Cell lines, reagents and instruments
Human NSCLC cell line A549 (catalogue number: TCHu150) was obtained from the Cell Bank of the Chinese Academy of Sciences and H1993 (catalogue number: ATCC®CRL-5909™) was obtained from the American Type Culture Collection (ATCC). Methyl-β-cyclodextrin (MβCD) was purchased from Meilun Biotechnology (Dalian, Liaoning, China). Antibodies against c-Met, c-Src and β-actin were purchased from Wanlei Biotechnology (Shenyang, Liaoning, China). Antibodies against phosphorylated (p)-c-Met and p-c-Src were obtained from Bioss Inc. (Woburn, Massachusetts, USA). Anti-flotillin-1 antibody was obtained from Boster Biotechnology (Pleasanton, CA, USA). Fluorescein isothiocyanate-conjugated-anti-cholera toxin subunit B was purchased from Sigma (St. Louis, Missouri, USA). Horseradish peroxidase-conjugated specific goat anti-rabbit secondary antibody, Cy3-labeled goat anti-rat c-Met antibody, Cy3-labeled goat anti-rat c-Src antibody, phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) lysis buffer, SDS, trypsin and a cell cycle analysis kit were purchased from Beyotime Biotechnology (Shanghai, China). A cell apoptosis analysis kit was purchased from Nanjing Keygen Biotechnology (Nanjing, Jiangsu, China).
The following instruments were used: a linear particle accelerator used for human radiotherapy (Clinac 600C/D; ONCOR-PLUS, Siemens, Germany); a flow cytometer (C6; BD Biosciences, Franklin lakes, New Jersey, USA); a low-temperature refrigerated centrifuge (H-2050R; Xiangyi Company, Changsha, Hunan, China); a dual-gel vertical protein electrophoresis apparatus (DYCZ-24DN; Beijing Liuyi Biotech, Beijing, China); a gel imaging system (WD-9413B; Beijing Liuyi Biotech, Beijing, China); a fluorescence microscope (BX3; Olympus, Japan); and a Beckman SW40 rotor (Beckman Coulter GmbH, Unterschleissheim-Lohhof, Germany).
Cell culture and treatment
A549 and H1993 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% carbon dioxide conditions. Cells were routinely subcultured in a monolayer, digested with 0.25% trypsin and stopped with DMEM when the cells covered 90% of the culture bottle. Then, the cells were cultured in FBS-free medium for another 24 h and prepared for various treatments.
MβCD is a cyclic polysaccharide containing a hydrophobic cavity that enables the extraction of cholesterol from cell membranes [7]. Cholesterol is the main component of lipid rafts. Therefore, MβCD is widely used as a lipid raft inhibitor. In this study, MβCD was dissolved in DMEM and used at final concentrations of 5 and 10 mM. In the experimental groups, cells were pretreated with MβCD for 1 h before irradiation. Control cells were treated with equal volumes of DMEM. As previous studies have shown, the survival fraction of A549 cells decreases when treated with increasing doses of X-ray irradiation (e.g., 0, 1, 2, 4, 6 and 8 Gy). This time, we exposed A549 and H1993 cells to conventional X-ray (0, 4, 8, 12 Gy; 3 Gy per min) emitted by a linear particle accelerator used for human radiotherapy operated at 6 MV and room temperature to obtain a proper radiation dose for our study.
Clonogenic survival assays
Clonogenic survival assays described by Franken et al. [8] were used to evaluate the proliferative ability of irradiated A549 and H1993 cells. Briefly, cells were treated with either DMEM (control) or MβCD (5 or 10 mM) for 1 h followed by X-ray irradiation to a discontinuous rising dose of 0, 4, 8 and 12 Gy, and then cells were counted. Every 200 cells were seeded in a 35-mm dish at 37 °C under 5% carbon dioxide conditions and incubated for 30 days to allow macroscopic colony formation. Colonies were fixed with 4% paraformaldehyde for 20 min and then stained with Wright-Giemsa stain for 5 to 8 min. The number of colonies formed in each group was counted, and colonies containing approximately 50 viable cells were considered representative of clonogenic cells. The clonogenic fraction was calculated using these formulas: colony-plating efficiency (PE) = (number of colonies/number of seeded cells) × 100%; survival fraction (SF) = (PE of MβCD treated cells/PE of control cells) × 100%.
Flow cytometry assays
Cell cycle and apoptosis analysis were performed with flow cytometry assays. Cells at a density of 2 × 106/ml were exposed to either control DMEM or 5 or 10 mM MβCD for 1 h followed by X-ray irradiation (8 Gy) or control irradiation (0 Gy) then cultured in fresh DMEM. Cells were harvested and fixed in ice-cold 70% ethanol (4 °C) after being cultured for 4, 8, or 24 h. For cell cycle assays, after staining with 25 μl propidium iodide (PI, 100 μg/ml), the samples were incubated with 10 μl RNase A for 30 min in the dark at 37 °C. Cell apoptosis assays were performed with 5 μl PI for 15 min in the dark at 37 °C after mixing with 5 μl Annexin V-FITC. Cell cycle and apoptosis were evaluated by flow cytometry (C6; BD Biosciences, Franklin lakes, New Jersey, USA), and the data were analyzed with BD Accuri C6 Software 1.0.264.21.
Immunofluorescence microscopy
Cells were plated on Lab-Tek chamber slides. After treatment with MβCD or control for 1 h followed by irradiation at 0 or 8 Gy, cells were fixed with 4% paraformaldehyde at 37 °C for 15 min, permeabilized with 0.5% Triton-X 100 after washing with PBS three times and then blocked with goat serum for 15 min. For lipid raft staining, cells were incubated with 0.05 mg/ml fluorescein isothiocyanate-conjugated-anti-cholera toxin subunit B for 1 h. For c-Met and c-Src staining, cells were incubated with anti-c-Met (Cy3-labeled) or anti-c-Src (Cy3-labeled) for 1 h then washed and blocked. 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) was used to stain the nuclei. Imaging was performed via fluorescence microscopy.
Western immunoblotting analysis
Western immunoblotting analysis was performed as previously described [9]. Briefly, A549 cells were treated with indicated reagents (DMEM or 10 mM MβCD for 1 h followed by irradiation at 0 or 8 Gy) then washed with ice-cold PBS three times and lysed in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and 0.1% SDS. Then, samples were centrifuged at 12000 rpm at 4 °C for 10 min in a low-temperature refrigerated centrifuge, and the supernatants were retained as protein lysates. For immunoblotting, 40 μg of protein lysates were subjected to electrophoresis on 4 to 10% SDS gels transferred to PVDF membranes, and blocked with 5% (w/v) skim milk in Tris-buffered saline-Tween 20 (0.05%, v/v; TTBS) for 1 h at 37 °C. Membranes were incubated overnight at 4 °C with primary antibodies against c-Met, p-c-Met, c-Src, p-c-Src and β-actin. After the overnight incubation, membranes were incubated with the appropriate horseradish peroxidase-conjugated specific goat anti-rabbit secondary antibody for 45 min and then washed with TTBS six times. The blots were developed by enhanced chemiluminescence followed by exposure to film, and the optical density values of target blots were analyzed with Gel-Pro-Analyzer software.
Biochemical lipid raft isolation
Biochemical lipid raft isolation was performed following established protocols [10, 11]. Briefly, all steps were performed at 4 °C. Cells were plated at a density of 1 × 107 cells in six 100-mm plates. Treated and untreated cells were washed twice with cold PBS, scraped into 2 ml of TNE solution [0.5% Triton-X-100, 1 mM PMSF, 150 mM NaCl, and 1 mM EDTA] and incubated for 40 min. The samples were scraped and homogenized completely by passing through a 5-ml needle 40 times. Homogenates were mixed with 2 ml of 90% (w/v) sucrose and placed at the bottom of a 15-ml ultracentrifuge tube. A 5–35% (w/v) discontinuous sucrose gradient was formed above the homogenate-sucrose mixture with a 4-ml layer of 35% sucrose followed by a 4-ml layer of 5% sucrose by adding sucrose solution along the tube wall gently and slowly while avoiding any shake during the whole process. Next, samples were centrifuged at 39000 rpm at 4 °C for 20 h in a Beckman SW40 rotor. Twelve 1-ml gradient fractions were collected from the top of the gradient. Each fraction with no MβCD treatment and no irradiation was separated via SDS-PAGE and established the expression of flotillin-1, c-Met, p-c-Met, c-Src, p-c-Src by Western blot analysis. Fractions 2–6 were determined to be lipid raft fractions due to the presence of the lipid raft-specific protein flotillin-1 (Fig. 1). Then, we examined the total expression of c-Met, p-c-Met, c-Src, and p-c-Src in fractions 2–6 treated with either control DMEM or 10 mM MβCD for 1 h followed by irradiation to dose at 0 or 8 Gy.
Statistical analysis
Student’s t-tests were performed utilizing the statistical software in GraphPad Prism version 5.0. Values of P < 0.05 were considered statistically significant. All the data expressed in our study are the mean ± SD from at least three independent experiments.