cells. These results demonstrated that compared with those in the control group, ECAR and OCR levels in the Britannin-treated groups were decreased in a dose-dependent manner (Fig. 3A and B). The lactic acid production and glucose consumption levels in the cell supernatant were next measured using assay kits, where the results demonstrated that with the increase in the treatment dose of Britannin, the lactic acid and glucose consumption levels were decreased in a dose-dependent manner (Fig. 3C and D). Compared with those in the untreated cells, lactic acid production was significantly decreased by all doses of Britannin treatment whereas the glucose consumption level was significantly decreased by 10 and 20 µM of Britannin.

KLF5 has been increasingly identified as an oncogene in lung cancer. A molecular docking strategy was used to study the interaction between ligand and receptor and to predict the binding mode and affinity between Britannin and KLF5. It was demonstrated that Britannin could target KLF5 (Fig. 4A). In addition, after Britannin treatment, the expression of the KLF5 protein in A549 cells was decreased significantly in a dose-dependent manner, compared with that in untreated cells (Fig. 4B).

The regulatory mechanism of Britannin in A549 cells was then explored. A KLF5 overexpression plasmid was constructed and the successful transfection of Oe-KLF5 was verified by RT-qPCR and western blotting (Fig. 5A). The cells were then divided into the following treatment groups: i) Control; ii) Britannin; iii) Britannin + Oe-NC; and iv) Britannin + Oe-KLF5. CCK-8, EdU staining and colony formation assays were used to analyze the degree of cell proliferation and the results demonstrated that compared with that in the Britannin + Oe-NC group, cell proliferation was found to be markedly increased in the Britannin + Oe-KLF5 group (Fig. 5B-D).

Wound healing and Transwell assay results demonstrated that the overexpression of KLF5 significantly reversed the inhibition of A549 cell migration caused by Britannin treatment (Fig. 6A and B). Western blotting results demonstrated that compared with those in the Britannin + Oe-NC group, the protein expression levels of E-cadherin, N-cadherin and Snail were significantly increased in the Britannin + Oe-KLF5 group (Fig. 6C).

The results of ECAR and OCR indices detected using the XF96 extracellular flux analyzer demonstrated that overexpression of KLF5 markedly reversed the inhibitory effect of Britannin on ECAR and OCR (Fig. 7A and B). In addition, lactate production and glucose consumption levels were significantly increased in the Britannin + Oe-KLF5 group compared with those in the Britannin + Oe-NC group (Fig. 7C and D). These results suggest that overexpression of KLF5 reversed the inhibitory effects of Britannin on the proliferation, migration and glycolysis of lung cancer cells.