Study On The Concentration-Dependent Mechanism Of Taxol-induced Anticancer Effect And It's Relationship With Spindle Checkpoint Protein Bub1

Partâ… :Research of the Effects of Taxol to Ovarian Cancer Cells and the Related Concentration-Dependent MechanismObjective To investigate the different mechanisms on the effect of taxol at different concentrations to ovarian cancer cells and the possible relativity of taxol-induced mitotic arrest with the spindle checkpoint protein Bub1. Method (1) human ovarian cancer cell lines A2780 were exposed to 50uM, 10uM , 1uM, 0.1uM and 0.01uM taxol. Fluorescence-activated cell sorting (FACS) was employed to examine the cell cycle distribution. (2) A2780 were exposed to 50uM, 1uM, 0.01uM taxol for 12, 24 and 24 hours respectively, the untreated cells was used as control. FACS was employed to examine cell cycle distribution. The differential expression of the Bub1 mRNA levels among the four groups was evaluated by RT-PCR. (3) After treated with 2mM thymidine for synchronization at G1-S early phase, A2780 cells were exposed to 0.01uM paclitaxel for 8 and 24 hours, followed by being analyzed with FACS for cell cycle distribution. (4) The ratio of the cells undergoing apoptosis or necrosis was analyzed by FACS with cells stained with Annexin V and PI to study the influence of 50uM taxol to cell necrosis. Result (1) when used at 10uM, 1uM, 0.1uM, taxol can cause a significantly higher percent of cells in G2/M than it's used at 50uM , 0.01uM (P50uM<0.001, P0.01uM<0.001). (2) The Bub1 protein level in A2780 cells treated with 1uM taxol is statistically higher than control and that in cells exposed to 50uM, 0.01uM taxol. (3) Neither G0/G1 arrest nor G2/M blockage was observed for a double time after synchronized A2780 cells were treated with 0.01uM paclitaxel. (4) The ratio of cells undergoing necrosis in A2780 cells treated with 50uM taxol is statistically higher than control and that in cells exposed to 1uM, 0.01uM taxol. Conclusion different concentrations of taxol employ different mechanisms to exert their anticancer effect. There's a positive correlation between taxol-mediated mitotic blockage and the spindle checkpoint protein Bub1. Partâ…¡: Dependence of Taxol-induced anticancer effect on Spindle Checkpoint Protein Bub1Objective To explore the role of spindle checkpoint protein Bub1 in taxol-induced mitotic arrest and anticancer effect. Method (1) A2780 and A2780/TS cells was exposed to a range of paclitaxel treatment concentrations for 24hr, followed by being analysed by FACS for the cell cycle distribution detected by MTT assay for cell survival. FACS was used to analyze apoptosis after A2780 and A2780/TS cells was exposed to 1uM for 24, 36, 48hr. (2) After synchronizing A2780 and A2780/TS cells with thymidine and taxol at G1/S early phase and G2/M phase, immunoblotting was employed to examine the differential expression of Bub1 at log phase, G1/S early phase and G2/M phase between A2780 and A2780/TS cells. (3) We constructed a small-hairpin-RNA-interfering plasmid targeting Bub1 (Bub1-shRNA) and applied this RNAi technique to achieve Bub1 knockdown of A2780 cells. RT-PCR and western-blot were utilized to identify the Bub1-knockdown effect. (4) Bub1-shRNA was transduced transiently to A2780 cells. Mitotic index was examined by staining cells with Hoechst 33342 to determine the integrity of the spindle checkpoint. The change of taxol-induced G2/M blockage and apoptosis after knockdown of Bub1 were analyzed by FACS and cell survival detected by MTT assays. Result (1) Taxol at 0.1, 1 and 10uM can induce a high percent of cells in G2/M phase and 1uM taxol induced the highest G2/M arrest among these three concentrations. At some concentrations taxol induced more cells in G2/M phase in A2780 cells than in A2780/TS cells. MTT assays and FACS analysis indicated that A2780 cells were significantly more sensitive than A2780/TS cells. (2) When A2780 andA2780/TS cells were synchronized at G2/M phase, the expressions of Bub1 in the two cells were higher than those of the control cells. Immunoblotting indicated that more Bub1 proteins were consistently detectable in A2780 cells than that of A2780/TS cells at log phase, G1-S early phase and G2/M phase. (3) The mRNA and protein level of Bub1 in cells transfected with Bub1-shRNA were significantly lower than control. (4) When A2780 cells were subjected to a down-regulation of Bub1 and followed by taxol treatment, the mitotic index diminished, the percent of cells in G2/M phase decreasedand the percent of cells in G1 and S phase raised, the proliferation inhibition rate was depressed,the taxol-induced apoptosis stained with PI reduced whereas the early apoptosis wasn't affected. Conclusion Taxol's induction of G2/M arrest is mediated by the spindle checkpoint protein Bub1. Knockdown of Bub1 can generate paclitaxel resistance.