Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture

Cis-diammine-dichloro-platinum II-based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non-small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin-treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin-treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20 passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post-cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy-related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15 proteins [glucose-regulated protein, 78 kDa (GRP78), heat shock protein 71, pre-mRNA processing factor 19, polypyrimidine tract binding protein 1, translationally controlled tumor protein, Cathepsin D, Cytochrome c, thioredoxin domain containing 5, MutS homolog (MSH) 6, Annexin A2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor 1A interacting protein, MSH2, protein phosphatase 2A 55 kDa regulatory subunit B, Rho glyceraldehyde-3-phosphate-dissociation inhibitor 1 and ANXA4] that were upregulated by >1.5-fold in heavy (H)- and light (L)-labeled A549R cells. In addition, 16 and 14 proteins were downregulated by >1.5-fold in the H- and L-labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin-resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin-resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti-apoptosis and/or autophagy promotion.