The Mechanisms Associated With Chemoresistance In Ovarian Cancer Cells

Background and ObjectiveThe spindle checkpoint, known as a surveillance mechanism, is crucial for ensuring the chromosome segregation process. Past studies indicated that the spindle checkpoint controls the cell cycle. The well-characterized components of the spindle checkpoint include Bub1, Bub2, Bub3, Mps1, Mad1, Mad2 and Mad3. The family of Mad (mitotic arrest deficient) and Bub (budding uninhibited by benzimidazole) proteins were identified by yeast mutagenesis screens for mutants unable to survive a temporary exposure to microtubule toxin. Bub1 is the paltform of spindle checkpoint. There are many controversial issues on the spindle checkpoint. The innner mechanisms are yet waiting to be explored. Paclitaxel succeeded in the treatment of solid tumors, including breast cancer, ovarian cancer and so on. Paclitaxel is a microtube stable reagent, which bloking cell cycle to kill the tumor. Some studies demonsteated that the killing effects depend on a functional spindle checkpoint. The mutant is rare, but the of spindle checkpoint often occour. There are no reports on the Bub1-mediated chemoresistance at home. Both of the efficiency and time length of siRNA is not enough. The pEGFPBub1-shRNA plasmid we used can obviously enhance the silent efficiency, supply powerful tool to probe the mechanisms of paclitaxel-resistance in ovarian cancer cells.Methods1. The immunohistochemistry staining of Bub 1;2. The construction of full-length and pEGFP-Bub1-shRNA plasmids;3. Apply paclitaxel with different concentration gradient to affect tumour cells, choose the concentration when the rate of apoptosis and G2/M was highest and without obvious apoptosis;4. Apply MTT and FACS method to test the apoptosis and cell cycle of tumour cells;5. Apply Hoechst33342 staining method to test the cell mitosis and apoptosis.ResultsAfter the treatment of certain paclitaxel, the rate of proliferation inhibition and apoptosis increased. The volume of apoptosis cell shrunk, the links dissolved that isolate from cells around, and following that, the density of cytoplasm increased and the nucleoplasm to condense. The cell cycle of apoptotic cells mainly in G2/M phase. The drug-sensitivity of A2780 and SKOV3 reduced after transfected with pEGFP-Bubl-shRNA plasmid.ConclusionBoth A2780 and SKOV3 cells could be induced into apoptosis by a certain dose paclitaxel. The cell apoptosis and arrest induced by paclitaxel could be reversed by transfecting with pEGFP-Bubl-shRNA plasmid. Background and ObjectiveRecent evidence has suggested that B2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and controlling the cell cycle. In addition, some scholars suggested that the PTX sensitivity dependent on a functional spindle assembly checkpoint. Bub1, as a platform protein, considered as an important component of spindle checkpoint. In the present study, we investigated the role of the B2/Bub1 cross-talking in apoptosis and cell cycle when exposure to PTX.MethodsIn order to explore the relationship between the Bubl and A/B pathway, recombinant expression plasmid WT-B2 and pEGFP-Bub1-shRNA were transfected into A2780 cells by lipofectamine2000, and then detected the expression level of Bub1,B2及p-B gene by using RT-PCR and western blot. Cell apoptosis and cycle were measured by flow cytometry and Hoechst33342 after treatment with PTX, AI and BI. In addition, we used CO-IP to find the ralated protein combined with Bibl.ResultsOur data showed that up-regulation of B2 contributed ovarian cancer cells to override PTX-induced G2/M arrest, and inhibited Bubl expression. The expression level of P-B is higher in pEGFP-Bubl-shRNA/A2780 cells than control cells. The result of CO-IP indicated that Bub1 protein may combine with p-B. Our findings will aid in understanding the molecular mechanism of PTX-induced resistance in ovarian cancer and facilitate the development of novel anti-neoplastic strategies.ConclusionThere are intimate relationships between Bub1 and B. This study has expanded the knowledge on regulation of cell cycle and apoptotic mechanisms, and may supply new targets. Background and ObjectiveMalignant tumors have almost individually unpredictable behavior and aggressiveness, therefore, a better insight in the molecular biological defects, which are responsible for initiation and progressive aggressiveness of malignant tumors, is necessary. Proteomics are an alternative to identify proteins which initiate carcinogenesis and can be useful to predict cancer prognosis. Today, the most commonly used technique for large-scale protein identification in clinical samples is two-dimensional electrophoresis (2-DE) in combination with image analysis and MS. Using these techniques, qualitative and quantitative information can be achieved regarding protein forms and post-translational modifications.Methods1. extracted total protein.2. Screened differential expression protein between C13K, OV2008, A2780, A2780/DDP,A2780/Taxol cells through two-dimensional gel electrophoresis proteomics technology. Repeated the experiment three times and picked out the special points for the follow-up MALDI-TOF-MS analysis.ResultsA total of 502±30 points were detected in C13K cells, while there were 430±21 points detected in OV2008 cells through two-dimensional gel electrophoresis. Selected 20 significantly differential expression protein spots for the follow-up MALDI-TOF MS analysis. ConclusionAs one of effective technique for isolating the different expression protein, two-dimensional gel electrophoresis can be successfully used for screening new chemoresistance-associated genes.