Abstract
BackgroundActivation 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.MethodsNSCLC 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).ResultsOur 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.ConclusionsX-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.
Highlights
Activation of Mesenchymal-epithelial transition factor (c-Met), a receptor tyrosine kinase, induces radiation therapy resistance in non-small cell lung cancer (NSCLC)
Within each cell line and each pretreatment group, there was a radiation dose-dependent decrease in colony-plating efficiency (PE) showing that higher radiation doses were significantly different from lower radiation doses except for A549 cells pretreated with DMEM followed by radiation of 4 Gy vs. 8 Gy, A549 cells pretreated with 5 mM MβCD followed by radiation of 0 Gy vs. 4 Gy, A549 cells pretreated with 10 mM MβCD followed by radiation of 0 Gy vs. 4 Gy, and H1993 cells pretreated with 5 mM MβCD followed by radiation of 0 Gy vs. 4 Gy
The results showed that X-ray irradiation induced the aggregation of lipid rafts and the clustering of c-Met and c-Src to lipid rafts
Summary
Activation of c-Met, a receptor tyrosine kinase, induces radiation therapy resistance in non-small cell lung cancer (NSCLC). C-Met, a receptor tyrosine kinase located in lipid rafts, promotes cancer cell migration and invasion and mediates resistance to current anticancer therapies, including radiotherapy. C-Src, a type of non-receptor tyrosine kinase, plays a vital role in a number of diverse cell signaling pathways, including cellular proliferation, cell cycle control, apoptosis, tumor progression, metastasis, and angiogenesis [5]. C-Src participates in radiation resistance [6] and might be the bridge to the activation of the downstream signaling pathway of 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 here that disturbing lipid raft integrity inhibits the activation
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