The Role Of Spindle Checkpoint And Cell Cycle Associated Genes In Acquired Paclitaxel-resistance Of Ovarian Carcinoma Cells

Despite significant initial response rates for advanced ovarian carcinoma using paclitaxel-platinum combination chemotherapy, the vast majority of patients relapse and develop a drug-resistant disease with an overall 5-year survival of <50% . Acquired resistance to paclitaxel is one of the most significant reasons for its failure in chemotherapy. Previous studies have demonstrated that paclitaxel resistance is attributable to various mechanisms: increased drug efflux that results from up-regulation of membrane transporters (e.g., P-gp); alterations in the expression of β-tubulin isotypes; and changes in apoptotic regulatory proteins (e.g., Bcl-2). However, limited achievements have been obtained in the development of means to combat resistance according to the possible mechanisms. Thus, the molecular mechanism of paclitaxel resistance has yet to be precisely defined.When paclitaxel stabilizes microtubules and interferes with normal formation of mitotic spindle, the spindle checkpoint is activated to make cells arrest at mitosis and cause cell death subsequently. The spindle checkpoint, also known as the mitotic spindle assembly checkpoint, is the major cell cycle control mechanism in mitosis, which ensures proper chromosome segregation during mitosis by producing inhibitors of the anaphase-promoting complex/cyclosome (APC/C). This E3 ubiquitin ligase complex is required for the ubiquitination and destruction of securin and cyclin B1, prerequisites for anaphaseonset and mitotic exit. Several evolutionary conserved proteins including Bub1, Bub3, BubR1, Mad1, Mad2 and Mps1 are required for spindle checkpoint function.Recently, spindle checkpoint has been associated with sensitivity to spindle damaging anticancer drugs. For example, Sudo and colleagues showed that loss of the spindle checkpoint by transient siRNA transfection in MCF-7 cells suppressed paclitaxel-induced cell death. However, by inactivating the spindle checkpoint, histone deacetylase inhibitor trichostatin A (TSA) can potentiate the lethal effects of microtubule-disrupting drugs. In addition, Tao et al found that cells that can sustain prolonged (>48hr) spindle checkpoint signaling (e.g., HT29) are less susceptible to KSPIA (kinesin spindle protein inhibiting agents) and taxol-mediated killing. These contradictory and limited data require further study to determine whether cancers with a weakened or defective spindle checkpoint are more or less suitable for treatment with antimitotic drugs.In addition, cancer is frequently considered to be a disease of the cell cycle. It is now becoming apparent that conventional chemotherapy drugs exert their function ultimately via the cellular machinery that controls the cell cycle and the molecular pathways that mediate the sequent cell death. Thus, alterations of cell cycle control may be associated to chemotherapy. An understanding of the cell cycle is critical to understanding how new cancer treatment strategies can be designed. Global transcriptome analysis can be performed through the use of focused cDNA microarrays of cell cycle gene.The goal in this study is to investigate the mechanisms of acquired paclitaxel-resistance in ovarian carcinoma cells. Firstly, a paclitaxel-resistant ovarian carcinoma cell subline, SKOV3-TR30, was developed by moderate concentration and intermittent action combined with low concentration and continuously exposure to paclitaxel; and the role of alteration in P-glycoprotein (Pgp) expression and β-tubulin isotypes in the mechanisms of acquired paclitaxel-resistance in ovarian carcinoma was investigated. Secondly, to demonstrate the relationship between spindle checkpoint and acquired paclitaxel-resistance, we determined the competence of spindle checkpoint and the expression of major components of spindle checkpoint (Mad2, BubR1) in SKOV3-TR30 cells in a big way. Finally, we investigated the difference of expression profile of cell cycle associated genes in paclitaxel-resistant ovarian carcinoma cell subline SKOV3-TR30 and its parental cell line SKOV3.Part IEstablishment of a paclitaxel-resistant cell subline of human ovarian carcinoma and preliminary observation of its biology Objective: To establish a new paclitaxel-resistant cell subline of human ovarian carcinoma for the study on mechanisms of drug resistance. The role of alterations in P-glycoprotein (P-gp) expression and β-tubulin isotypes in the mechanisms of acquired paclitaxel-resistance in ovarian carcinoma cells were investigatedMethods: A paclitaxel-resistant human ovarian carcinoma cell subline (SKOV3-TR30) was induced by moderate concentration and intermittent action combined with low concentration and continuously exposure to paclitaxel. The resistance index was determined by MTT assay, the cell cycle was detected by flow cytometry. Cell morphology was observed using HE staining and electron microscopy. The expression of Pgp and β-tubulin isotypes of SKOV3-TR30 and its parental cell line were determined by immunocytochemistry and fluorescent real-time semiquantitative RT-PCR, respectively. Results: The SKOV3-TR30 cells were 27.5-fold resistant to paclitaxel compared with the parental cell line. Various cross-resistance to docetaxel, topotecan, adriamycin, and nocodazole, but not carboplatin, was shown. The doubling time, cell cycle and cell morphology of SKOV3-TR30 were inequable to those of the parental cell line. Comparing with SKOV3 cells, SKOV3-TR30 cells did not show any significant changes in the expression level of P-gp. The expression of β_I, β_II, β_III, β_IVb -tubulin mRNA isotypes were increased in SKOV3-TR30 cells When compared with the paclitaxel-sensitive parental cells, however the statistical difference was only seen in the alteration of β_I- and β_II -tubulin isotype. While the expression of β_IVa -tubulin mRNA isotype was decreased slightly.Conclusions: Paclitaxel-resistant cell subline SKOV3-TR30 is a good experimental model in vitro for the study on mechanisms of drug resistance and the screening of anti-tumor drug. Altered expressions of β-tubulin isotypes are associated with acquired paclitaxel-resistance in ovarian carcinoma cells. While P-gp does not play a major role in drug resistance of this paclitaxel-resistant cell subline.Part IIWeakened spindle checkpoint with reduced BubR1 expression inpaclitaxel-resistant ovarian carcinoma cell subline SKOV3-TR30Objective: Paclitaxel sensitivity has recently been associated with the spindle checkpoint. The aim of our study is to investigate the status of spindle checkpoint and the alteration of its major components in phenotype with acquired paclitaxel-resistance in ovarian carcinoma.Methods: The competence of spindle checkpoint in paclitaxel-resistant and paclitaxel-sensitive ovarian carcinoma cells was evaluated by mitotic index calculation, flow cytometric analysis, chromosome spread analysis and western blot determination of APC substrates. The expressions of the major spindle checkpoint proteins (BubR1 and Mad2) in SKOV3-TR30 and SKOV3 cells were investigated by western blot and immunocytochemistry.Results: 1) About 60% of SKOV3 cells were found to arrest (i.e. mitotic index 60%) in M-phase by 24 hr of treatment with paclitaxel. By contrast, more than 70% of SKOV3-TR30 cells failed to arrest (i.e. mitotic index <30%) in M-phase and exited mitosis. Similar results were observed after treatment with nocodazole, which prevents microtubule attachment to the kinetochores. 2) Consistently, flow cytometric analysis of SKOV3-TR30 cells showed a lack of G₂-M phase arrest after treatment with paclitaxel or nocodazole for 24 hr, in contrast to SKOV3 cells, which showed a clear G₂-M phase block. 3) After 5 hr treatment of paclitaxel and nocodazole, SKOV3 cells displayed a low rate of premature anaphases (4.7% and 5.3%, respectively). In contrast, SKOV3-TR30 cells showed much higher proportions of premature anaphases (30.5% and 26.8%, respectively). 4) After 24 hr of treatment with 100 nM paclitaxel or 50 nM nocodazole, western blot analysis revealed that SKOV3-TR 30 cells had significantly lower expression of APC substrates securin and cyclin B1 when compared with SKOV3 cells. 5) Western blot analysis showed that the expression of BubR1 protein was evidently lower in SKOV3-TR30 cells than that in SKOV3 cells (22.6%±10.2% vs 76.4%±9.1%, as ratio to β-actin, P<0.01). Immunocytochemistry also revealed that the BubR1 expression wassignificantly decreased in SKOV3-TR30 cells when compared with the paclitaxel-sensitive parental cells. However, there was no significant difference in Mad2 expression between SKOV3-TR30 and SKOV3 cells. 6) The expression levels of BubR1 in both SKOV3 and SKOV3-TR30 cells were significantly elevated after 24 hr exposure to 30 nM of paclitaxel.Conclusions: paclitaexl-resistant SKOV3-TR30 cell subline has a weakened spindle checkpoint in response to spindle poisons.Our result suggests that reduced BubR1 expression contributes to the weakened spindle checkpoint in SKOV3-TR30 cells. Weakening of spindle checkpoint induced by down-regulation of BubR1 may be one of the mechanisms of acquired paclitaxel-resistance in ovarian carcinoma cells. Part IIIThe difference of expression profile of cell cycle associated genes inSKOV3-TR30 cellsObjective: To investigate the difference of expression profile of cell cycle associated genes in paclitaxel-resistant ovarian carcinoma cell subline SKOV3-TR30 and its parental cell line SKOV3.Methods: By cDNA array chip containing 283 cell cycle associated genes, the gene expression pattern, of which paclitaxel-resistant and the parental ovarian cancer cell lines, were compared and analyzed. The expressions of different genes were validated by fluorescent real-time semi-quantitative RT-PCR.Results: A total of 41 genes expressed differentially were screened out in paclitaxel-resistant ovarian carcinoma cell subline. Among these genes, 6 were up-regulated and the other 35 were down-regulated. Three genes were identified as markedly differentially expressed (>5 fold), including CCNA1 (NM₀03914), GRB10 (NM₀05311), and GSK-3a (NM₀19884). Real-time RT-PCR proved that the expression level of GSK-3a in SKOV3-TR30 cell was markedly higher ( â–³ Ct 7.85±0.30 vs 10.96±1.00, P=0.007) than that in SKOV3 cell, and the expression level of GRB10 inSKOV3-TR30 cell was appreciably lower (â–³ Ct 9.85±0.56 vs 9.07±0.84, P=0.251) than that in SKOV3 cell. Western blot analysis showed that the expression of GSK-3 protein was evidently higher in SKOV3-TR30 cells than that in SKOV3 cells (172.2±36.2 vs 52.3±14.4, as ratio to β-actin, P=0.006). The expressions of GSK-3a and GSK-3β proteins were both significantly increased in SKOV3-TR30 cells when compared with SKOV3 cells (71.7±20.9 vs 19.4±13.8, P=0.022; 127.1±13.9 vs 51.0±9.0, P=0.001, respectively). Conclusions: There are differently expressed genes associated with cell cycle between paclitaxel-resistant ovarian carcinoma cell subline SKOV3-TR30 and its parental cell line SKOV3. Up-regulation of GSK-3 is a novel mechanism of of acquired paclitaxel-resistance in ovarian cancer cells.