Differential Proteomic Analysis Of Esophageal Carcinoma Cell Line Eca-109 Before And After Irradiation

ObjectiveTo research the radiobiological features of esophageal carcinoma cell line Eca-109 by MTT experiment and flow cytometer.Utilizing the methods and techniques of Proteomies to analyze differential proteomic expression of esophageal carcinoma cell line Eca-109 before and after irradiation.The purpose of the experiment is to research deeply the mechanism of radiotherapy in protein level,to find out the new potential molecular targets which influence the effectiveness of radiotherapy.Through the research we try to find out the new potential molecular target to increase the radiosensitivity of the tumors,and offer rationales for radiotherapy.Methods1.The cell growth curves of esophageal carcinoma cell line Eca-109 after irradiation with different doses were made through MTT experiment.The changes of apoptosis and cell stages were detected by flow cytometer.2.The proteome of esophageal carcinoma cell line Eca-109 before and after irradiation were separated and identified using immobilized pH gradient(IPG). Isoelectric focusing(IEF)electrophoresis was run as the first dimensional electrophoresis,and then horizontal SDS-PAGE as the second dimensional electrophoresis.After coomassie blue staining,images were captured by scanner,and then the images were edited and matched using PDQuest analysis software.3.The differentially expressed proteins were identified by Matrix-assisted laser desorption inoization-time of flight-time of light mass spectrometry (MALDI-TOF/TOF-MS)and SWISS-PROT or BLAST nr database searching.And the function of proteins was searched through internet.Results1.The cell growth suppression of Eca-109 was positive correlation with the radiation dose.The inhibition ratios of 2,4,6,8Gy groups were 32.81%,35.65%,49.48%,87.13%.Followed with the close increase the cell growth was more suppressed.And radiation can change the cell cycle.Followed with the dose increase the cells in S stage were decreased,the cells in G₂/M stage were increased,and the cells in G₁ stage had been little changed.That means radiation can make G₂/M stage arrest.The apoptosis induced by radiation was little.The apoptosis rates observed in 0,2,4,6,8Gy groups were 0.53%,0.73%,0.77%,0.88%,1.26%. But the proportion of the apoptosis in all groups was low.2.By trying different loading quantity,procedure of isoelectfic focusing electrophoresis,time of equilibrium,density of SDS-PAGE gel and other important procedures of two-dimensional electrophoresis,the images were analyzed successfully and good reproducibility were obtained.Analyzing by PDQuest software,there were 388 protein spots detected in radiation group and 339 protein spots in control group.21 protein spots were found differentially expressed in Eca-109 before and after irradiation.16 protein spots were up-regulated in the radiation group,and 5 protein spots were up-regulated in the control group.3.The peptide mass spectrometry maps and amino acid sequences were obtained successfully by the MALDI-TOF/TOF-MS technique.And the 14 proteins were identify ed through the database search,they were Actin-3,Cofilin-1,Lamin-A/C, Actin-related protein 3,hnRNP C,hnRNP A2/B1,HSP70,HSP60,cytokeratin-18, calreticulin,Protein mago nashi homolog,Protein disulfide-isomerase A3,Ornithine aminotransferase.Most of the proteins were related with apoptosis regulation,cell cycle regulation,RNA transcription,signal transduction,cytoskeleton component and stress reaction induced by radiation.Conclusion1.The cell growth suppression of Eca-109 is positive correlation with the radiation dose.Radiation can change the cell cycle and make G₂/M stage arrest.Apoptosis induced by radiation is a main road to treat tumors.The proportion of the apoptosis induced by radiation in esophageal carcinoma cell line Eca-109 was low,we can induce apoptosis by other methods to increase the radiosensitivity of Eca- 109.2.Using immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis(2-DE),good reproducibility and images could be obtained to separate and identify the proteome in Eca-109 before and after irradiation.21 proteins were found differentially expressed.3.By mass spectrum identification and bioinformatics analysis,Most of the proteins were related with apoptosis regulation,cell cycle regulation,RNA transcription, signal transduction,cytoskeleton component and stress reaction induced by radiation. Those proteins may be the new potential target to increase the radiosensitivity. Proteomics offers new research threads and methods for radiotherapy.