Reagents and antibodies. EA was purchased from Nanjing Spring & Autumn Biological Engineering Co. Ltd., with a purity >98% and dissolved in DMSO (the concentrations used in vitro were 10−6 M and 10−5 M; and those used in vivo were 25 and 50 mg/kg via intraperitoneal injection). Rabbit cyclin D polyclonal antibody (1:1,000; cat. no. ABE52), rabbit β-actin polyclonal antibody (1:5,000; cat. no. SAB3500350), rabbit anti-PI3K (1:1000; cat. no. SAB5500162), rabbit anti-partitioning defective 3 homolog (Par3) antibodies (1:1,000; cat. no. SAB1401772), rabbit anti-Bcl-2 (1:1,000; cat. no. SAB4301440), rabbit anti-Bax (1:1,000; cat. no. SAB5700840), rabbit anti-cleaved caspase-3 (1:1,000; cat. no. AB3623), rabbit anti-mTOR (1:1,000; cat. no. T2949), rabbit anti-Akt (1:1,000; cat. no. SAB4500797) and rabbit anti-p-Akt (1:1,000; cat. no. SAB4301497) were purchased from Sigma-Aldrich; Merck KGaA. The ECL chemiluminescence system was purchased from Thermo Fisher Scientific, Inc.
Cells and cell culture. The A549 cell line was purchased from The American Type Culture Collection (cat. no. CRM-CCL-185). Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), in a carbon dioxide incubator in which conditions were maintained at 37˚C and 5% CO2. The cells were sub-cultured and seeded in 6- or 24-well plates. The cells were divided into following groups: The control; EA(L), treated with 10−6 M EA; EA(H), treated with 10−5 M EA; and the vehicle, treated with DMSO. Cells were treated with EA for 48 h. Finally, the cells were digested using 0.2% trypsin prior to mRNA or protein analysis.
Establishment of NSCLC tumor xenografts in nude mice. All animal protocols were approved by the Medical Animal Ethics Committee of Lanzhou University Second Hospital (Gan X2020J011), and were all performed according to the Federation of Laboratory Animal Science Associations (FELASA) guidelines for the definition of humane endpoints and the Arrive guidelines for animal care and protection (14). For metastatic tumor models, the specific humane endpoints were as follows: Rapid weight loss or emaciation characterized by anemia, hunched posture, an ungroomed appearance and lethargy. A total of 20 healthy male athymic BALB/c (nu/nu) nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were 6 weeks of age and weighed 18-20 g. The mice were housed and fed in a specific pathogen-free (SPF) environment with a 12-h light/dark cycle at 22-25˚C, with 50-65% humidity and were provided with sterile food and water day/night. All procedures were performed under aseptic conditions. Prior to the animal experiments, the mice were fed ad libitum for 1 week in their new environment.
Cultured cells were washed with phosphate-buffered saline (PBS), and 0.2 ml (1x106 cells in total) of cell suspension was injected into the skin of the right underarm region of 5 nude mice (per group). The groups were as follows: The control; EA(L), treated with 25 mg/kg EA; EA(H), treated with 50 mg/kg EA; and the vehicle, treated with corn oil. EA was administered to the mice via intraperitoneal injection from the first day at a dose of 25 mg/kg or 50 mg/kg body weight per day, and corn oil served as the vehicle. The tumor sizes were measured every 5 days. After 4 weeks, the mice were sacrificed and the tumors were collected and fixed with 4% paraformaldehyde. For the tumor models, the specific humane endpoints were the following: Rapid weight loss or emaciation characterized by anemia, hunched posture, ungroomed appearance and lethargy. At the end of the study, tumor volume (mm3) was calculated according to the following formula: Tumor volume = (length x width2)/2. All mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (30 mg/kg) until they lost consciousness and then sacrificed by exsanguination.
Cell Counting Kit-8 (CCK-8) assay. A CCK-8 kit (Dojindo China Co., Ltd.) was used to measure the proliferation of the A549 cells. A total of 1,000 cells in a volume of 100 µl per well were cultured in five replicate wells in a 96-well plate in medium containing 10% FBS. CCK-8 reagent (10 µl) was then added to 90 µl DMEM to generate a working solution, of which 100 µl was added per well followed by incubation at 37˚C for 1.5 h. This assay was performed at 0, 12, 24 and 36 h.
5-Ethynyl-2’-deoxyuridine (EdU) assay. The Cell-Light™ EdU staining kit (Guangzhou Ribobio Co., Ltd.) was used for the in vitro labelling of the nuclei of dividing cells. A549 cells were treated with 2 µM tetrafluorophenyl (TFP) and subsequently with 10 µM EdU. After 16 h, the cells were fixed with 4% paraformaldehyde. The cells were then examined under a laser scanning confocal microscope (Zeiss GmbH).
Cell cycle measurement. Propidium iodide (PI) staining was used for cell cycle measurements according to the instructions provide with the commercial kit (P4170; Sigma-Aldrich; Merck KGaA). Briefly, a cell suspension was prepared in PBS, centrifuged at 500 x g at room temperature for 5min, fixed with 70% alcohol at -20˚C overnight, centrifuged at 300 x g at 4˚C for 5 min and collected, and then washed with PBS. PI solution was added to yield a final concentration of 50 µg/ml. This suspension was kept in the dark for 30 min. DNA binding was measured using flow cytometry, and the data were analyzed using FlowJo software (Version 9, B&D America).
Cell invasion assay. For the cell invasion assay, a Transwell assay was used with Matrigel® as previously described (15). Briefly, A549 cells (5x105/ml) were re-suspended in serum-free DMEM containing 0.01% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) for an additional 24 h. The upper chamber of the Transwell plate was covered with serum-free DMEM supplemented with 50 mg/l Matrigel, and was then air-dried at 4˚C for 15 min. A total of 50 µl fresh serum-free DMEM containing 10 g/l BSA was subsequently added followed by incubation for 30 min at 37˚C. Following the removal of this medium, 200 µl cell suspension were added to the upper chamber of the Transwell plate (Sigma-Aldrich; Merck KGaA), and 600 µl complete DMEM culture supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) were added to the lower chamber. Following 48 h of incubation at 37˚C in 5% CO2, the Transwell plate was then washed with PBS to remove the cells on the upper side of the microporous membrane, followed by fixation in ice-cold alcohol for 30 min. The non-migratory cells were removed from the upper surface of the filter using a cotton swab. Finally, the cells were stained with 0.1% crystal violet (cat. no. C0775; Sigma-Aldrich; Merck KGaA) for 30 .min at room temperature, and then decolorized with 33% acetic acid. The absorbance of eluents was observed at an optical density of 570 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Cell migration assay. For the cell migration analysis, a wound healing assay was performed as previously described (10). Briefly, A549 cells (1x105) were seeded in 24-well plates and grown overnight to achieve confluence (80%). The monolayer of cells was scratched using a 20-µl pipette tip in order to create the wound. Floating cells were removed by washing twice with PBS. Subsequently, serum-free DMEM was added to permit wound healing. The rate of wound closure was assessed using images captured after 24 and 48 h. Image analysis was performed under a fluorescence microscope (magnification, x 200) using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.).
Hoechst 33258 staining assay. After 4 weeks, the mice were sacrificed and the tumors were collected and fixed with 4% paraformaldehyde for 10 min at room temperature. Following washing with PBS two times, the tissues were stained with 0.5 ml of Hoechst 33258 (Beyotime Institute of Biotechnology) for 5 min, and then again washed twice with PBS. The stained nuclei were observed under an Olympus 1X73 fluorescence microscope (Olympus Corporation) using 350 nm for excitation and 460 nm for emission.
Detection of apoptotic cells in tissues. A TUNEL assay kit was used to detect the apoptotic cells. Tissue sections (thickness, 5 µm) were excised and fixed with 4% paraformaldehyde in PBS at room temperature for 24 h. Fixed tissues were embedded in paraffin and stained using a TUNEL kit (cat. no. 11684795910; Roche Diagnostics), according to the manufacturer’s protocol, and 6 sections were analyzed for each mouse. The numbers of apoptotic cells and total tumor cells in each section were counted in three randomly selected fields (magnification, x400) using a fluorescence microscope (Nikon TE-2000 U; Nikon Corporation). The apoptosis index (AI) was expressed as the mean percentage of apoptotic cells within the total number of tumor cells for each animal.
DAPI staining. DAPI staining was performed as follows: Briefly, A549 cells were seeded into 6-well plates with 140,000 cells/well and treated with EA for 24 h. The cells were fixed with 4% polyoxymethylene at room temperature for 10 min. The coverslips were equilibrated in PBS, following which 300 µl DAPI (300 nM; cat.no. D9542; Sigma-Aldrich; Merck KGaA) staining solution was added to the coverslips and incubated for 5 min at room temperature. The coverslips were rinsed two times in PBS and viewed using a fluorescence microscope (Nikon TE-2000 U; Nikon Corporation). The normal cell nuclei were faint staining (cells were alive). The apoptotic cell nuclei were brightness
Western blot analysis. The A549 cells from each group were lysed on ice with a tissue or cell protein extraction reagent (cat.no. P0013; Beyotime Institute of Biotechnology) containing a cocktail of 0.1 mM dithiothreitol and proteinase inhibitor. The protein concentration was determined using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 100 µg protein fractions were separated via SDS-PAGE (12% gel), and then transferred onto nitrocellulose membranes (EMD Millipore) in Tris-glycine buffer at 100 V for 55 min. The membranes were then blocked with 5% (w/v) non-fat milk powder in Tris-buffer with 0.05% (v/v) Tween-20 (TBST) at room temperature for 2 h. Following overnight incubation at 4˚C with the appropriate primary antibodies [cyclin D, cat. no. ABE52; Par3, cat. no. SAB1401772; PI3K, cat. no. SAB5500162; AKT, cat. no. SAB4500797; mTOR, cat. no. T2949; p-mTOR, cat. no. SAB4504476; Bax, cat. no. B8429; Bcl-2, cat. no. SAB4503899; caspase-3, cat. no. ABC495; cleaved caspase-3, cat. no. AB3623; proliferating cell nuclear antigen (PCNA), cat. no. WH0005111M2; all 1:1,000; all from Sigma-Aldrich; Merck KGaA] and rabbit β-actin polyclonal antibody (1:5,000; cat. no. SAB3500350; Sigma-Aldrich; Merck KGaA), the membranes were washed three times with TBST, incubated with secondary antibodies (1:5,000; cat. no. ab205718; Abcam) for 2 h at room temperature, and then washed again three times with TBST. The western blot bands were imaged using an enhanced chemiluminescence solution (Beyotime Institute of Biotechnology). The protein expression levels were visualized with Image Lab version 2 Software (Bio-Rad Laboratories, Inc.).
Measurement of mRNA expression. Reverse transcription-quantitative PCR (RT-qPCR) was used to analyze the mRNA levels. Total RNA was extracted from the cells using TRIzol® reagent Takara Biotechnology Co., Ltd; the concentration and purity of RNA were determined by spectrophotometry (Thermo Fisher Scientific Varioskan LUX). A total of 200 ng RNA from each sample was used for the reverse transcription reaction, which was conducted according to instructions for a commercial transcription kit (DRR037A; Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used: Initial denaturation at 95˚C for 30 sec; followed by 40 cycles of denaturation at 95˚C for 5 sec, and annellation and extension at 60˚C for 31 sec. Using qPCR, the mRNA expression levels of Par3 (forward, 5′-CAATTACTATTCAAGCCGTTTTGCCCCTGACAGCCAGTAT-3′ and reverse, 5′-CAATTACTATTCAAGCCGTTTTGACCCTGACAGCCAGTAT-3′; ) and cyclinD (forward, 5’-GTCGCTGGAGCCCGTAA-3’ and reverse, 5’-GAGTTGTCGGTGTAG-3’) were quantified using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) and ABI 7300. Data analysis was performed using ABI software (Version 2.3; Thermo Fisher Scientific, Inc.) using the 2−∆∆Cq method (16).
Statistical analysis. SPSS software (version 19.0; SPSS, Inc.) was used for the statistical analysis. Data are expressed as the mean ± standard error of the mean. Differences in measured values among the multiple groups were evaluated using one-way ANOVA with Bonferroni’s multiple comparisons correction. P<0.05 was considered to indicate a statistically significant difference.