Abstract
Project I: High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease Hyperglycemia can cause a variety of abnormal disorders in liver cells, one of these abnormalities is diabetic liver disease. Previous study has shown that high glucose concentration in blood can damage liver cells via glycoxidation. However, the details of molecular mechanisms underlying the effects of high glucose concentration in blood in the development of diabetic liver disease have not to be elucidated. In this study, we incubated a liver cell line (Chang liver cell) in mannitol-balanced 5.5 mM, 25 mM and 100 mM glucose media and assessed protein expressional levels and redox-regulations. We identified 141 proteins that showed significant alterations in protein expression and 29 proteins that showed significant alterations in thiol reactivity in response to high glucose concentration. According to the proteomic results, several proteins involved in transcription-control, signal transduction, redox regulation, and cytoskeleton regulation have showed significant alterations in expression. However, proteins involved in protein folding and gene regulation have displayed significant alterations in redox regulations, related to the thiol reactivity. Further analyses by utilizing clinical plasma specimens confirmed that the proteins showed type 2 diabetic liver disease-dependent changes, such as galectin-3, GRP-78, GSTP1, etc. In conclusion, in this study we used a comprehensive hepatocyte-based proteomic approach to identify the high glucose concentration-induced alterations in protein expressional level and to identify redox-associated diabetic liver disease markers. Several identified proteins were validated with clinical samples and might serve as potential targets for the prognosis and diagnosis of diabetic liver disease. Project II: Proteomic analysis of proteins responsible for the development of pemetrexed resistance in human lung adenocarcinoma Lung cancer occupies the top 7 leading causes of death in the world and is the top one deadliest cancer in domestic. However, cancer drug resistance is one of the major reasons which cause the failure of chemotherapy in lung cancer. To comprehend the more detailed mechanisms of drug resistance for lung cancer, we used pemetrexed-sensitive-A549 cells and pemetrexed-resistant A549/PEM cells to examine the pemetrexed-resistance-dependent cellular responses and to identify the potential therapeutic targets for drug resistance. We combined two-dimensional differential gel electrophoresis (2D-DIGE) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the global protein expression alterations induced by pemetrexed treatment and pemetrexed resistance. A proteomic study indicated that pemetrexed-exposure changed the expressions of 81 proteins in A549 cells, whereas no significant response took place in treated A549/PEM cells, indicating these proteins are associated with drug specific resistance. Moreover, 72 proteins demonstrated differentially expressional levels between A549 cells and A549/PEM cells regarding as baseline resistance. Further studies have used siRNA silencing to against calreticulin, flavin reductase and membrane-associated progesterone receptor component 1 (PGRMC1) proteins, to examine and evaluate their potency in the formation of pemetrexed resistance. The proteomic approach allowed us to identify numerous proteins which are involved in a variety of drug-resistance-forming mechanisms. In this study, we provide useful therapeutic candidates and diagnostic markers for the treatment of pemetrexed-resistant lung cancer.