| Gastric cancer is one of the most common malignant tumors.As most of patients suffer from progressive cancer,chemotherapeutics plays an important role in comprehensive therapics.5-Fluorouracil(5-FU)and its derivants are widely used in clinic to treat progressive gastric cancer. However,a significant number of tumors often fail to respond to chemotherapy,because tumors become resistant to 5-FU and several other anticancer drugs after consecutive treatments.Therefore,it is very important to understand mechanism of 5-FU resistance of gastric cancer and find new resistance modifying agents(RMAs)for better treatment of tumors.Proteomics has been widespreadly implanted into every field of life science and medicine as an important part of post-genomics era research. The development of theory and technology in proteomics has provided new ideas and research fields for tumor multiple drug resistance.Two-dimensional gel electrophoresis with immobilized pH gradient was used to display and compare the differential expression of multidrug-resistant proteins in gastric cancer,cells BGC823 and BGC823/5-FU,which is in order to find the new multidrug-resistant proteins and make the foundation for further studying the mechanism of gastric cancer multidrug resistance.The major research procedures and results are as follows:1.Establishment of 5-fluorouracil-multidrug-resistant cell line BGC823/5-FU and its biological characteristicsTo investigate the mechanism of 5-FU resistance of gastric cancer,a 5-fluorouracil-resistant cell line from a Chinese gastric cancer was established by intermittent high dose 5-Fluorouracil induced,and it showed not only resistance to 5-FU but also cross- resistance to ADR,MMC and DDP.Compared with its parent cells,the morphological changes were observed by inverted phase contrast microscope.Flow cytometry was used to detect the expression of P-glycoprotein on the surface of the cells and the intracellular concentration of DNR,The expression of P-glycoprotein was increased and the accumulation of the intracellular DNR was decreased in BGC823/5-FU cell line;the expression of mdr1 gene was examined by reverse transcriptase-polymerase chain reaction(RT-PCR).Compared with its parent cells,the expression of mdr1 gene was significantly up-regulated in BGC823/5-FU cell line,suggesting that this subline was classic multidrug resistance and could be good experimental modal for study the mechanism of gastric cancer multidrug resistance.Apoptosis associated proteins were examined by Western-blotting. Compared with its parent cells,expressions of Bax and Caspase-3 were significantly down-regulated and bcl-2,survivin were obviously upregulated in BGC823/5-FU cell line.The present study might conduce to researches of the mechanism of 5-FU resistance and pave the way to exploit drug related to these genes and proteins.2.Diferencial display of proteins in gastric cancer cells BGC823 and 5-Fluorouracil-resistant BGC823 cel ls(BGC823/5-FU)The total proteins of BGC823 and BGC823/5-FU were extracted by chemistry lysis method and Bradford method was used to detect their concentrations.The total proteins of the two cell lines were separated by immobilized pH-gradient-based 2-DE and the spots of proteins were visualized by silver staining.The differentially expressed proteins were analyzed using ImagingMaster 2D Melanie 5.0 software. Results:(1282±36)and(1305±29)proteins pots were identified in the2-DE gel of 8GC823 and BGC823/5-FU individually.Thirty two proteins were seen to be unique high in one region or the others(27 in BGC823/5-FU cells,5 in BGC823 cells).There were 33 over expressed spots in the BGC823/5-FU cells,and 17 over expressied spots in the BGC823 cells. The whole-cell proteins of gastric cancer cells BGC823 and BGC823/5-FU could be well separated and displayed by two-dimensional electrophoresis. The results suggest that these diferential proteins be related to the 5-Fluorouracil-resistant mechanism in human gastric cancer cell line BGC823/5-FU.
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