| Gastric cancer is one of the most prevalent malignancies, and chemotherapy remains the conventional method in treating it. However, long-term exposure to chemotherapeu-tic drugs often confers tumor cells resistance to a number of diverse natural product drugs that do not share a common structure or target, which is termed as multidrug resistance (MDR). Resistance to chemotherapeutic drugs is the toughest problem in cancer management. MDR-related molecules identified so far (such as P-gp, MRP, LRP, BCRP, GST- , PKC and TopoII) can not explain all the MDR types of gastric cancer, indicating that there are unknown MDR-related molecules and mechanisms in gastric cancer. In our previous work, We found that the CacyBP gene was expressed higher in the gastric cancer drug-resistant cells than in the sensitive cells on mRNA level by subtractive hybridization.It was not clear whether the CacyBP played the role in the formation of MDR in the gastric cancer cells. It might be a novel modulator of MDR in the gastric cancer cells. So, the exploration of its function would help understanding the mechanisms of MDR and designing the methods to reverse the MDR in the gastric cancer.Objective: To express the recombinant CacyBP in E. coli and raise the polycloned antibody against CacyBP from mice, identify the expression and distribution of CacyBP in the SGC7901 and the SGC7901/ADR cells and investigate the function and possible mechanisms of CacyBP in the drug resistance. To study the expression of CacyBP in the gastric cancer and normal tissues.Methods: 1) The cDNA fragments of CacyBP gene were subcloned into prokaryotic expression vector; 2) The recombinat CacyBP protein was expressed under IPTG induction in E coli; 3) Prokaryotic expressed protein products were purified by Ni-NTA; 4) Using purified CacyBP as immunogen, mouse polyclonal antibody against CacyBP was prepared; 5) The antisera were characterized with purified CacyBP by Western blot; 6) The different expression of CacyBP in SGC7901 cells and SGC7901/ADR cells was detected by Western blot ; 7) The distribution of CacyBP in SGC7901cells and SGC7901/ADR cells was detected by immunofluorescence; 8) The Cytosolic Free Ca2+was measured by laser scanning cofocal microscope; 9) Tissue arrays of normal and malignant stomach tissues were stained with polyclonal antibody against CacyBP by immunohistochemical (IHC) method to study the expression and distribution patterns of CacyBP.Results: 1) The fusion protein was expressed in E. Coli and that comprises 10% of total bacterial protein; 2) The polyclonal antibody against CacyBP was successfully prepared with the purified recombinant CacyBP and recognized a special protein band of CacyBP; 3) The result of Western blot showed that the expression of CacyBP was higher in the SGC7901/ADR cells than in the SGC7901 cells in protein level; 4) Immunofluorescence show that CacyBP was present in cytoplasm of SGC7901 cells, while in SGC7901/ADR cells, CacyBP was present in nucleus; 5) The resting Ca2+ concentration was relatively lower in SGC7901 cells but was higher in SGC7901/ADR cells; 6) The results of IHC showed that 52 of 82 gastric cancer samples were stained positively by antibody against CacyBP(63%) and 46 of 82 normal stomach tissue samples were stained positively (56%). The difference of CacyBP expression between normal and cancertissues was of statistical significance (R<0. 05); 7) The difference of expression of CacyBP was not statistical significance in different pathological parameter, which included sex, age, tissue type, stage and differentiation.Conclusion: In the process of the formation of MDR in the gastric cancer cells, the expression level and subcellular localization of CacyBP have been changed. This change suggested that CacyBP might play the role in the formation of MDR in the gastric cancer cells and the function of CacyBP was related with variation of the Cytosolic Free Ca2+ The difference of CacyBP in the normal tissues and the gastric cancer tissues indicated that it might be a potent...
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