Studies Of Role And Mechanisms Of Sorcin On Multidrug Resistance Of Gastric Cancer

Gastric cancer is one of the leading causes of mortality in China. Chemotherapy is one of the main treatments of gastric cancer.However, dispite of the emergence of new reagents and schemes of chemotherapy, the clinical outcomes of chemotherapy have not made much progress. Multidrug resistance(MDR)in cancer represents a major obstacle to successful chemotherapies.To overcome MDR is a critical task in cancer chemotherapy.Successful development of effective MDR reversal agent depends on our understanding to the various molecules/pathways involved in the emergence of MDR phenotype.It is well known that some drug-resistanct-related molecules play important roles in MDR of gastric cancer,such as MDR1/P-gp,MRP, BCRP,Topoâ…¡and GST-Ï€.However,treatment of MDR cells with inhibitors of these molecules did not restore full sensitivity of gastric cancer cells to chemo-therapeutic drugs,which indicates that other mechanisms might be involved in the development of resistance to the drugs and it is necessary to detect new protein related to chemoresistance. We had examined the differences of the overall protein expression pattern between vincristine(VCR)-induced multidrug-resistant gastric cancer cells SGC7901/VCR and its parent SGC7901 cells using proteomics approaches.The result revealed that the expression of Sorcin,a calcium-binding protein,in SGC7901/VCR cells was significantly higher than that in its parent SGC7901 cells.Supression of Sorcin expression by Sorcin antisense oligonuceotides could enhance VCR chemosensitivity in SGC7901/VCR cells.These results suggested that high expression of Sorcin was tightly associated with chemoresistance of SGC7901/VCR. Sorcin was demonstrated to be overexpressed in MDR cell lines derived from selection against a variety of chemotherapeutic drugs.While the mechanisms of Sorcin in MDR have yet to be identified.Based on our previous studies,this study aim to elucidate the function and mechanisms of Sorcin encoded gene in MDR of gastric cancer,which will help us to fully understand the mechanisms of MDR in gastric cancer.ObsjectiveTo construct the FLAg-Sorcin fussion expression system;To explore the role of Sorcin in the development of MDR in human gastric cancer cell line SGC7901 and study the mechanisms of Sorcin encoded gene in MDR of gastric cancer.METHODS1.Construction of FLAg-Sorcin fussion expression plasmidThe full length Sorcin cDNA was isolated by reverse transcriptionpolymerase chain reaction(RT-PCR).The pcDNA3.1-Sorcin-FLAG plasmid was constructed using DNA recombination technique and verified by DNA sequencing.2.Establishment of Sorcin-overexpressing SGC7901 cell line and studyon the role of Sorcin on MDR of SGC7901 cellspcDNA3.1-Sorcin-FLAG plasmid was transfected into SGC7901 cells mediated by Lipofectamine.The mRNA and protein expression levels of Sorcin in stable clones were detected by RT-PCR and Western blot.The sensitivity of Sorcin-trasfected SGC7901 cells(SGC-F-Sor)to chemo-therapeutic drugs was detected using MTT assay.Then SGC-F-Sor cells were transfected with Sorcin antisense oligonucleotides (ASO).The VCR-sensitivity of ASO-transfected SGC-F-Sor cells was determined by MTT assay. 3.Investigation of mechanisms of MDR mediated by Sorcin in gastric cancer cellsThe level of P-glycoprotein(P-gp)expression in SGC7901-F-Sor cells was detected by Western blot and the reversing effection of verapami(VRP)was determined by MTT assay.The intracellular concentration of VCR and ADM in SGC-F-Sor cells and SGC7901 cells with or without VRP was measured by high performance liquid chromatography(HPLC).The role of Sorcin on apoptosis in SGC7901 cells was screened by FACS.RESULTS1.Construction of FLAg-Sorcin fussion expression plasmidThe full-length sorcin cDNA(616 bp)was amplified by RT-PCR and was subcloned to pcDNA3.1-p53-FLAGplasmid.DNA sequencing confirmed that the cloned gene segment was 100%homologous to the published sorcin sequence in genebank with a inserted FLAG sequence. pcDNA3.1-Sorcin-FLAG plasmid was constructed successfully.2.Establishment of Sorcin-overexpressing SGC7901 cell line and studyon the role of Sorcin on MDR of SGC7901 cellspcDNA3.1-Sorcin-FLAG plasmid was successfully transfected into SGC7901 cells mediated by lipofectamine.The mRNA and protein levels of sorcin were up-regulated in Sorcin-transfected SGC7901 cells, indicated that Sorcin-overexpressing SGC7901 cell line was established. MTT assay revealed that overexpression of Sorcin produced 8.87 folds of VCR-resistance,6.13 folds of adriamycin(ADM)-resistance,6.67 folds of taxol-resistance,and 2.80 folds of 5-fiolouracil(5-FU)-resistance. However,when SGC-F-Sor cells were transfected with Sorcin ASO, down-regulation of Sorcin expression and increased sensitivity to VCR were observed.3.Investigation of mechanisms of MDR mediated by Sorcin in gastric cancer cellsAs revealed by western blot,the protein level of P-gp in SGC7901 was increased after transfection of Sorcin gene.Results of verapamil block assay showed that the decreased sensitivity to chemotherapy drugs of SGC7901 cells transfected with Sorcin could be reversed by verapamil partially.Compared with the parent SGC7901 cells,the concentration of VCR and ADM in SGC-F-Sor cells was decreased by 76.89%and 80.62%,respectively.But when treated by VRP,the concentration of VCR increased by 2.41 times.The FACS results showed that overexprssion of Sorcin supressed the VCR-induced apoptosis of SGC7901 cells.CONCLUSIONSThe pcDNA3.1-Sorcin-FLAG plasmid was constructed successfully and Sorcin-overexpressing SGC7901 cell line was established. Overexpression of Sorcin could induce low-middle level of MDR in SGC7901 cells,indicating that Sorcin play an important role in the drug-resistant phenotype of human gastric cancer cells.The role of Sorcin in MDR of gastric cancer is associated with P-gp.Overexpression of Sorcin by gene transfection in SGC7901 cells resulted in decreased intracellular concentration of VCR.Furthermore,overexpression of Sorcin surpress the apoptosis of SGC7901 cells.