A total of 20 paired NSCLC tissues and their adjacent non-tumor tissues were collected from patients who underwent curative surgical resection in the department of thoracic surgery, The First People's Hospital of Yunnan Province. All patients were diagnosed and confirmed by experienced pathologists, and none of the patients underwent adjuvant chemotherapy or radiation treatment prior to surgery. This study was approved by Ethics Committee of Kunming University of Science and Technology and informed consent was obtained from all patients.
Antibodies and reagents
Dic was obtained from Chengdu Herbpurify Co., Ltd. (Chengdu, China), and was prepared by dissolving the compound in ethyl alcohol and diluted in DMSO. All antibodies used in Western blotting or IHC were shown in Additional file 1: Table S1. Polyvinylidene fluoride membrane (IPVH00010) was from Millipore (Merck Millipore Ltd., County Cork, Ireland). Cell Counting Kit-8 (C0039), Apoptosis and Necrosis Assay Kit (C1056), EdU Cell Proliferation Kit (C0088S) were purchased from Beyotime (Beyotime Biotechnology, Shanghai, China). TRIzol reagent (180411) was obtained from Ambion (Thermo Fisher Scientific, Inc. Waltham, MA, USA). FastStart Essential DNA Green Master (32597600) was supplied by Roche (Roche Diagnostics, Shanghai, China). RevertAid™ First Strand cDNA Synthesis Kit (K1622) was obtained from Thermo (Thermo Scientific, Waltham, MA, USA).
A549 (human non-small cell lung adenocarcinoma) cell line was obtained from Kunming Institute of Zoology, Chinese Academy of Sciences. Human lung carcinoma cell lines H1299 and PC9, liver cancer cell line HepG2, renal cell carcinoma cell line ACHN and cervical carcinoma cell line HeLa were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells mentioned above were maintained in the RPMI-1640 (Hyclone, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco, Invitrogen) and antibiotics (10 mg/mL streptomycin sulfate, and 100 mg/mL penicillin G). To establish a Gefitinib-resistant cell line, the PC9 cells were initially exposed to Gefitinib at 0.2 µM for about 7 days. Then the cells were transferred to and cultured in medium without Gefitinib for about 14 days. Following the above steps, the exponential growth cells were harvested and seeded in 96-well microtiter plates, the concentration of Gefitinib was increased from 0.2 µM to 5 µM. After about six months, the Gefitinib-resistant PC9 cells (PC9-GR) were established.
The Gene Expression Profiling Interactive Analysis (GEPIA) browser (http://gepia.cancer-pku.cn/) is a friendly web-based tool to explore customizable functionalities based on the data provided by TCGA (https://tcga-data.nci.nih.gov/tcga/) and the Genotype-Tissue Expression project (GTEx, https://www.gtexportal.org/home/index.html). GEPIA was used to identify the transcriptional expression of c-Met between lung cancer and normal tissues. The prognostic value of c-Met expression in lung cancer patients was analyzed by using the Kaplan-Meier plotter online database (http://kmplot.com/analysis/).
CCK-8 cell proliferation assay
The viability of cancer cells was determined by using the CCK-8 assay according to the manufacturer's protocol. Cancer cells were seeded in 96-well plates (5 × 103 cells per well) and cultured for 24 h. The cells were treated with varying concentrations of Dic (0, 50, 100, 200 µM) for 2, 24, 48 and 72 h. Then 10 µL CCK-8 (Beyotime Biotechnology, Shanghai, China) was added to each well, and incubated at 37 °C for 1.5 h. The absorbance of each well was measured at a wavelength of 450 nm by using a microplate reader.
EdU cell Proliferation assay
A549 cells cultivated in 96-well plates (5 × 103 cells per well) were treated with different concentrations of Dic (0, 50, 100, 200 µM) for 24 and 48 h. After the administration of 30 µM 5-ethynyl-2’-deoxyuridine (EdU; Beyotime Biotechnology, Shanghai, China) to each well, the cells were continually incubated for 2 h at 37 °C. Then the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After the removal of the fixative, A549 cells were washed with PBS and permeabilized with 0.3% Triton X-100 for another 15 min, the cells were incubated with 100 µL 0.3% hydrogen peroxide solution for 20 min. After washing three times with 3% BSA in PBS, 50 µL Click Reaction Solution was added to each well and the cells were incubated for 30 min at room temperature. The Click Reaction Solution was discarded and the washing repeated, 20 µL Streptavidin-HRP was added and incubated for another 30 min. After washing with 3% BSA in PBS, 100 µL TMB was added to each well and incubated for about 30 min, the absorbance of each well was measured at 650 nm.
In order to measure cell death and apoptosis, cells were treated with Dic (0, 50, 100, 200 µM) for 24 h and then collected for Hoechst 33342/propidium iodide (PI) staining with an apoptosis and Necrosis Assay Kit (Beyotime Biotechnology, Shanghai, China). The stained cells were observed and photographed under a confocal microscope (Nikon A1, Tokyo, Japan), and the images analyzed using the ImageJ software program (NIH, Bethesda, MD). The percentage of dead cells was then calculated according to the following: PI positive cells/total cells × 100.
Plate colony formation assay
For colony formation assays, 500 cells in the logarithmic phase were plated into 12-well plates with Dic (0, 50, 100, 200 µM) and incubated for 14 days. The cell culture medium was refreshed every three days. When the cells grew to form visible colonies, the supernatants were discarded and the colonies were fixed with 4% paraformaldehyde for 30 min, and then washed with PBS and stained with crystal violet for 15 min. Cell colonies were counted under an inverted microscope using the Image-Pro Plus software (Media Cybernetics, Inc. Rockville, MD). The assays were performed five times independently.
Transwell migration and matrigel invasion analysis
The migration and invasion assay were carried out using the transwell chambers (Corning Costar, Cambridge, Massachusetts, pore size 8 µm). A549 cells were treated with Dic (0, 50, 100, 200 µM) for 24 h. Cells were then trypsinized, washed, and resuspended in serum-free RPMI-1640. Migration assays and invasive assays were performed using the uncoated transwell chamber and the chamber coated with Matrigel (BD Sciences, Franklin Lakes, NJ, USA), respectively. The Matrigel was diluted with DMEM medium (3:1) and dropped onto the chamber and incubated at 37 °C for 2 h. A total of 100 µL cell suspension (5 × 105/mL) was inoculated into the upper chamber, the bottom compartment contained 600 µL RPMI-1640 supplemented with 10% FBS to serve as a chemoattractant. After incubation for 36 h, transwell inserts were washed 3 times with PBS, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The invading cells on the lower surface of the transwell insert were visualized with a light microscope, and counted in 5 randomly selected visual fields. The assays were performed five times independently.
Wound healing assay
The effects of Dic on the migratory behavior of lung cancer cells were assayed by means of a wound healing assay using a culture-insert (ibidi GmbH, Am Klopferspitz 19, Martinsried, Germany) according to the manufacturer’s instructions. 200 µL cell suspension (1.5 × 105 cells/mL) was applied to the Culture–Insert of each well and allowed to grow to a confluent monolayer at 37 °C and 5% CO2 for 24 h. The insert was gently removed using sterile tweezers, and the cells were washed twice with PBS to remove any loose cells and fresh medium containing mitomycin was added. A549 cells were treated with Dic (0, 50, 100, 200 µM) and Photographs were taken at 0, 24 and 48 h to assess cell migration into the wound using a light microscope.
Sphere formation assay
To investigate the effect of Dic on lung cancer stem cells, sphere formation assays were performed. Approximately 1 × 104 A549 cells were seeded into ultra-low attachment 6-well plates (Corning Inc, Corning, NY, USA) and cultured in serum-free DMEM/F-12 (1:1 ratio) media supplemented with B27 supplements (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL bFGF and 20 ng/ml rhEGF. Cells received treatment with various concentrations of Dic for 48 h at 72 h after cell seeding. The medium was exchanged every 3 days. After 2 weeks, cell spheres with diameters ≥ 50 µm were counted using a microscope.
Cell adhesion assay
A549 cells were treated with Dic (0, 50, 100, 200 µM) for 24 h and were then seeded into 96-well plates pre-coated with 10 µg/mL fibrinogen, 10 µg/mL collagen type IV, and cultured at 37 °C for 30 min. Wells coated with 100 µg/mL poly-L-lysine were employed as a control condition. Subsequently, the medium was discarded, and the non-adherent cells were carefully removed by washing the cells three times with PBS. The attached cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Excess crystal violet was washed away using PBS and 100 µL of acetic acid was added to each well with gentle shaking for 10 min to dissolve the purple crystals. Absorbance was measured at 590 nm by a Microplate Reader. The quantified cellular adhesion results were normalized to control cells and were represented as relative absorbance.
Quantitative real-time PCR analysis
Total RNA was extracted from cells using TRIzol regent. The RNA (5 µg) was then reverse transcribed into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The level of mRNA was measured by real-time PCR using FastStart Essential DNA Green Master Mix (Roche Diagnostics, Shanghai, China) and was normalized to GAPDH. The sequences of primers used in the experiment were listed in Additional file 1: Table S2. The reaction was performed using the LightCycler® 96. The results of q-PCR were expressed relative to threshold cycle (CT) values and fold changes were calculated using the 2−ΔΔCT method.
Western blot analysis
Western blotting was used to analyze the protein levels. Cells were collected and lysed with RIPA buffer. The nuclear protein and cytoplasmic Protein were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). Proteins of cell lysates were quantified by Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of cell lysate were separated on an SDS-PAGE gel and transferred to the PVDF membrane. After being blocked with 5% non-fat milk, the membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies were as follows: anti-c-Met, anti-phospho-c-Met, anti-PI3K, anti-phospho-PI3K, anti-AKT, anti-phospho-AKT, anti-m-TOR, anti-phospho-m-TOR, anti-STAT3, anti-phospho-STAT3, anti-B-Raf, anti-phospho-B-Raf, anti-ERK, anti-phospho-ERK, anti-GSK-3β, anti-phospho-GSK-3β, anti-β-catenin, anti-c-Myc, anti-cyclin D1, anti-CDK1, anti-IL-1β, anti-GAPDH, anti-Actin, anti-Tubulin, anti-H2A. The information of antibodies was listed in Additional file 1: Table S1. After subsequent washing with TBST, the membranes were then incubated with corresponding secondary antibodies for 1.5 h at room temperature. Protein bands were visualized by a chemiluminescence system (GeneGnome, Syngene, UK).
Cellular Thermal Shift Assay (CETSA)
CETSA was performed using cell lysates and intact cells as previously described [6, 7]. For a CETSA in A549 cell lysates, A549 cells were lysed with lysis buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 0.2% NP-40, 5% glycerol, 25 mM NaF and 1 mM Na3VO4) supplemented with 0.1% protease inhibitor cocktail. The cell suspensions were rapidly frozen and thawed three times using liquid nitrogen, and incubated on ice for 30 min, the lysates were centrifuged at 20000 × g for 20 min at 4 °C, supernatants were transferred to new tubes and protein concentrations were measured by BCA Protein Assay Kit. The cell lysates were diluted with appropriate volume of lysis buffer, and 50 µL of the lysates at 0.6 mg/mL were incubated with DMSO or Dic at different concentrations for 2 h at room temperature and then heated individually at different temperature for 3 min in a PCR machine (Applied Biosystems). The heated lysates were centrifuged to separate the precipitated proteins from the soluble fractions. The equal portions of the supernatants were loaded onto SDS-PAGE gels followed by Western blot analysis. For a CETSA in intact A549 cells, the cells cultured in 12-well plates (3.0 × 105 cells/well) were treated with different concentrations of Dic for 2 h, the control cells were exposed to an equal volume of DMSO. After incubation, the cells were washed with PBS and 500 µL of lysis buffer was added to each well, then the lysates were heated and analyzed by Western blotting as described above.
Drug affinity responsive target stability (DARTS)
DARTS was used to assess the binding affinity of Dic to c-Met using protease protection from pronase digestion (Roche Applied Science, Inc.) as previously described . A549 cells were washed with cold PBS and lysed using cold M-PER lysis buffer (Thermo Scientific, Inc.) supplemented with a protease inhibitor cocktail and phosphatase inhibitors. The cell lysates were centrifuged at 20000 × g for 10 min at 4 °C, and the supernatants were diluted with 10 × TNC buffer (500 mM Tris-HCl pH 8.0, 500 mM NaCl and 100 mM CaCl2). The lysates in 1 × TNC buffer were incubated with DMSO or Dic at the indicated concentrations for 2 h at room temperature. After incubation, each sample was proteolyzed with 1 µg pronase to every 3000 or 4000 µg of total protein in lysate for 20 min at room temperature. Proteolysis was stopped by adding 5 × SDS sample loading buffer (250 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 0.5% bromophenol blue and 5% β-mercaptoethanol) and boiling at 100 °C for 5 min. An equal portion of each sample was loaded onto SDS-PAGE gels and analyzed by Western blotting.
Molecular modeling of Dic with c-Met
The crystal structure of c-Met was obtained from the Protein Data Bank (PDB: 4IWD). AutoDock Tool was utilized to prepare the input PDBQT file for c-Met and set the size and the center of the grid box. Hydrogen atoms were added and all water molecules were removed from the structure of c-Met. The center of the grid box was set with the dimension (X= -8.833, Y = 12.926, and Z= -16.482) using 0.1 nm spacing and a box size of 20 × 20 × 20. The three-dimensional structure of Dic was generated using Open Babel software. Molecular docking of Dic at the active site of c-Met was achieved using AutoDock Vina. The best pose on the basis of binding affinity and docking was selected and post-docking results were analyzed using LigPlot+.
Subcutaneous xenografts in nude mice
All animal experiments were approved by our Institutional Animal Care and Treatment Committee. Four to five weeks-old female BALB/c nude mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China) and maintained under specific pathogen-free conditions with controlled light and humidity, receiving food and water ad libitum. Before inoculating into mice, A549 cells were washed twice and counted, approximately 5 × 106 cells suspended in 100 µL PBS/animal were inoculated subcutaneously into the right armpits of mice. The mice were randomly assigned into 2 groups, an experimental group and a control group, each of 6 mice. Treatment with Dic began 9 days after inoculation of the cells when the tumors reached a volume of 100–150 mm3. The experimental group was treated with i.p. injections of Dic (50 mg/kg body weight) for the next 15 days, while the control group received an equal volume of vehicle. All mice were weighted daily during the treatment time, and tumor volume was calculated using the following formula: 0.5 × length × (width)2. One day after the last treatment mice were sacrificed, and then the tumors were dissected, weighed and subjected to hematoxylin-eosin (HE) staining combined with immunohistochemistry (IHC) analysis.
Hematoxylin-eosin, immunohistochemistry and fluorescence staining
Solid tumors from control and treatment groups were fixed with 4% paraformaldehyde and embed in paraffin for H&E staining and IHC. Tissue sections were deparaffinized in xylene, dehydrated in graded ethanols and finally hydrated in distilled water, the sections were then counterstained with HE. For IHC, epitope retrieval was performed by directly boiling the slides in 10 mM sodium citrate (pH 6.0) for 30 min, immunohistochemistry was performed according to the manufacturer’s guidelines and recommendations. Briefly, the sections were treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase and non-specific binding was blocked with 5% normal goat serum/TBST (Cell Signaling Technology, Cambridge, USA #5425). The slides were incubated overnight at 4ºC with anti-Ki67 antibody (rabbit polyclonal antibody, diluted 1:200, Merck-Millipore, Darmstadt, Germany #AB9260). After incubation, the tissues were incubated with Signalstain Boost IHC detection reagent (HRP, rabbit, Cell Signaling Technology, Cambridge, USA #8114) for 30 min at room temperature and detected with the Signalstain DAB substrate kit (Cell Signaling Technology, Cambridge, USA #8059), followed by counterstaining with hematoxylin. Apoptotic cells in tumors were detected using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay according to the manufacturer's protocol (KeyGEN BioTECH, Nanjing, China #KGA700). Images were taken using an Olympus microscope and quantified by the image analyzer ImageJ.
All quantitative Data were presented as the mean ± standard deviation (SD). Statistically significant differences were obtained using an unpaired two-tailed student’s t-test or by one-way ANOVA. A P value of less than 0.05 was considered statistically significant.