Background: Activation of c-Met, a receptor tyrosine kinase, induces radiation therapy resistance in non-small cell lung cancer (NSCLC). The activated residual of c-Met is located in lipid rafts (Duhon et al. Mol Carcinog 49:739-49, 2010). Therefore, we hypothesized that disturbing the integrity of lipid rafts would restrain the activation of the c-Met protein and reverse radiation resistance in NSCLC. In this study, a series of experiments was performed to test this hypothesis. Methods: NSCLC A549 and H1993 cells were incubated with methyl-β-cyclodextrin (MβCD), a lipid raft inhibitor, at different concentrations for 1 h before the cells were X-ray irradiated. The following methods were used: clonogenic (colony-forming) survival assays, flow cytometry (for cell cycle and apoptosis analyses), immunofluorescence microscopy (to show the distribution of proteins in lipid rafts), Western blotting, and biochemical lipid raft isolation (purifying lipid rafts to show the distribution of proteins in lipid rafts). Results: Our results showed that X-ray irradiation induced the aggregation of lipid rafts in A549 cells, activated c-Met and c-Src, and induced c-Met and c-Src clustering to lipid rafts. More importantly, MβCD suppressed the proliferation of A549 and H1993 cells, and the combination of MβCD and radiation resulted in additive increases in A549 and H1993 cell apoptosis. Destroying the integrity of lipid rafts inhibited the aggregation of c-Met and c-Src to lipid rafts and reduced the expression of phosphorylated c-Met and phosphorylated c-Src in lipid rafts. Conclusions: X-ray irradiation induced the aggregation of lipid rafts and the clustering of c-Met and c-Src to lipid rafts through both lipid raft-dependent and lipid raft-independent mechanisms. The lipid raft-dependent activation of c-Met and its downstream pathways played an important role in the development of radiation resistance in NSCLC cells mediated by c-Met. Further studies are still required to explore the molecular mechanisms of the activation of c-Met and c-Src in lipid rafts induced by radiation. Keywords: Lipid rafts, Mesenchymal-epithelial transition factor (c-met), C-Src, Radiation resistance, NSCLC Background [1, 2]. c-Met, areceptortyrosinekinaselocated in lipidrafts, Radiotherapy alone or combined with chemotherapy is promotes cancer cell migration and invasion and mediates the foundation for treating various solid tumors. However, resistance to current anticancer therapies, including radio- radiation resistance greatly limits the curative effect of therapy. Studies have demonstrated that the activated radiotherapy, which becomes one of the most important residual of c-Met is located in lipid rafts [3, 4]. c-Src, a reasons for local recurrence and metastasis. Therefore, type of non-receptor tyrosine kinase, plays a vital role reversing the resistance of radiotherapy and increasing the in a number of diverse cell signaling pathways, including radiosensitivity become the toughest challenge in cancer cellular proliferation, cell cycle control, apoptosis, tumor treatment. progression, metastasis, and angiogenesis . c-Src partic- Lipid rafts are special microdomains in the plasma ipates in radiation resistance  and might be the bridge membrane that influence cell proliferation, apoptosis, to the activation of the downstream signaling pathway of angiogenesis, immunity, cell polarity, and membrane fusion c-Met. Whether and how lipid rafts are involved in the radio-resistance of non-small cell lung cancer (NSCLC) mediated by c-Met has not been established. We reveal * Correspondence: firstname.lastname@example.org here that disturbing lipid raft integrity inhibits the activation The First Oncology Department, Shengjing Hospital affiliated with China Medical University, Shenyang 110004, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zeng et al. BMC Cancer (2018) 18:611 Page 2 of 11 of c-Met and its downstream pathways, increases the cell membranes . Cholesterol is the main component of sensitivity of NSCLC cells to radiotherapy, enhances lipid rafts. Therefore, MβCD is widely used as a lipid raft the therapeutic ratio, and thus provides a new strategy inhibitor. In this study, MβCD was dissolved in DMEM to address the radio-resistance of NSCLC cells. and used at final concentrations of 5 and 10 mM. In the experimental groups, cells were pretreated with MβCD for Methods 1 h before irradiation. Control cells were treated with equal Cell lines, reagents and instruments volumes of DMEM. As previous studies have shown, the Human NSCLC cell line A549 (catalogue number: survival fraction of A549 cells decreases when treated with TCHu150) was obtained from the Cell Bank of the Chinese increasing doses of X-ray irradiation (e.g., 0, 1, 2, 4, 6 and Academy of Sciences and H1993 (catalogue number: 8 Gy). This time, we exposed A549 and H1993 cells to ATCC®CRL-5909™) was obtained from the American Type conventional X-ray (0, 4, 8, 12 Gy; 3 Gy per min) emitted Culture Collection (ATCC). Methyl-β-cyclodextrin (MβCD) by a linear particle accelerator used for human radiother- was purchased from Meilun Biotechnology (Dalian, apy operated at 6 MV and room temperature to obtain a Liaoning, China). Antibodies against c-Met, c-Src and proper radiation dose for our study. β-actin were purchased from Wanlei Biotechnology (Shenyang, Liaoning, China). Antibodies against phosphory- lated (p)-c-Met and p-c-Src were obtained from Bioss Inc. Clonogenic survival assays (Woburn, Massachusetts, USA). Anti-flotillin-1 antibody Clonogenic survival assays described by Franken et al.  was obtained from Boster Biotechnology (Pleasanton, CA, were used to evaluate the proliferative ability of irradiated USA). Fluorescein isothiocyanate-conjugated-anti-cholera A549 and H1993 cells. Briefly, cells were treated with toxin subunit B was purchased from Sigma (St. Louis, either DMEM (control) or MβCD (5 or 10 mM) for 1 h Missouri, USA). Horseradish peroxidase-conjugated spe- followed by X-ray irradiation to a discontinuous rising cific goat anti-rabbit secondary antibody, Cy3-labeled goat dose of 0, 4, 8 and 12 Gy, and then cells were counted. anti-rat c-Met antibody, Cy3-labeled goat anti-rat c-Src Every 200 cells were seeded in a 35-mm dish at 37 °C antibody, phenylmethanesulfonyl fluoride (PMSF), radio- under 5% carbon dioxide conditions and incubated for immunoprecipitation assay (RIPA) lysis buffer, SDS, trypsin 30 days to allow macroscopic colony formation. Colonies and a cell cycle analysis kit were purchased from Beyotime were fixed with 4% paraformaldehyde for 20 min and then Biotechnology (Shanghai, China). A cell apoptosis analysis stained with Wright-Giemsa stain for 5 to 8 min. The kit was purchased from Nanjing Keygen Biotechnology number of colonies formed in each group was counted, (Nanjing, Jiangsu, China). and colonies containing approximately 50 viable cells The following instruments were used: a linear particle were considered representative of clonogenic cells. The accelerator used for human radiotherapy (Clinac 600C/ clonogenic fraction was calculated using these formulas: D; ONCOR-PLUS, Siemens, Germany); a flow cytometer colony-plating efficiency (PE) = (number of colonies/number (C6; BD Biosciences, Franklin lakes, New Jersey, USA); a of seeded cells) × 100%; survival fraction (SF) = (PE of low-temperature refrigerated centrifuge (H-2050R; Xiangyi MβCD treated cells/PE of control cells) × 100%. Company, Changsha, Hunan, China); a dual-gel vertical protein electrophoresis apparatus (DYCZ-24DN; Beijing Liuyi Biotech, Beijing, China); a gel imaging system Flow cytometry assays (WD-9413B; Beijing Liuyi Biotech, Beijing, China); a Cell cycle and apoptosis analysis were performed with fluorescence microscope (BX3; Olympus, Japan); and a flow cytometry assays. Cells at a density of 2 × 10 /ml BeckmanSW40rotor (Beckman CoulterGmbH, were exposed to either control DMEM or 5 or 10 mM Unterschleissheim-Lohhof, Germany). MβCD for 1 h followed by X-ray irradiation (8 Gy) or control irradiation (0 Gy) then cultured in fresh DMEM. Cell culture and treatment Cells were harvested and fixed in ice-cold 70% ethanol A549 and H1993 cells were cultured in DMEM supple- (4 °C) after being cultured for 4, 8, or 24 h. For cell cycle mented with 10% fetal bovine serum (FBS) at 37 °C under assays, after staining with 25 μl propidium iodide (PI, 5% carbon dioxide conditions. Cells were routinely sub- 100 μg/ml), the samples were incubated with 10 μl cultured in a monolayer, digested with 0.25% trypsin and RNase A for 30 min in the dark at 37 °C. Cell apoptosis stopped with DMEM when the cells covered 90% of the assays were performed with 5 μl PI for 15 min in the culture bottle. Then, the cells were cultured in FBS-free dark at 37 °C after mixing with 5 μl Annexin V-FITC. Cell medium for another 24 h and prepared for various cycle and apoptosis were evaluated by flow cytometry (C6; treatments. BD Biosciences, Franklin lakes, New Jersey, USA), and MβCD is a cyclic polysaccharide containing a hydropho- thedatawereanalyzedwithBD AccuriC6Software biccavitythatenables theextractionof cholesterol from 1.0.264.21. Zeng et al. BMC Cancer (2018) 18:611 Page 3 of 11 Immunofluorescence microscopy the homogenate-sucrose mixture with a 4-ml layer of Cells were plated on Lab-Tek chamber slides. After 35% sucrose followed by a 4-ml layer of 5% sucrose by treatment with MβCD or control for 1 h followed by adding sucrose solution along the tube wall gently and irradiation at 0 or 8 Gy, cells were fixed with 4% parafor- slowly while avoiding any shake during the whole maldehyde at 37 °C for 15 min, permeabilized with 0.5% process. Next, samples were centrifuged at 39000 rpm Triton-X 100 after washing with PBS three times and at 4 °C for 20 h in a Beckman SW40 rotor. Twelve 1-ml then blocked with goat serum for 15 min. For lipid raft gradient fractions were collected from the top of the staining, cells were incubated with 0.05 mg/ml fluorescein gradient. Each fraction with no MβCD treatment and no isothiocyanate-conjugated-anti-cholera toxin subunit B for irradiation was separated via SDS-PAGE and established 1 h. For c-Met and c-Src staining, cells were incubated the expression of flotillin-1, c-Met, p-c-Met, c-Src, p-c-Src with anti-c-Met (Cy3-labeled) or anti-c-Src (Cy3-labeled) by Western blot analysis. Fractions 2–6weredetermined for 1 h then washed and blocked. 4′,6-Diamidine-2′- to be lipid raft fractions due to the presence of the lipid phenylindole dihydrochloride (DAPI) was used to stain raft-specific protein flotillin-1 (Fig. 1). Then, we examined the nuclei. Imaging was performed via fluorescence the total expression of c-Met, p-c-Met, c-Src, and p-c-Src microscopy. in fractions 2–6 treated with either control DMEM or 10 mM MβCD for 1 h followed by irradiation to dose Western immunoblotting analysis at 0 or 8 Gy. Western immunoblotting analysis was performed as previ- ously described . Briefly, A549 cells were treated with Statistical analysis indicated reagents (DMEM or 10 mM MβCD for 1 h Student’s t-tests were performed utilizing the statistical followed by irradiation at 0 or 8 Gy) then washed with software in GraphPad Prism version 5.0. Values of P < ice-cold PBS three times and lysed in RIPA lysis buffer 0.05 were considered statistically significant. All the data containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% expressed in our study are the mean ± SD from at least NP-40, and 0.1% SDS. Then, samples were centrifuged at three independent experiments. 12000 rpm at 4 °C for 10 min in a low-temperature refrig- erated centrifuge, and the supernatants were retained as Results protein lysates. For immunoblotting, 40 μgof protein MβCD suppressed proliferation of A549 and H1993 cells lysates were subjected to electrophoresis on 4 to 10% SDS with or without X-ray irradiation gels transferred to PVDF membranes, and blocked with MβCD was used to disrupt lipid rafts in cell membranes 5% (w/v) skim milk in Tris-buffered saline-Tween 20 via depletion of cholesterol from the plasma membrane (0.05%, v/v; TTBS) for 1 h at 37 °C. Membranes were . To assess a potential role for MβCD in suppressing incubated overnight at 4 °C with primary antibodies proliferation, we exposed A549 and H1993 cells pretreated against c-Met, p-c-Met, c-Src, p-c-Src and β-actin. After with either DMEM or MβCD to a rising dose of X-ray the overnight incubation, membranes were incubated with irradiation (0, 4, 8 and 12 Gy, respectively). The results of the appropriate horseradish peroxidase-conjugated specific clonogenic survival assays were shown in Additional file 1: goat anti-rabbit secondary antibody for 45 min and then Table S1 and Additional file 2: Table S2 and Fig. 2.Within washed with TTBS six times. The blots were developed by each cell line and each pretreatment group, there was a enhanced chemiluminescence followed by exposure to radiation dose-dependent decrease in colony-plating film, and the optical density values of target blots were efficiency (PE) showing that higher radiation doses were analyzed with Gel-Pro-Analyzer software. significantly different from lower radiation doses except for A549 cells pretreated with DMEM followed by radi- Biochemical lipid raft isolation ation of 4 Gy vs. 8 Gy, A549 cells pretreated with 5 mM Biochemical lipid raft isolation was performed following MβCD followed by radiation of 0 Gy vs. 4 Gy, A549 cells established protocols [10, 11]. Briefly, all steps were per- pretreated with 10 mM MβCD followed by radiation of formed at 4 °C. Cells were plated at a density of 1 × 10 0 Gy vs. 4 Gy, and H1993 cells pretreated with 5 mM cells in six 100-mm plates. Treated and untreated cells MβCD followed by radiation of 0 Gy vs. 4 Gy. As shown in were washed twice with cold PBS, scraped into 2 ml of Additional file 1: Table S1 and Additional file 2:Table S2 TNE solution [0.5% Triton-X-100, 1 mM PMSF, 150 mM and Fig. 2, the PEs of A549 and H1993 cells decreased in a NaCl, and 1 mM EDTA] and incubated for 40 min. The radiation dose-dependent way. The PEs of A549 and samples were scraped and homogenized completely by H1993 cells in each group pretreated with the same passing through a 5-ml needle 40 times. Homogenates concentration of MβCD irradiated with 8 Gy or 12 Gy were mixed with 2 ml of 90% (w/v) sucrose and placed X-ray compared with control group were significantly at the bottom of a 15-ml ultracentrifuge tube. A 5–35% different. But this trend was not shown in each group (w/v) discontinuous sucrose gradient was formed above irradiated with 4 Gy compared with control group. The Zeng et al. BMC Cancer (2018) 18:611 Page 4 of 11 Fig. 1 Lipid rafts were separated by a sucrose density gradient centrifugation procedure, and immunoblotting was performed for c-Met, p-c-Met, c-Src, p-c-Src and flotillin-1. Blots are representative of at least three independent experiments. Fractions 2–6 were determined to be lipid raft fractions due to the presence of the lipid raft-specific protein flotillin-1. c-Met was mainly distributed in fractions 1 and 3–8; p-c-Met was mainly distributed in fractions 2–8; c-Src was mainly distributed in fractions 1–8; and p-c-Src was mainly distributed in fractions 2–6 and 10 PEs of A549 and H1993 cells pretreated with either respectively; Fig. 3). The combination of 10 mM MβCD DMEM or MβCD and irradiated with 12 Gy were too and radiation (8 Gy) markedly increased the apoptosis rate low to continue the remaining experiments; therefore, we when compared with that of radiation alone at 4, 8 and chose 8 Gy as the proper radiation dose in our further 24 h, and the differences were statistically significant experiments. Our results showed that the PEs of A549 (at 4, 8 and 24 h in A549 cells: P = 0.0026, P = 0.0013, cells in each group pretreated with 10 mM MβCD and P = 0.0016; at 4, 8 and 24 h in H1993 cells: P = compared with DMEM followed by the same radiation 0.0038, P = 0.0020, and P = 0.0002, respectively; Fig. 3). dose (0, 4, 8 and 12 Gy, respectively) were significantly These results showed that the combination of MβCD different, but the PEs were not significantly different in each and radiation resulted in additive increases in the apop- radiation group pretreated with 5 mM MβCD vs. DMEM tosis of A549 and H1993 cells. or 5 mM MβCD vs. 10 mM MβCD (Additional file 1: Table S1 and Fig. 2). This trend was also shown in X-ray irradiation induced the redistribution of c-met and H1993 cells (Additional file 2:Table S2 and Fig. 2). c-Src in lipid rafts Therefore, we chose 10 mM as the proper concentration of To investigate the impact of X-ray irradiation on the MβCD for our further experiments. These results showed redistribution of c-Met and c-Src in lipid rafts, A549 cells that MβCD suppressed the proliferation of A549 and were treated with 10 mM MβCD or control (DMEM) for H1993 cells whether followed by X-ray irradiation or not. 1 h followed by X-ray irradiation to a dose of 0 or 8 Gy. Sixteen hours later, the distribution of c-Met and c-Src in The combined treatment of MβCD and radiation resulted in lipid rafts was determined (Fig. 4). The results showed that additive increases in apoptosis of A549 and H1993 cells X-ray irradiation alone induced the aggregation of lipid In this study, we aimed to ascertain whether the com- rafts and clustering of c-Met and c-Src to lipid rafts. bination of MβCD and radiation had an additive or Through destroying the integrity of lipid rafts, MβCD supra-additive effect on the apoptosis of A549 and pretreatment blocked both the aggregation of lipid rafts H1993 cells. Notably, pretreatment with 5 mM MβCD and clustering of c-Met and c-Src to lipid rafts. alone produced little apoptosis in both A549 and H1993 cells (Fig. 3). Increasing the concentration of MβCD to The activation of c-met and c-Src and the accumulation of 10 mM greatly increased the apoptosis rate in both cell c-met and c-Src to lipid rafts were restrained by MβCD lines (Fig. 3). The combined treatment of 5 mM MβCD In agreement with the sucrose density gradient centri- and X-ray irradiation (8 Gy) did not significantly differ fugation procedure [10, 11], the original location of from that of X-ray irradiation alone at 4, 8, or 24 h in c-Met, p-c-Met, c-Src and p-c-Src in A549 cells with no A549 cells or at 4 h in H1993 cells with respect to an MβCD treatment and no X-ray irradiation was revealed additive effect on apoptosis (at 4, 8 and 24 h in A549 (Fig. 1): c-Met was mainly distributed in fractions 1 and cells: P = 0.1124, P = 0.0650, P = 0.1110; at 4 h in H1993 3–8; p-c-Met was mainly distributed in fractions 2–8; cells: P = 0.7438; respectively; Fig. 3); however, the com- c-Src was mainly distributed in fractions 1–8; and bined treatment of 5 mM MβCD and X-ray irradiation p-c-Src was mainly distributed in fractions 2–6 and 10. As (8 Gy) significantly increased apoptosis at 8 and 24 h in we mentioned before, fractions 2–6 were determined to H1993 cells (at 8 h and 24 h: P = 0.0071, P = 0.0010, be the lipid raft fractions. Zeng et al. BMC Cancer (2018) 18:611 Page 5 of 11 Fig. 2 MβCD suppressed proliferation of A549 and H1993 cells whether followed by X-ray irradiation or not. Cells were pretreated with either control (DMEM) 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. Then, cells were incubated for 30 days to allow macroscopic colony formation. The results showed that exposing A549 and H1993 cells to a rising dose of radiation either pretreated with DMEM or 5 or 10 mM MβCD inhibited cell proliferation in a radiation dose-dependent manner (a1 represents PE(%) of A549 cells under different conditions. b1-d1 represents PE(%) of A549 cells pretreated with the same concentration of MβCD (0, 5 or 10 mM, respectively) followed by different doses of X-ray(0, 4, 8 and 12 Gy). e1-h1 represents PE(%) of A549 cells pretreated with different concentration of MβCD (0, 5 or 10 mM) followed by the same doses of X-ray(0, 4, 8 and 12 Gy, respectively). a2 represents PE(%) of H1993 cells under different conditions. b2-d2 represents PE(%) of H1993 cells pretreated with the same concentration of MβCD (0, 5 or 10 mM, respectively) followed by different doses of X-ray(0, 4, 8 and 12 Gy). e2-h2 represents PE(%) of H1993 cells pretreated with different concentration of MβCD (0, 5 or 10 mM) followed by the same doses of X-ray(0, 4, 8 and 12 Gy, respectively). “no statistical significance” is shown as “ns”, “P < 0.05” is shown as “*”, “P < 0.01” is shown as “**”, and “P < 0.001” is shown as “***”) The expression levels of c-Met, p-c-Met (activated pretreatment with MβCD (c-Met, p-c-Met, c-Src, and c-Met), c-Src and p-c-Src (activated c-Src) in the whole-cell p-c-Src: P = 0.0033, P = 0.0005, P = 0.0012, and P = samples were significantly increased after X-ray irradiation 0.0024, respectively; Additional file 3: Table S3 and (c-Met, p-c-Met, c-Src, and p-c-Src: P = 0.0406, P = 0.0012, Fig. 5). The sucrose density gradient centrifugation re- P = 0.0085, and P = 0.0045, respectively; Additional file 3: sults showed that the accumulation of c-Met, p-c-Met, Table S3 and Fig. 5) when compared with those of the con- c-Src and p-c-Src to lipid rafts was significantly induced trol group. However, this up-regulation of c-Met, p-c-Met, by X-ray irradiation (c-Met, p-c-Met, c-Src, and p-c-Src: c-Src and p-c-Src in the whole-cell samples was blocked by P < 0.0001, P < 0.0001, P = 0.0030, and P = 0.0051, Zeng et al. BMC Cancer (2018) 18:611 Page 6 of 11 Fig. 3 The apoptosis rate of A549 and H1993 cells in each group pretreated with 10 mM MβCD compared with DMEM followed by the same radiation dose were significantly different but not significantly different in each group pretreated with 5 mM MβCD vs. DMEM or 5 mM MβCD vs. 10 mM MβCD (a1 represents the apoptosis rate of A549 cells under different conditions. b1-d1 represents the apoptosis rate of A549 cells after treatment for 4, 8, 24 hours respectively. a2 represents the apoptosis rate of H1993 cells under different conditions. b2-d2 represents the apoptosis rate of H1993 cells after treatment for 4, 8, 24 hours respectively. “no statistical significance” is shown as “ns”, “P <0.05” is shown as “*”, “P <0.01” is shown as “**”,and “P <0.001” is shown as “***”) respectively; Additional file 4: Table S4 and Fig. 6). p-c-Src expressed in lipid rafts out of those expressed in Moreover, this accumulation of c-Met, p-c-Met, c-Src and the whole-cell samples were obviously decreased in the p-c-Src to lipid rafts was blocked by pretreatment with combined group when compared with the MβCD alone MβCD (c-Met, p-c-Met, c-Src, and p-c-Src: P < 0.0001, group (Additional file 5: Table S5 and Fig. 7). P < 0.0001, P = 0.0028, and P = 0.0082, respectively; Additional file 4: Table S4 and Fig. 6). Discussion Interestingly, compared with the MβCD alone group, Lung cancer is the leading cause of cancer death world- the combined treatment groupshowedsignificantly wide, and NSCLC accounts for approximately 85% of increased expression of p-c-Met and p-c-Src in the the total number of lung cancer diagnoses. Although whole-cell samples. However, there was no significant significant progress in diagnosis and treatment has change in the accumulation of p-c-Met or p-c-Src to lipid been made over the past several years, it is still too late rafts. More importantly, the percentages of p-c-Met and for most NSCLC patients to have radical surgery at Fig. 4 X-ray irradiation induced the aggregation of lipid rafts and clustering of c-Met and c-Src to lipid rafts in A549 cells. MβCD blocked both the aggregation of lipid rafts and clustering of c-Met and c-Src to lipid rafts(C: control group, R: radiation only group, M: MβCD only group, M + R: MβCD and radiation combined group, LR: lipid raft marker) Zeng et al. BMC Cancer (2018) 18:611 Page 7 of 11 Fig. 5 Expression of c-Met, p-c-Met, c-Src and p-c-Src in the whole-cell samples was significantly increased by X-ray irradiation in A549 cells. However, this up- regulation was blocked by pretreatment with MβCD (a-d presents the expression of c-Met, p-c-Met, c-Src and p-c-Src in the whole-cell samples under different conditions respectively. “no statistical significance” is shown as “ns”, “P <0.05” is shown as “*”, “P <0.01” is shown as “**”,and “P <0.001” is shown as “***”) their first diagnosis. Radiotherapy is one of the basic STAT pathways . The activation of c-Met and its treatments for unresectable NSCLC, but the resistance to downstream signaling pathways has been shown to induce radiation greatly limits the curative effect of radiotherapy. invasion and migration of cancer cells . Fan et al. Currently, cellular survival pathways that regulate DNA showed that the activation of c-Met protected tumor cells damage repair after radiotherapy have been heavily from DNA damage caused by radiation and led to radi- researched to reveal the mechanism of NSCLC radiation ation resistance . Overexpression of c-Met has been resistance . Many clinical studies have shown that the noted in various tumors, and c-Met activation appears to radiotherapy resistance of various solid tumors is associ- be associated with increased tumor differentiation, shorter ated with the overexpression of c-Met [14–16]. c-Met is a survival times and an overall worse prognosis in patients 170-kDa transmembrane protein that can be activated by with NSCLC [21, 22]. c-Src, a non-receptor tyrosine binding hepatocyte growth factor (HGF) to its extracellular kinase, is localized to intracellular membranes. c-Src is region . De Bacco et al. demonstrated that irradiation overexpressed or highly activated in a number of human directly induced the overexpression and activity of the Met malignancies, including carcinomas of the breast, lung, oncogene and activated c-Met signaling through the colon, esophagus, skin, parotid, cervix, and gastric tissues, ATM-NF-κB signaling pathway. In turn, the activated as well as in the development of cancer and progression c-Met signaling triggered the activation of downstream to distant metastases . Recent studies have shown that signaling, mainly through the PI3K/Akt, MAPK, and c-Src enhances DNA damage repair and induces NSCLC Zeng et al. BMC Cancer (2018) 18:611 Page 8 of 11 Fig. 6 Expression of c-Met, p-c-Met, c-Src and p-c-Src in lipid rafts was significantly increased by X-ray irradiation in A549 cells. However, this up-regulation was blocked by pretreatment with MβCD (a-d presents the expression of c-Met, p-c-Met, c-Src and p-c-Src in lipid rafts under different conditions respectively. “no statistical significance” is shown as “ns”, “P <0.05” is shown as “*”, “P <0.01” is shown as “**”,and “P <0.001” is shown as “***”) radiation resistance through ERK, AKT, and NF-κBpath- proliferation, resisting apoptosis, evading growth suppres- ways . c-Met activates the PI3K/Akt, ERK, and NF-κB sors, enabling replicative immortality, inducing angiogen- pathways via c-Src in cervical cancer cells [24–26]. c-Src esis, activating invasion and metastasis, reprogramming of might be the bridge by which the c-Met signaling pathway energy metabolism and evading immune destruction . induces radiation resistance. A growing body of evidence has shown that lipid raft The plasma membrane is the structural basis for signal microdomains provide signaling platforms that regulate transduction. Lipid rafts are small (10–200 nm), heteroge- a variety of cellular signaling pathways through which neous, highly dynamic, sterol- and sphingolipid-enriched tumors can be initiated and developed [29–31]. Recent domains that compartmentalize cellular processes. Smaller studies have shown that the activated residual of c-Met lipid rafts can stabilize their structure and form a larger located in lipid rafts, which serve as a huge signaling platform through protein-protein and protein-lipid inter- platform for the activation of c-Met and its downstream actions . As a “highly dynamic platform”,the lipid raft pathways . Localization of c-Src to lipid rafts has environment plays an important role in cell proliferation, been demonstrated in a variety of cancer cell lines . apoptosis, and functional activities through regulating We hypothesized that disturbing the integrity of lipid various cell signal transduction mechanisms. Hanahan et rafts would block the activation of the c-Met signaling al. summarized that the occurrence and development of pathwayand reversethe radiationresistanceofNSCLC tumors is closely connected with uncontrolled cell cells in some way. Zeng et al. BMC Cancer (2018) 18:611 Page 9 of 11 Fig. 7 The percentages of c-Met, p-c-Met, c-Src and p-c-Src expressed in lipid rafts out of the whole samples in A549 cells In this study, the clonogenic survival assays showed that irradiation induced the aggregation of lipid rafts and the X-ray irradiation inhibited the proliferation of A549 and clustering of c-Met and c-Src to lipid rafts. The results H1993 cells in a radiation dose-dependent manner regard- also demonstrated that destroying the integrity of lipid less of MβCD pretreatment. Our results further confirmed rafts restrained both the aggregation of lipid rafts and the that inhibiting the integrity of lipid rafts suppressed the clustering of c-Met and c-Src to lipid rafts. These results proliferation of A549 and H1993 cells whether followed indicate that X-ray irradiation-induced redistribution of by X-ray irradiation or not. Furthermore, we found the c-Met and c-Src in lipid rafts might result in radiation proper concentration of MβCD (10 mM) and the proper resistance in NSCLC cells. radiation dose (8 Gy) for our remaining experiments. Western blotting results showed that X-ray irradiation Next, we found that pretreating A549 and H1993 cells significantly increased the expression of c-Met, p-c-Met, with 10 mM MβCD alone obviously increased the apop- c-Src and p-c-Src in the whole-cell samples, but this up-- tosis rate in both control (0 Gy) and irradiated cells (8 Gy) regulation was blocked by pretreatment with MβCD. The but not for 5 mM MβCD alone. Our results also showed sucrose density gradient centrifugation analysis showed that that the combined treatment of MβCD and radiation sig- X-ray irradiation significantly induced the accumulation of nificantly increased the apoptosis rates of A549 and H1993 c-Met, p-c-Met, c-Src and p-c-Src to lipid rafts. Further- cells when compared with those of radiation alone at 4, 8 more, the accumulation of these four proteins to lipid rafts and 24 h, but this effect was not significant for the combin- was blocked by pretreatment with MβCD. Interestingly, we ation of 5 mM MβCD and radiation (8 Gy) compared with also found that the expression levels of p-c-Met and p-c-Src radiation alone. These results suggest that disturbing the in the whole-cell samples were significantly increased in the integrity of lipid rafts by MβCD sensitized A549 and H1993 combined group compared with those in the MβCD alone cells to radiotherapy in both time-dependent and group. However, there was no significant change in the concentration-dependent manners. Our findings also accumulation of these two proteins to lipid rafts. The indicate that lipid rafts play an important role in percentages of p-c-Met and p-c-Src expressed in lipid increasing the radiation sensitivity of NSCLC cells, and rafts out of the whole-cell samples was obviously decreased the combination of MβCD and radiation may provide a in the combined treatment group when compared with new effective therapeutic strategy for the treatment of those in the MβCD alone group. Collectively, these results radiation-resistant NSCLC. show that X-ray irradiation might activate c-Met and c-Src To investigate the impact of X-ray irradiation on the through both lipid raft-dependent and lipid raft-independent redistribution of c-Met and c-Src in lipid rafts, A549 mechanisms. cells were treated with 10 mM MβCD or DMEM for 1 h By analyzing the percentages of c-Met, p-c-Met, c-Src, followed by X-ray irradiation to a dose of 0 or 8 Gy, and and p-c-Src proteins expressed in lipid rafts out of the the distribution of c-Met and c-Src in lipid rafts was whole-cell samples, we found that in A549 cells, the determined 16 h later. The results showed that X-ray expression of p-c-Met and p-c-Src in lipid rafts induced Zeng et al. BMC Cancer (2018) 18:611 Page 10 of 11 by X-ray irradiation was significantly higher than that of Acknowledgements The following individuals and institutions participated in this study: Juan c-Met and c-Src. Furthermore, the inhibition of p-c-Met Zeng, Heying Zhang, Yonggang Tan, Cheng Sun, Yusi Liang, Jinyang Yu, and p-c-Src expressed in lipid rafts was more obvious than Huawei Zou, Shengjing Hospital affiliated with China Medical University, that of c-Met and c-Src by the destruction of lipid rafts. Shenyang, China. We are grateful to all the library staff of Shengjing Hospital affiliated with China Medical University for helping us with data collection, In summary, this study confirmed that MβCD sup- sorting, verification and analysis. pressed the proliferation of human NSCLC cell lines A549 and H1993 with or without X-ray irradiation, and the Funding This study has received a major funding from national natural science combination of MβCD and radiation resulted in additive foundation of China, and the award number is 81472806. This foundation increases in the apoptosis of A549 and H1993 cells. X-ray does not affect the study design, analysis and interpretation of data, and the irradiation induced the aggregation of lipid rafts and the writing the manuscript. clustering of c-Met and c-Src to lipid rafts through both Availability of data and materials lipid raft-dependent and lipid raft-independent mecha- The datasets used and/or analyzed during the current study are available nisms. Our results also demonstrated that destroying the from the corresponding author on reasonable request. integrity of lipid rafts significantly inhibited the aggregation Authors’ contributions of c-Met and c-Src to lipid rafts. More importantly, the JZ drafted the manuscript. JZ, HYZ, YGT, CS, YSL, JYY, HWZ planned, expression of p-c-Met and p-c-Src in lipid rafts induced by coordinated, and conducted the study. YGT contributed to data management. HYZ, CS and YSL conducted the statistical analysis. JZ, YGT X-ray irradiation was notably higher than that of c-Met and HWZ participated in revising the manuscript. All authors read and and c-Src. Furthermore, the inhibition of p-c-Met and approved the final manuscript. p-c-Src expressed in lipid rafts was more obvious than that Ethics approval and consent to participate of c-Met and c-Src by destruction of lipid rafts. Not applicable. Competing interests Conclusions The authors declare that they have no competing interests. Taken together, we draw a conclusion that lipid rafts serve as the signaling platforms for the lipid raft-dependent acti- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in vation of c-Met and c-Src induced by X-ray irradiation. The published maps and institutional affiliations. lipid raft-dependent activation of c-Met and its downstream pathways play an important role in radiation resistance of Received: 11 October 2017 Accepted: 11 May 2018 NSCLC cells mediated by c-Met. Destroying the integrity of lipid rafts can reverse these signaling pathways and improve References the radiosensitivity of NSCLC cells, which can provide a 1. Patra SK, Bettuzzi S. Epigenetic DNA methylation regulation of genes coding for lipid raft-associated components: a role for raft proteins in cell new strategy for developing radiation sensitizing agents and transformation and cancer progression. Oncol Rep. 2007;17:1279–90. for improving the therapeutic effect of radiotherapy. 2. Brown DA, London E. Structure and function of sphingolipids- and Further studies are still required to explore the molecular cholesterol-rich membrane rafts. J Biol Chem. 2000;275:17221–4. 3. Duhon D, Bigelow RLH, Coleman DT, Steffan JJ, Yu C, Langston W, et al. The mechanisms of the activation of c-Met and c-Src in lipid polyphenol epigallocatechin-3-gallate affects lipid rafts to block activation rafts induced by radiation. of the c-Metreceptor inprostate cancer cells. Mol Carcinog. 2010;49:739–49. 4. Coleman DT, Bigelow R, Cardelli JA. Inhibition of fatty acid synthase by luteolin post-transcriptionally down-regulates c-met expression Additional files independent of proteosomal/lysosomal degradation. Mol Cancer Ther. 2009; 8(1):214–24. 5. Aleshin A, Finn RS. SRC: a century of science brought to the clinic. Additional file 1: Table S1. Colony-plating efficiency (PE) of A549 cells Neoplasia. 2010;12:599–607. treated with either control or MβCD followed by irradiation. (DOC 28 kb) 6. Shimm DS, Miller PR, Lin T. Effects of v-src oncogene activation on radiation Additional file 2: Table S2. Colony-plating efficiency (PE) of H1993 cells sensitivity in drug-sensitive and in multidrug-resistant rat fibroblasts. Radiat treated with either control or MβCD followed by irradiation. (DOC 29 kb) Res. 1992;129(2):149–56. Additional file 3: Table S3. Expression of proteins in the whole-cell 7. Radhakrishnan A, Anderson TG, McConnell HM. Condensed complexes, rafts, samples under different conditions in A549 cells. (DOC 28 kb) and the chemical activity of cholesterol in membranes. Proc Natl Acad Sci U S A. 2000;97:12422–7. Additional file 4: Table S4. Expression of proteins in lipid rafts under 8. Franken NA, Rodermond HM, Stap J, Haveman J, van BC. Clonogenic assay different conditions in A549 cells. (DOC 28 kb) of cells in vitro. Nat Protoc. 2006;1(5):2315–9. Additional file 5: Table S5. The percentage of protein expressed in 9. Bigelow RLH, Cardelli JA. The green tea catechins, (−)-Epigallocatechin-3- lipid rafts out of the whole-cell samples in A549 cells. (DOC 28 kb) gallate (EGCG) and (−)-Epicatechin-3-gallate (ECG), inhibit HGF/met signaling in immortalized and tumorigenic breast epithelial cells. Oncogene. 2006;25:1922–30. Abbreviations 10. Macdonald JL, Pike LJ. A simplified method for the preparation of c-Met: Mesenchymal-epithelial transition factor; DAPI: 4′,6-Diamidine-2′-phenylindole detergent-free lipid rafts. J Lipid Res. 2005;46:1061–7. dihydrochloride; HGF: Hepatocyte growth factor; MβCD: Methyl-β-cyclodextrin; 11. Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP. Co- NSCLC: Non-small cell lung cancer; PE: Colony-plating efficiency; PI: Propidium purification and direct interaction of Ras with caveolin, an integral iodide; PMSF: Phenylmethanesulfonyl fluoride; RIPA: Radioimmunoprecipitation membrane protein of caveolae microdomains. Detergent-free purification of assay; SF: Survival fraction caveolae microdomains. J Biol Chem. 1996;271:9690–7. Zeng et al. BMC Cancer (2018) 18:611 Page 11 of 11 12. Yancey PG, Rodrigueza WV, Kilsdonk EP, Stoudt GW, Johnson WJ, Phillips MC, et al. Cellular cholesterol efflux mediated by cyclodextrins. Demonstration of kinetic pools and mechanism of efflux. J Biol Chem. 1996; 271:16026–34. 13. Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 2011;11(4):239–53. 14. Yu H, Li X, Sun S, Gao X, Zhou D. C-met inhibitor SU11274 enhances the response of the prostate cancer cell line DU145 to ionizing radiation. Biochem Biophys Res Commun. 2012;427:659–65. 15. Li B, Torossian A, Sun Y, Du R, Dicker AP, Lu B. A novel selective c-met inhibitor with radiosensitizing effects. Int J Radiat Oncol Biol Phys. 2012;84: e525–e31. 16. Buchanan IM, Scott T, Tandle AT, Burgan WE, Burgess TL, Tofilon PJ, et al. Radiosensitization of glioma cells by modulation of met signalling with the hepatocyte growth factor neutralizing antibody. AMG102. J Cell Mol Med. 2011;15:1999–2006. 17. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003;4(12):915–25. 18. De Bacco F, Luraghi P, Medico E, Reato G, Girolami F, Perera T, et al. Induction of MET by ionizing radiation and its role in radioresistance and invasive growth of cancer. J Natl Cancer Inst. 2011;103:645–61. 19. Qian LW, Mizumoto K, Inadome N, Nagai E, Sato N, Matsumoto K, et al. Radiation stimulates HGF receptor/c-met expression that leads to amplifying cellular response to HGF stimulation via upregulated receptor tyrosine phosphorylation and MAP kinase activity in pancreatic cancer cells. Int J Cancer. 2003;104:542–9. 20. Fan S, Wang JA, Yuan RQ, Rockwell S, Andres J, Zlatapolskiy A, et al. Scatter factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents. Oncogene. 1998;17:131–41. 21. Nakamura Y, Niki T, Goto A, Morikawa T, Miyazawa K, Nakajima J, et al. C- met activation in lung adenocarcinoma tissues: an immunohistochemical analysis. Cancer Sci. 2007;98:1006–13. 22. Masuya D, Huang C, Liu D, Nakashima T, Kameyama K, Haba R, et al. The tumour-stromal interaction between intratumoral c-met and stromal hepatocyte growth factor associated with tumour growth and prognosis in non-small-cell lung cancer patients. Br J Cancer. 2004;90:1555–62. 23. Yeatman TJ. A renaissance for SRC. Nat Rev Cancer. 2004;4:470–80. 24. Kim MJ, Byun JY, Yun CH, Park IC, Lee KH, Lee SJ. C-Src-p38 mitogen- activated protein kinase signaling is required for Akt activation in response to ionizing radiation. Mol Cancer Res. 2008;6(12):1872–80. 25. Ishizawar R, Parsons SJ. C-Src and cooperating partners in human cancer. Cancer Cell. 2004;6(3):209–14. 26. Funakoshi-Tago M, Tago K, Andoh K, Sonoda Y, Tominaga S, Kasahara T. Functional role of c-Src in IL-1-induced NF-kappa B activation: c-Src is a component of the IKK complex. J Biochem. 2005;137(2):189–97. 27. Pike LJ. Rafts defined: a report on the keystone symposium on lipid rafts and cell function. J Lipid Res. 2006;47:1597–8. 28. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. 29. Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME. Molecular ordering of the initial signaling events of CD95. Mol Cell Biol. 2002;22:207–20. 30. Bang B, Gniadecki R, Gajkowska B. Disruption of lipid rafts causes apoptotic cell death in HaCaT keratinocytes. Exp Dermatol. 2005;14:266–72. 31. Li HY, Appelbaum FR, Willman CL, Zager RA, Banker DE. Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses. Blood. 2003;101:3628–34. 32. Arcaro A, Aubert M, Espinosa del Hierro ME, Khanzada UK, Angelidou S, Tetley TD, et al. Critical role for lipid raft-associated Src kinases in activation of PI3K-Akt signalling. Cell Signal. 2007;19:1081–92.
BMC Cancer – Springer Journals
Published: May 30, 2018
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