2.1 Materials and reagents
The human NSCLC A549 and PC9 cell lines were obtained from the Hunan Fenghui Biotechnology Co., Ltd (Hunan China), RPMI 1640 and fetal bovine serum were from Thermo Fisher Scientific and penicillin/streptomycin was from Solarbio (Beijing, China).GA with purity above 98% was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). IH was kindly provided by Zhejiang Beta Pharma Co., Ltd. (Zhejiang, China). Fetal bovine serum (FBS) and RPMI-1640 medium were purchased from Gibco-BRL (Grand Island, NY, USA). The cell apoptosis detection kit with Annexin V-fluorescein Isothiocyanate (FITC)/Propidiumiodide (PI), was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Primary antibodies against Yap, p-Yap, Cappase-3, and β-actin were purchased from SantaCruz Biotechnology, Inc. (Santa Cruz, CA, USA).
6 cases of primary lung cancer and 6 cases of paracancers from June 2019 to September 2019 were collected from the Department of Pathology, Chinese PLA General Hospital. There were 4 males and 2 females, who aged from 42 to 68 years old, with an average age of (59.24 ±5.76) years. Histological type was adenocarcinoma; All selected patients underwent radical resection without chemotherapy or radiotherapy before operation. The study was approved by the hospital ethics committee.
2.3 Cell Culture
Human NSCLC A549 and PC9 cell lines were cultured in RPMI-1640 medium containing 10% FBS at 37°Cin a humidified 5% CO2 incubator. The medium was changed 2~3 times every week and the cells were used in their logarithmic growth phase.
2.4 Human lung tissue immunohistochemistry
Tissue specimens were fixed in 10% neutral formalin solution for 48h, embedded in conventional paraffin. 3 raffin in convent were baked in 60°C oven for 30min, followed by dewaxing, antigen retrieval, antigen exposure, sealing and dripping peroxide. The operation steps were carried out according to the operating instructions of the Roche automatic immunohistochemistry instrument. The primary antibody dripped manually, and finally, it was counterstained with artificial hematoxylin, blue back, and sealed with a neutral gum sheet. The primary antibody of immunohistochemistry was rabbit anti-human caspase-3, YAP, p-YAP polyclonal antibody (1:200, Santa Cruz, USA). Immunohistochemical staining scoring was judged by the intensity of nuclear staining or cytoplasm staining in tumor tissues and paracancers. The widely accepted German semi-quantitative scoring system was used to score staining intensity and extent in different areas. Each specimen was assigned a score according to the intensity of the nuclei, cytoplasmic, and membrane staining (no staining¼0; weak staining¼1, moderatestaining¼2, and strong staining¼3) and the extent of stained cells (0–5%¼0,5–25%¼1, 26–50%¼2, 51–75%¼3 and 76–100%¼4). The final immunoreactivity score was determined by multiplying the intensity score by the score for the extent of stained cells, generating a score that ranged from 0 (the minimumscore) to 12 (the maximum score).
2.5 Cell viability assay
According to the manufacturer’s instructions, cell viability was evaluated using MTT (Solarbio, Beijing,China). Briefly, cells were seeded into 96-well plates at 8×103cells per well and cultured for 48 hours (control group, GA group, IH Group, GA+IH group). Ten microlitres of MTT (concentration: 5mg/ml) solution was added into the medium in each well. After a 4 hr incubation, the supernatants were removed from each well, and 100μl DMSO was added to each well. The plate was incubated with shaking at room temperature for 15 minutes and red by a microplate reader (Bio-Tek Company,Winooski, VT) at a wavelength of 570nm. Each timepoint was repeated in three wells, and the experiment was independently performed three times.
2.6 Drug combination research
To evaluate the combined index (CI) of the effect of GA with IH on the growth of A549 and PC9 cells, the inhibitory effect of varying concentration of IH combined with varying concentrations of GA on the growth of A549 and PC9 cells was analyzed. CalcuSyn software (version2.0) was used to calculate the CI of GA combined with IH. CI > 1.1: antagonism; 0.9＜ CI ＜ 1.1: anadditive effect; 0.7< CI < 0.9: low synergy; 0.3＜ CI ＜ 0.7: synergy; 0.1＜ CI ＜ 0.3: strong synergy.
2.7 Cell apoptosis assay
Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China). Briefly, cells were seeded into 24-wellplates at 5×105cells per well and cultured for 48 hr (control group, GA group, IH Group, GA+IH group). Then the cells were detached by trypsinization, washed twice in PBS (2000 r.p.m., 5 min), and resuspended in 500µl binding buffer. A volume of 5μl Annexin V-FITC and 10µl propidium iodide was added and mixed gently, and the cells were stained in the dark for 10 min at room temperature. The cells were analyzed immediately by flow cytometry (BD FACSCalibur, BD. Bioscience, San Diego, CA) and analyzed using FLOWJO software (FlowJo, Ashland, OR). The experiment was repeated three times.
2.8 Transwell migration assay
The ability of cell migration was measured by Transwell assay. The cells density was adjusted to 5×105 cells/ml. In addition, 200μL cell suspension was put into the upper chamber of Transwell cells, and then 600μL complete medium (control group, GA group, IH Group, GA+IH group) was added to the lower chamber of 24-well culture plate. The culture plate was placed in a CO2 incubator at 37°C for 48 hr. It was took out the Transwell, and washed twice with PBS. In addition, the matrix glue and the cells in the chamber were wiped off with a cotton swab. Moreover, it was soaked in 70% methanol for 30min, and dyed with crystal violet. After crystal violet staining, the stained migrating cells can be decolorized with 33% acetic acid, crystal violet can be completely eluted, and then the OD value of the eluent at 570nm was determined by microplate reader, which indirectly reflected the number of migrating cells. The experiment was repeated at least three times.
2.9 Real-time qPCR (qPCR)
A549 and PC9 cells, passaged in a 6-well plate at 2×105/L, were cultured in an incubator for 24 hr, and different intervention drugs were added (control group, GA group, IH Group, GA+IH group). After 48 hr of treatment, cells were collected, total RNA was extracted with RNA Isolation Kit (SinoGene, Beijing, Chaina) and reverse transcribed using The First Strand Synthsis Kit (SinoGene, Beijing, China). The qPCR was performed using SYBR MasterMix Kit (SinoGene, Beijing, China). ACTB was used as an internal reference gene to detect the expression of Caspase-3 and YAP (Primers in the Table 1).The2-△△Ct method was used to caclulate the relative mRNA expression.
2.10 Western blot
Cells were treated with GA, IH, and GA+IH for 48 hours and then harvested and lysed in RIPA buffer (SinoGene, China) containing 1% phosphatase for total protein extraction. 30 µg proteins per samples were separated via SDS-PAGE and transferred to PVDF membranes after quantification by the Pierce BCA protein assay Kit (Thermo Fsiher Sceinetifc, USA) and boiled for 10 minutes with protein loading buffer. Protein Marker (SM26616, Thermo Fermentas, USA) was loaded at the first lane. the membranes were incubated with different primary antibodies anti-caspase-3, YAP, p-YAP (1:1000) at room temperature for 1 hour and then exposed to HRP-conjugated secondary antibody (1:3000) for 1 hr. Finally, membranes were exposed to films with ECL luminescent solution (29050, Engreen, China) in the darkroom. Beta-actin (1:3000, AC028, Abclonal, China) was as loading control.
2.11 Animal study
A total of 24 BALB/C nude mice (4 weeks old, 18–22 g) were purchased from the Experimental Animal Center. The study was approved by the Research Ethics Committee oftheChinease PLA General Hospital. Under sterile conditions, 0.2 ml (PC9) of cell suspension (l×107cells/mL) was pipetted with a 1 mL disposable syringe and slowly injected into the right armpit of the nude mice. The long diameter of the tumor block >1 mm and felted lumps was indicative of the successful establishment of the NSCLC mouse model. After successful modeling, animals were divied into 4 groups: control group, GA group (30μg/kg), IH group (100mg/kg), and combined treatment group (30μg/kg+100mg/kg) and thenadministration for 14 d (1 time/d). During the administration, the long diameter and short diameter of the transplanted tumors were measured by vernier calipers every 3d, repeated in triplicate to obtain the average values. The tumor volume was then calculated: V=long diameter × short diameter2/2, and the growth curve of the transplanted tumors were plotted based on the values obtained. Each mouse was heparinized by intraperitoneal injection of 0.5% heparin (0.5mL), and then eye blood were collected to measure the levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine (Cr) after the last administration on the 14th day. In addition, the animals were euthanized with carbon dioxide asphyxiation, the tumors were removed, and the volumes of the tumors were measured after weighing the animals. In addition, protein expressions of caspase-3, YAP and p-YAP in tumors was detected by immunohistochemical staining, and the percentage of caspase-3, YAP and p-YAP protein positive staining was quantified. A microscope (IX51Olympus, Japan) and Image Pro Plus software were used to take photos and analyze slides.
2.12 Statistical analysis
The quantitative experiments were performed in triplicates, and the data were represented as the mean ± standard deviation (S.D.). A statistical comparison of the data was conducted using One-way ANOVA followed by Tukey’s honest significant difference (HSD) test in post-hoc comparisons. Statistical significance between two groups was defined as P <0.05.