Correlation Of S100A6 To Multidrug Resistance Of Human Gastric Cancer Cell Line SGC7901

Background:Gastric carcinoma is the commonest malignant tumor of digestive tract and the average 5 year survival rate is about 15-20%. Chemotherapy,regarding as one of the chief therapeutic methods to the patients with gastric carcinoma of intermediate and advanced stage,relapse and metastasis after operation ,and with remnant gastric cancer, plays the important role in tumor therapy. But with the appliance of chemo- ther- apeutants, the multidrug resistance enhances increasingly.The occur- rence of multidrug resistance of cancer cells becomes the main factor that hinders the success of chemotherapy and will explore the wide perspective for tumor therapy. 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, the MDR mechanism could be studied from the whole protein level of tissue and cell.In order to find out new MDR related proteins of gastric cancer, two-dimentional gel electrophoresis(2-DE)was used to separate the total proteins of Adriamycin-resistant human gastric cancer cell line SGC7901/ADR,its parental cell line SGC7901,and the reversed cell line by Salvia miltiochiga.PDQuest software was applied to analyze 2-DE images, And the differential protein spots among the three cell lines were identified by HPLC-ESI-MS/MS.We found that,S100A6 is highly expressed in SGC7901,down-regulated in SGC7901/ADR,and up-regulated again after the reveral by Salvia miltiochiga.In this study,immunocytochemistry method,Western-blot analysis and RT-PCR technique were applied to detect the expressional levels of S100A6 in SGC7901 and SGC7901/ADR cells,which was to confirm the differential expression result found by pro- teomics and was also to further explore the role of S100A6 in the deve- lopment of MDR in human gastric cancer.Objective:Verify and detect the differential expression of S100A6 in SGC7901 and SGC7901/ADR , deeply explore the role of S100A6 in the development of MDR in human gastric cancer.Methods: First, the cell-chip technology was used to climb a certain concentration of cells cultured planting onto 0.8×0.8cm~2 pretreated covers- lips which was just in size to be put in 24-well plates and cultured for 48 hours, rapid immunohistochemistry method was adopted to observe the differential expression level of S100A6 in SGC7901 and SGC7901/ADR preliminarily; Chemistry lysis methods extracted the total proteins and RNA of SGC7901 and SGC7901/ADR, Western-blot analysis and quantitative RT-PCR technology were used from the protein level and the RNA level separately to detect the differences specifically, when to verify the results of comparative proteomics, meanwhile, also to further explore the correla- tion of S100A6 to multi-drug resistance of gastric cancer.Results: Immunocytochemistry method detected that S100A6 is showed strongly positive expression of brown-yellow color in SGC7901 cytoplasm and showed weak positive expression of light-yellow color in SGC7901/ADR cytoplasm; Western-blot result:The protein straps were analyzed for its IOD by BandScan5.0 software,the result is, SGC7901- S100A6 IOD (X|-):110,236,SGC7901/ADRS100A6 IOD(X|-):62.701,the aver- age expression amount of S100A6 in SGC7901 was 4.356±0.759,in SGC7901/ADR was 2.243±0.089,T-test was applied to analyze the dif- ferences,P﹤0.05;Semi-quantitative RT-PCR: The RT-PCR product was subjected to 2% agarose gel,BandScan5.0 Sofeware detected the gene straps IOD,SGC7901S100A6 mRNAIOD(X|-):16979,SGC7901/ADRS100A6 IOD(X|-): 11682,the average expression amount of S100A6 mRNA in SGC7901 is 0.659±0.017,in SGC7901/ADR is 0.582±0.06.T-test was adopted to analyze the differential expression,P﹥0.05.Conclusions:1. There is differential expression between SGC7901 and SGC7901/A DR on S100A6. It is highly expressed in drug-sensitive cell , and lowly ex- pressed in multidrug-resistant cell.2. No differential expression on S100A6 mRNA occurs between SGC- 7901 and SGC7901/ADR.The differential expression of S100A6 protein between the two cells begins from the post-translation level,which may be associated with the more degradation of S100A6 in SGC7901/ADR.3. The down-regulation of S100A6 protein in SGC7901/ADR may be associated with MDR of SGC7901/ADR.