Department Of Thoracic Surgery, Union Hospital Of Huazhong University Of Science And Technology

Background:Lung cancer has highest incidence rate and mortality rate of all malignancy cancers in China. About 70% of the patients have been developed to advanced stage at their diagnosis, and have lost the opportunity of surgical resection. Chemotherapy has become a very important part in the comprehensive treatment of lung cancer, but the resistance of tumor cells to chemotherapy drug often fails the chemotherapy. In recent years, many research has been done to reveal the drug-resistant mechanism of tumor cells.Cisplatin has long been the foundation stone of the chemotherapy of lung cancer. Platinum-based regime is the standard of advanced NSCLC chemotherapy. Almost all first-line NSCLC chemotherapy regime is cisplatin application solutions. The mechanism of cisplatin is cross-coupling with DNA after entering lung cancer cells, founding stable cisplatin-DNA complex, blocking DNA duplication, and causing lung cancer cells death. Recent research has shown that the cisplatin-resistant mechanism of lung cancer cells includes lower drug concentration and higher degradation metabolism in tumor cells, and more importantly the enhancement of DNA damage repair. The mechanism is a complex and multi-gene involved event, coming down to several pathways and many proteins. It involves the classical pathways mediated by transmembrane transporters, multidrug resistance associated protein, lung resistance protein, and non-classical pathways mediated by non-transmembrane mechanism such as apoptosis, enzyme activity, intracellular PH. However, the cisplatin-resistant mechanism of lung cancer cells is still not fully understood. It's of great importance to keep looking for new drug resistance related genes and continue in-depth research to elucidate the resistant mechanism of lung cancer cells, and then we shall find ways of reversing the resistance. Objective:Our study is to identify differently expressed proteins before and after cisplatin resistance of human lung adenocarcinoma cell A549 by using proteome analysis, so as to find new targets for the treatment of lung cancer, reveal drug-resistant mechanism, and provide clues for ways to reverse resistance.Methods:Cisplatin-resistant cell strain A549/CDDP was established from their parental human lung adenocarcinoma cell line A549 by combining gradually increasing concentration of cisplatin with big dosage impact. Comparative proteome analysis of A549 and A549/CDDP were carried out by means of two-dimensional gel electrophoresis. The differentially expressed proteins were analyzed and identified by MALDI-TOF mass spectrometry. The siRNA for the gene of the identified protein was transfect to A549 and A549/CDDP cells by lentivirus. Western blot and methyl thiazolyl tetrazolium (MTT) assay were applied to explore the influence of silencing the gene expression on lung cancer cell biological behavior, and elucidate the function of the gene.Results:82 differentially expressed proteins were screened by analyzing the electrophoretic maps of A549 and A549/CDDP.6 differential proteins were analyzed by peptide mass fingerprinting. Glusose regulating protein 75 (GRP75), ribosomal protein S4, mitochondrial ATP synthase Fl complex beta subunit, and immunoglobulin heavy chain variable region were identified. All four differentially expressed proteins were over-expressed in A549/CDDP, whereas low-expressed or no-expressed in A549. GRP75siRNA was successfully transfected to A549 and A549/CDDP. After transfection GRP75 protein in A549/CDDP dramatically decreased, bcl-2 protein decreased, p53 protein increased. And the sensitivity to cisplatin of the transfected cells was increased compared with A549/CDDP.Conclusion:Our study Successfully established cisplatin-resistant A549/CDDP cell strain by combining gradually increasing concentration of cisplatin with big dosage impact. The differentially expressed proteins give some clues to elucidate the mechanism of lung cancer cell resistant of cisplatin, and provide the basis of searching for potential target of chemotherapy of lung cancer. The fact that cisplatin resistance of lung cancer cells may be associated with the over-expression of GRP75 gene gives priority to overcome the drug resistance of lung cancer cells.