The Study Of Radiobiological Characteristics And Proteomic Of Human Lung Adenocarcinoma Cell Line

Objective (1) To observe radiobiological characteristics of A549 and A549/DDP cell lines, and investigate the impact of the drug resistance on the radioresistance in human lung adenocarcinoma cell line. (2) To establish 2-dimensional electrophoresis(2-DE) maps of A549 and A549/DDP cell lines, to screen Multidrug Resistance (MDR) and radioresistance related proteins in human lung adenocarcinoma.Methods (1) Two tumor cell lines A549 and A549/DDP were irradiated by 6MV-X ray, then the radiobiological characteristics of the two lines were analyzed by the method of colony-forming assay According to radiosensitivity parameters Do, Dq, N and SF2. Compare the radiosensitivity of the two tumors cell lines. Flow cytometry (FCM)was used to detect the cell cycle distribution and the apoptosis index of tumor cells. The relationship between radiosensitivity and the cell cycle,radiation-induced apoptosis in two tumor cell lines were analyzed. (2)The total proteins of A549 and A549/DDP cells were obtained, and were extracted and separated by two-dimensional gel electrophor-esis(2-DE). The images of the gels were acquired by the scanner and then analyzed by using PDquest7.1 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots was incised from the gels and digested by trypsin.The peptide mass fingerprintings(PMF) was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database. Western blot and immunohistochemical method were used to determine the differential expression levels of the 3 proteins.Results (1) Do and Dq and N and SF2 were ranked in the following descending order:A549 A549/DDP. G2 phase arrest was detected by FCM after irradiation of 6Gy in both groups, the mean percentage of G2 phase cells was higher in A549 group than in A549/DDP group (P<0.05) at 24h,48 h and 72h. The result of FCM assay detected obvious apoptosis peak. The apoptosis peak of the two tumor cell lines:A549> A549/DDP, in accord with the height of radiosensitivity. (2)The proteins were separated successfully for proteome research,2-DE maps of total proteins with high resolution and reproducibility from A549 and A549/DDP were established. A total of 45 differential protein spots in the 2 cell lines were found, and 27 differential expression proteins were identified by MALDI-TOF-MS. The differential expressional proteins could be divided into six main groups based on their functions:①metabolic enzymes:ATP synthase subunit beta, mitochondrial,Egl nine homolog 1,Delta(3, 5)-Delta(2,4)-dienoyl-COA isomerase, mitochondrial,Triosephosphate isomerase,Proteasome subunit alpha type-6,Cytochrome b5 type B,Fatty acid-binding protein,epidermal,Hemoglobin subunit beta.②proteins relative to signal transduction:Cofilin-1,Keratin,typeⅠcytoskeletal 18,Keratin,typeⅡcytoskeletal 1.③proteins involved in drug detoxification or Translation:Peroxiredoxin-4,Elongation factor 1-delta,Membrane-associated progesterone receptor component 2,Putative RNA-binding protein 3,Probable ATP-dependent RNA helicase DDX6.④chaperones:Heat shock protein beta-1,10 KDa heat shock protein, mitochondrial.⑤Proteins related to cellular structure:Vimentin,Tubulin beta chain,Tubulin beta-2A chain,PDZ and LIM domain protein 1,Myosin regulatory light chain MRLC2,Histidine triad nucleotide-binding protein 1,Histone H2B type 1-C/E/F/G/I.⑥calcium- binding Proteins:Annexin A2,Annexin A4. Western blot and immunohistochemical method showed that Heat shock protein beta-1,Annexin A4,Vimentin were differential expression proteins in A549 and A549/DDP, which was consistent with the results from the comparative proteomic analysis.Conclusion (1) Drug resistant human lung adenocarcinoma cell line have reduced G2/M period percentage and the rate of apoptosis, and then increased radioresistance. (2) 27 differential expression proteins in human lung adenocarcinoma are useful for studying the multidrug resistance and radioresistance mechanism of lung adenocarcinoma.