| Background: gastric cancer is still the second most common cause of death from cancer in Asia and worldwide. It remains a great clinical challenge, due to its very poor prognosis. There are reports indicated that the prognosis of gastric cancer is not only relevant to proliferation, but also apoptosis of tumor itself. The poorly differentiated gastric carcinomas have escape apoptosis mechanism, and apoptosis inhibited may exert important role in invasion and metastasis of gastric carcinomas. Our preceding research suggested that there was a rapid proliferation and expansion of the GA in gastric mucinous adenocarcinomas with high secretion of hydratase and liability to invasion and metastasis, while it's hard to see GA in gastric tubular adenocarcinomas with no or little secretion of hydratase. Another report shows that the Golgi has altered structure and abnormal glycosylation in colorectal cancer. In addition, the transportation of Golgi appeared functional disturbance in gastric cancer. So we carried out our study using gastric cancer cells from the point of Golgi and hoped to find out the function of Golgi played in gastric cancer from protein level.The Golgi complex is a major organelle of the secretory pathway. The key function of it is to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. Nevertheless the Golgi complex has many other complex functions, such as cell polarization and differentiation, mitosis and intracellular signaling, as well as cell division. Recent datas show that the Golgi also plays an important role in apoptosis. These unresolved questions regarding nonclassical functions of the Golgi have stimulated us to do more research on this organelle with the goal of comprehensively identifying its protein components.Proteome studies have so far been developed with the technology. It includes two main technology approaches, one of which is using 1- or 2-DE for protein separation and MS for protein identification, and the other is the multidimensional protein identification technology (MudPIT) or'shotgun'proteomics approach. The second one enabled more comprehensive high-throughput profiling strategies of enriched organellar fractions, resulting in hundreds of proteins identifications. It is very feasible to build up a holistic picture of research material and discover new proteins. However the traditional and the most used method is the first one, because the reproducible two-dimensional (2D) gel systems can give the direct picture about the protein complements. Highly reproducible 2D gels allow the one-time identification of a protein spot from a complex or organelle without the requirement of any prior knowledge about the protein being examined. Furthermore, it is possible to begin to address functional questions by comparative analyses of proteomes obtained for organelles during the change of functional states.In the present study,we used the organellar proteomic analysis to study gastric cancer cells with different differentiation for the first time. The protein complements of purified golgi fractions isolated from well-differentiated gastric cancer cell line MKN-28 and poorly differentiated gastric cancer cell line SGC-7901 respectively were separated in 2-DE gels first, then about 100 proteins different expressed in both pictures were found, and 39 proteins were indentified using MS at last. These proteins have different functions, involving in cell division, transportation, apoptosis, and play a role in the differentiation and development of gastric cancer and so on. It is the first time to open the door which would guide us to understand the relationship between Golgi and gastric cancer and it would help us to do more research for better knowing the function of Golgi in gastric cancer.The research includes three parts:Part 1: The Isolation and Assessment of Golgi Apparatus from Gastric Cancer CellsObjective: To isolate and purify the Golgi apparatus from Gastric Cancer Cells.Methods: A large number of gastric cancer cells SGC7901 were cultivated in vitro, then Golgi apparatus were isolated from the cells by differential centrifugation combined with sucrose density gradient ultra-centrifugation. Its purity was characterized biochemically by enzymatic assays, morphologically by electron microscopy (EM) and neutral red supravital staining.Results: Electron microscopy detection showed that the majorities of product were Golgi apparatus. The fraction was stained red by neutral red. Marker enzyme analysis demonstrated that TPP activity increased to 97-fold, and the relative specific activity of galactosyltransferase was 107-fold enriched over the homogenate.Conclusion: The first successful isolation of Golgi apparatus from gastric cancer cells by using ultra- centrifugation will lead to do more research into the function of GA.Part 2: Two-dimensional gel electrophoresis of Golgi apparatus isolated from the well-, and poor-differentiated gastric cancer cells respectively.Objective: To establish a relatively perfect, reproducible and high-resolution technique of two-dimensional electrophoresis (2-DE) for Golgi apparatus. Compare these two pictures to find out the main proteins expressed differently between these two cells.Methods: To Optimize the condition of two-dimensional electrophoresis(2-DE) by choosing the best parameters of sample preparation,the amount of sample, the focal condition, and the focal setting instrument, and so on. The proteins of GA were seperated by 2-DE. Electrophoresis patterns were analyzed by imagemaster software.Results: High-resolution electrophoregrams were obtained and were repeatable. Using pH 3-10 non-liner strip and 12% SDS-PAGE, almost 800 spots can be obtained from 2-DE map of GA. After silver stained, 769±22 spots are detected in the GA of well-differentiated gastric cancer cells, while 837±43 spots are identified in the GA of poorly-differentiated gastric cancer cells. Compare these two pictures, we can see that the spot distribution patterns are similar between them, but the differences are obvious between them, ans the differentiated spots are numbered.Conclusion: Protein patterns of GA from well-, and poor-differentiated gastric cancer cells were obtained by using ultra-centrifugation and 2-DE. The first installment of the proteins' profile of GA from gastric cancer cells will benefit further investigation on Golgi function in gastric cancer.Part 3: The main differentiated spots of GA between the well-, and poor-differentiated gastric cancer cells were identified by MALDI—TOF MS and the results were verified by Western bolt.Objective: To identify the differential expression proteins by using mass spectrum. These identified proteins have close relationship with proliferation and differentiation of gastric cancer cells, which will help explaining the initiation and development mechanism of gastric cancer at Golgi apparatus level and providing another way to do more reaserch on GA in gastric cancer. Methods: The differentiated spots are excised from the gels, digested by trypsin, and analyzed by MALDI-TOF MS. The peptide mass fingerprint (PMF) of the differentiated spots was obtained, and these datas were searched in MASCOT on line. At last 39 proteins differentially expressed were identified by tandem mass spectrometry. The TCP-1 and Annexin 2 were verified by Western bolt.Results: 39 proteins differentially expressed in GF between SGC-7901 and MKN-28 were identified by tandem mass spectrometry. Based on the main functions of these proteins, they were classified as protein synthesis- and folding-related proteins, membrane fusion protein, cytoskeleton proteins, metabolic enzymes, apoptosis/tumorgenesis-related proteins, transcription-and translation-related proteins, angiogenesis/metastasis-related proteins, and cell communication/signal transduction-related proteins, and et cl. To affirm the result of identification, TCP-1 and Annexin 2 were verified by Western bolt. And the results of Western bolt were perfect matched with the results of 2-DE.Conclusion: by a serize of proteomic methods, we found that the contant of apoptosis proteins was higer in GA of well-differentiated gastric cancer cells compared with poor-differentiated gastric cancer cells, while the proteins relate to cell differentiation and proliferation were expressed highly in GA of poor-differentiated gastric cancer cells. Such proteins change on the GA, suggested that regulation of these proteins by GA can influence the differentiation and proliferation of gastric cancer. So this fist report on proteomic analysis of GA isolated between well-, and poor-differentiated gastric cancer cells will benefit further investigation on Golgi function in gastric cancer. |
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