| Background and objective:Gastric cancer (GC) is one of the most common cancer worldwide.The effect of chemotherapy in advanced GC is only 20%-40%. Multidrug resistance (MDR) is one of the major problems preventing the chemotherapeutic efficacy of GC. To over-come MDR is a critical task in cancer chemotherapy. Even though lots of mechanisms of MDR have been found recent years, some new moleculars related to MDR have been found, such as ribosome protein S13, ZNRD1 and so on, the mechanisms of MDR have not been fully elucidated yet; treatment of MDR cells with inhibitors of the known molecules did not restore full sensitivity of gastric cancer cells to chemotherapeutic drugs, which indicates that there is some new mechanisms of MDR in gastric cancer. So it is necessary to detect new mechanisms and proteins related to chemoresistance.We had confirmed that the expression of Sorcin, a Calcium-binding protein, in SGC7901/VCR cells was significantly higher than that in its parent SGC7901 cells for the first time. Supression of Sorcin expression by Sorcin antisense oligonuceotides could partially reverse MDR. However, the exact mechanisms of Sorcin in MDR is still unknown. With the development of Proteomics, more and more research indicated that cellular and molecular functions and vital movements are almost carried out through complex or even network referred a great deal of proteins. Protein-protein interactions have been researched extensively recent years. Based on our previous studies, this study aim to identify and validate proteins interacting with Sorcin, elucidate the mechanisms of Sorcin encoded gene in MDR of gastric cancer, and to search some new targets related to chemoresistance meanwhile.Methods:(1) Firstly, pcDNA3.1-Sorcin-FLAG plasmid was transfected into HEK293T cells mediated by Lipofectamine. The mRNA and protein expression levels of Sorcin were detected by RT-PCR and Western blotting. Then, the proteins extracted from these cells were purified by anti-FLAG M2 affinity gel, prefractionated by one dimensional SDS-polyacrylamide gel electrophoresis (1D-SDS-PAGE). The fractionated proteins were trypsined and then analyzed by ESI-Q-TOF/MS; (2) Coimmunoprecipitation coupled with Western blotting were done to confirm the partial Sorcin-interacting proteins, such as HSP70,GSTP1,Tubulin, in the SGC7901/VCR cells; The mRNA and protein expression levels of HSP70 in SGC7901/VCR and SGC7901 cells were detected by RT-PCR and Western blotting; Suppressing HSP70 expression in SGC7901/VCR by HSP70 small interfering RNA(siRNA), then deal with VCR of different concentration, Proliferation of cells was detected by MTT assay; cell apoptosis was detected by Hoechest 33258 staining and flow cytometry; The mRNA expression levels of bcl-2 and bax were detected by RT-PCR.Results:(1) A total of 72 Sorcin-interacting proteins were identified, which could be classified into seven categories based on their functions: metabolic enzymes, molecular chaperone, enzyme related to biological oxidation, cytoskeleton organization, proteins relative to signal transduction, proteins related to enzymolysis and proteins of unknown functions; (2) HSP70,Tubulin,GSTP1 were detected in the immune complex of Sorcin, which were consistent with the results of mass spectrum analysis; The mRNA and protein levels of HSP70 were up-regulated in SGC7901/VCR cells compared with SGC7901; The mRNA and protein levels of HSP70 were obviously down-regulated in SGC7901/VCR cells indicated that HSP70-suppressing SGC7901/VCR cell line was established; Compared with control groups, after treated by Vincristine (VCR), cell apoptosis of HSP70 siRNA group was increased detected by MTT assay, Hoechest 33258 staining and flow cytometry; In SGC7901 and HSP70-suppressing SGC7901/VCR cell line groups, bcl-2 mRNA was down-regulated by 42.7% and 28.6% respectively, while the expression of bax mRNA did not change significantly after VCR treatment detected by RT-PCR.Conclusions:(1) Successfully separate and identified 72 proteins interacting with Sorcin; (2) The result of coimmunoprecipitation coupled with Western blotting confirmed that HSP70, GSTP1 and Tubulin were interacted with Sorcin, which were consistent with MS data; (3) HSP70 was up-regulated in SGC7901/VCR cells compared with SGC7901; The ratio of the expression of bax/Bcl-2 was up-regulated accordingly with suppression of HSP70 in SGC7901/VCR by HSP70 siRNA, which increased the VCR-induced apoptosis and the sensibility to anti-cancer drug of SGC7901/VCR cells. HSP70 is hopefully to be a new target protein for reversing MDR.
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