Comparative Proteomics Analysis Of Associated Molecular Changes Of Human Lung Adenocarcinoma A549 Cells Induced By Cigarette Smoking Extract

Aims:Cigarette smoking is the most established risk factor for lung carcinogenesis, it also have an important impact on prognosis and treatment of lung cancer. Current-smokers lung cancer patients tend to have a significantly worse survival rate than non-smokers, especially in adenocarcinoma patients, however, the cigarette smoking effects on the progression of lung cancer are still unclear. Harmful substances in cigarette smoke not only induce the bronchial epithelial cells to lung cancer, but also continue stimulating the cancer cells and cause the changes in expression level and structure of some genes in lung cancer cells. This changes is bound to affect changes in lung cancer cell proteomics, and further affect the biological characteristics of lung cancer cells. The proteomics approach provided us a new tool to study the prognosis mechanism of lung cancer, the current studies were focused on smoking affect a single or a few gene expressions in lung cancer patients, researches from the overall cell protein level were conducted less. Here we use proteomic tools to analysis the mechanism of cigarette smoking on the progression of lung cancer in vitro.Chapter one:Comparative proteomics analysis of associated molecular changes of human lung adenocarcinoma A549 cells induced by benzo(a)pyreneMethods:Levels of intracellular reactive oxygen species (ROS) and lipid peroxidation were investigated in A549 cells treated with varying concentrations of B(a)P. Comparative proteomics involving two-dimensional gel electrophoresis (2-DE) and MALDI-TOF Mass Spectrometry (MS) was performed on total proteins extracts from B(a)P-A549 and its control cell line DMSO-A549. Then the expression of heat shock protein 27(HSP27) and Mn superoxide dismutase (Mn SOD), two of the identified differential expressed protein, was analyzed using western blot. After 0.1?M,1?M,10?M B(a)P treatment, the Mn SOD expression levels were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, the total antioxidant capacity, total activity of SOD, catalase activity(CAT) and glutathione reductase activity (Gr) were determinned also.Results:Our results demonstrated that 1?M B(a)P could induce ROS generation and lead to lipid peroxidation in A549 cells. Comparative two-dimensional gel electrophoresis (2-DE) technology was performed to separate the total protein of B(a)P-A549 and DMSO-A549, respectively. The well-resolved, reproducible 2-DE patterns of B(a)P-A549 and DMSO-A549 were established. Then, PDQuest software was used to analyze 2-DE images, and the 23 differential expression proteins between the two cell lines were identified by peptide mass fingerprint (PMF) based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry). Western blot confirmed that B(a)P decreased heat shock protein 27 and Mn SOD expression levels in A549 cells. After 0.1?M, 1?M,10?M B(a)P treatment on A549 cells, the expression level of Mn SOD and SOD activity were significantly induced after 0.1?M B(a)P exposure and then reduced after 1?M and 10?M B(a)P exposure. With the increased concentration of B(a)P, the catalase activity, the total antioxidant capacity and the glutathione reductase activity increased.Conclusions:These findings indicate that HSP27 and Mn SOD may be related to the B(a)P mediated cell-injury of oxidative stress in A549 cells, and may serve as molecular biomarkers to monitor B(a)P cell-injury of oxidative stress. CAT and Gr may play a important role to antioxidant injury induced by B(a)P.Chapter two:Comparative proteomics analysis of associated molecular changes of human lung adenocarcinoma A549 cells induced by cigarette smoking extractMethods:We use cigarette smoking extract (CSE) treat A549 cells for 16 weeks to model the lung cancer patients who continue smoking (Current-smoking group), after 16 weeks of CSE treatment, the A549 cells were cultured in CSE-free medium for 8 weeks which were used to model the lung cancer patients who quit smoking (Ex-smoking group), and at the same time, we use the A549 cells were cultured in CSE-free medium to model the lung cancer patients who never smoking (Non-smoking group). Comparative proteomics involving two-dimensional gel electrophoresis (2-DE) and MALDI-TOF Mass Spectrometry (MS) was performed on total proteins extracts from Current-smoking group, Ex-smoking group and Non-smoking group cell lines. The influence of CSE on migration of A549 was evaluated by transwell and wound-healing assay. The expression levels of partial differential expressional proteins were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.Results: The well-resolved, reproducible 2-DE patterns of Current-smoking group, Ex-smoking group and Non-smoking group cell lines were established. Then, PDQuest software was used to analyze 2-DE images, and the 40 differential expression proteins between the three cell lines were identified by peptide mass fingerprint (PMF) based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry)). Among the 41 differential expression proteins, the expression levels of 21 proteins in Ex-smoking group did not return to the control level after remove CSE-treatment for 8 weeks, the expression levels of the remaining 19 proteins in Ex-smoking group return to the control level after remove CSE-treatment for 8 weeks. By bioinformatic analysis, we chose three differential expression proteins for further studies, including nm23-H1,14-3-3a and vimentin, which were related to the metastasis ability of cancer cells. Western-blot analysis and RT-PCR results show that the expressiong level of vimentin increased, nm23-H1 and 14-3-3?decreased after CSE treatment for 16 weeks. After CSE-free medium treatment for 8 weeks, the expression level of vimentin still in a high level, nm23-H1 and 14-3-3?still in a low level. These results were identical with the proteome analysis. Transwell and wound-healing assay results show that the invasion and metastasis ability increased in Current-smoking group and Ex-smoking group.Conclusions:We identified 40 differential expressional proteins by 2-DE in combination with MALDI-TOF-MS which may play important roles in the mechanism of cigarette smoking on the progression of lung cancer. Nm23-Hl and 14-3-3?down-regulation, vimentin up-regulation may related to the increased invasion and metastasis ability of A549 cells after CSE treatment.Chapter three:Clinicopathologic signicances of partial differential expressional proteins in non-small cell lung cancerMethods:Immunohistochemistry was performed to detect the expression levels of the partial differential expressional proteins in 125 cases of paraffin-embedded archival lung cancer tissue specimens, including 74 cases of squamous cell carcinoma and 51 cases of adenocarcinoma, and the correlation of their expression level with clinicopathologic features and clinical outcomes were evaluated.Results:Immunohistochemistry results show that the expression levels of vimentin, nm23-H1 and 14-3-3?were correlated with the smoking status, the expression levels of vimentin, nm23-H1 and 14-3-3?were correlated with the lymph node metastasis status (N-status). Kaplan-Meier survival curves show that patients with nm23-H1 and 14-3-3?down-regulation, vimentin up-regulation had a poor prognosis.Conclusions:Our results showed that three differential proteins (vimentin, nm23-H1 and 14-3-3?) are potential biomarkers for metastasis and prognosis of lung cancer.Overall conclusions:1) We identified 23 differential expressional proteins by proteomic analysis after treatment of B(a)P on A549 cells, HSP27 and Mn SOD may serve as molecular biomarkers to monitor B(a)P cell-injury of oxidative stress.2) With the increased concentration of B(a)P, the catalase activity, the total antioxidant capacity and the glutathione reductase activity increased. CAT and Gr may play a important role to antioxidant injury induced by B(a)P. 3) We identified 40 differential expressional proteins by proteomic analysis after treatment of CSE on A549 cells, these differential expressional proteins may play important roles in the mechanism of cigarette smoking on the progression of lung cancer.4) Our results showed that three differential proteins (vimentin, nm23-Hl and 14-3-3?) are potential biomarkers for metastasis and prognosis of lung cancer.