| Objective:In this study, we assess the activities of lobaplatin alone or in combination with paclitaxel against human gastric cancer SGC- 7901 cell,and explore the underlying molecular mechanisms. The second purpose of this study was to investigate the expression AKAP95,cyclinE1,cyclinD1 and Cx43 in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins. At last,We identified whether the LMW-cyclin E2 is the real cyclinE.Methods:1. Using MTT method to detect lobaplatin and paclitaxel alone and in combination cytotoxic effect on gastric cancer SGC-7901 cell.2. The effect of lobaplatin and paclitaxel alone and in combination on gastric cancer SGC-7901 cell cycle distribution was determined by FCM analysis.3. Using Western Blot method to detect the expressions of cyclinD1, cyclinE1,cyclinB1, CDK2, CDK4, Rb and pRb.4. The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method.5. Using molecular cloning techniques to build cyclin E2 overexpression plasmid and silence plasmid.6. Using the methods of western blot and immunofluorescence assay to detect the location of cyclinE2 and LMW-cyclinE2,and detect both antibodies specific to proteins encoded by CCNE2.7. Using the methods of Co-IP, silver staining, MS / MS to identified cyclin E2(A-9) antibody protein corresponding to whether the protein encoded by the gene CCNE2.Results:1. Labaplatin combination with paclitaxel on gastric cancer SGC-7901 has a synergistic effec,CI<1.2. Labaplatin combination with paclitaxel increase the expression of cyclinE1,CDK2, Rb and pRb(P<0.05), reduce the expression of cyclinB1(P<0.05).3. The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30%(P<0.05);The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%,which was lower than that of ovarian pericarcinoma tissues expressing as 75%.4. The expression of cyclinD1 in ovarian cancer tissues was related to the type of tissues(P<0.05) while it showed no correlation with the degree of differentiation(P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the type of tissues(P>0.05).5. Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other(P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other(P<0.05). However, AKAP95 and cyclinD1 showed no correlation(P>0.05).6. cyclinE2 mainly located in cytoplasm, little expression in the nucleus, while the LMW-cyclinE2 mainly localized in the nucleus.7. LMW-cyclinE2 instesd of cyclinE2 is specific to the protein encoded by the gene CCNE2.8. The amino acid sequence encodedt by CCNE2 was not detected,using the antibody of cyclinE2(A-9) for Co-IP assy.Conclusion:1. Labaplatin combination with paclitaxel on gastric cancer SGC-7901 has a synergistic effect caused by the increasing cell cycle arrest.2. AKAP95, cyclinD1 and cyclinE1 played an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened.3. The cyclinE2 antibody(A-9) recognizes a unknown protein, this protein may not LMW-cyclinE2, cyclinE2 antibody(40890) recognized the real cyclinE2 protein. |
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