Studies On The Influence Of Aurora Kinase Inhibitor VX-680 On The Proliferation And Apoptosis In Human Bladder Carcinoma Cell Line T24

Studies on the Influence of Aurora kinase inhibitor VX-680 on the proliferation and apoptosis in Human Bladder Carcinoma Cell Line T24Background:Urinary bladder cancer ranks ninth in worldwide cancer incidence, while it ranks seventh in male cancer incidence and ranks seventeenth in female. More than 90% of UBC is bladder transitional cell carcinoma(BTCC), from which the patients families and society suffer heavy burden. In china, the incidence of the UBC has been increasing in some cities, that is no less than it in rural areas of china and is no more than western countries in the world. Superficial bladder cancer have a tendency to recurrence after posteroperative chemotherapy and immunotherapy and progression to muscle-invasive bladder cancer. Sugery therapy, radical cystoectomy, is associated severe complications, while neoadjuvant chemotherapy and radiotherapy have a poor outcome. To explore the new treatment to UBC is always a focus in cancer research.Proliferation is organic modulation of cell. Mitotic is dependent on spindle checkpoint, which inspection of spindle shape,centromere connection to microtubule and tension,alignment of chromosome, to ensure that sister chromosome can separate into daughter cell accurately. It is proved that faults of mitotic checkpoint result to aneuploid in daughter cell, which can lead to carcinogenesis in various cancers. Aurora A is one of three serine/threonine kinases(A, B and C) that are evolutionally conserved and regulate mitotic progression in various organisms, Aurora kinases is overexpressed in various cancers. To date, a growing number of inhibitors of Aurora kinases have been described including Hesperadin, ZM447439, and VX-680, whereas VX-680 inhibits all three family members. VX-680 can block cell-cycle progression and induces apoptosis in diverse range of human tumor types, such as breast cancer, lymphoma, prostate cancer, pancreatic cancer. So VX-680 may be a potent therapy of carcinoma. We evaluated the in vitro effects and mechanism on T24 cells of VX-680 involved in proliferation and apoptosis, and provided a new idea to therapy of tumor. Part IExpression of Aurora A, CyclinDl,CyclinBl and Bcl-2 and its correlation in BTCCObjective To investigate the expression of Aurora A, CyclinDl, CyclinB 1 and Bcl-2 in BTCC with regard to the clinical significance.Methods Expressions of Aurora A, CyclinD1, CyclinB 1 and Bcl-2 were detected by immmunohistochemical staining in 56 cases of BTCC and 15 cases of normal bladder tissues as controls. The relevance between the expressions of Aurora A, CyclinD1, CyclinB 1 and Bcl-2 and the clinical pathology of the patients of BTCC were studied.Results Immmunohistochemical staining demonstrated that the positive rate of Aurora A, CyclinD1,CyclinB 1 and Bcl-2 respectively was 51.8%(29/56),46.4%(26/56),75.0%(42/56) and 62.5%(35/56)in BTCC, which is significantly higher than normal bladder tissues as 13.3%(2/15), 6.7%(1/15),33.3%(5/15) and 6.7%(1/15) (P<0.01) respectively. The positive rate of Aurora A, CyclinB 1 and Bcl-2 were significantly positive correlated with the grade of the tumor (P<0.05), but CyclinD1 with negative correlation. And no correlation was found between The positive rate of CyclinB 1,Bcl-2 and clinical staging, but Aurora A with positive correlation and Bcl-2 with negative one. There is a significantly verse relationship between the positive rate of Aurora A and CyclinD1(P< 0.01), but a positive correlation between Aurora A and CyclinB1,Bcl-2(P＜0.01).Conclusion1. The expression of Aurora A, CyclinD1, CyclinB 1 and Bcl-2 was significantly increasing in BTCC tissues compared to normal bladder tissues.2. The expression of Aurora A, CyclinD1, CyclinB1 and Bcl-2 significantly correlated with the grade of the tumor, Aurora A, CyclinB1 and Bcl-2 with positive correlation and CyclinDl with negative one.3. The expression of CyclinB1 and Bcl-2 was not relevant with the clinical staging, but Aurora A with positive relation and CyclinD1 with negative one. PartⅡEffects of VX-680 on proliferation and apoptosis of human bladder cancer cell line T24Objective To explore the effects of VX-680 on biological behavior of T24 cells, including proliferation inhibition and cell apoptosis.Methods The effect of VX-680 on human bladder cancer T24 cells was evaluated with various concentration of VX-680 treatment for various phase by MTT assay. The cell apoptosis of human bladder cancer T24 cells was determined with flow cytometry and the apoptosis index was detected. The shape change of T24 cell was inspected by hoechst staining.Results The dose-dependent manner and time-dependent manner was found, significantly, between the detection of proliferation inhibition,apoptosis,Hochest staining of T24 cell and Aurora kinases inhibitor VX=680 (P< 0.05).Both effects of VX-680 can reach to the climax in a concentration of 500nM or in a time of 72h. The IC50 of VX-680 may be 761.222nM in 72h. The survival rate, significantly decreasing with increasing concentration in 72h (P< 0.05), of T24 cell, 100nM:0.790±0.038; 300nM:0.737±0.033; 500nM:0.532±0.013. The apoptosis index, significantly increasing with elevating concentration in 72h(P<0.01), of T24 cell, OnM:(0.493±0.033)%, 100nM: (6.608±0.550)%,300nM:(12.225±1.232)%,500nM:(18.275+4.986)%. Observed by hoechst staining, the apoptotic cells is gradually increasing with elevating concentration of VX-680, the apoptotic body found in high concentration.Conclusion Our data suggested VX-680 treatment lead to proliferation inhibition and induces apoptosis of the human bladder T24 cell line, in a significant dose-dependent and time-dependent manner. PartⅢStudy on the molecular biological mechanism of VX-680 in human bladder cancer cell line T24Objective To investigate the molecular biological mechanism of VX-680, by which cell apoptosis and proliferation inhibition found in human bladder cancer cell line T24.Methods The effect of VX-680 on human bladder cancer T24 cells was assessed with varying concentration of VX-680 treatment for different phase. Semi-quantitive RT-PCR and Western blot were used to detect the expression of Aurora A, CyclinD1, CyclinB1 and Bcl-2 respectively.Results The dose-dependent manner and time-dependent manner was found, significantly, in down-regulated expression of Aurora A,CyclinD1,CyclinB1 and Bcl-2 related to the various concentration(OnM, 100nM,300nM,500nM) of VX-680 or treatment time (12h,24h,48h, 72h) by Semi-quantitive RT-PCR and Western blot. The expression ratio of Aurora A mRNA(Aurora A/GAPDH) in 72h was 0.749±0.012(0nM), 0.638±0.014(100nM),0.272±0.011(300nM),0.253±0.003(500nM), VX-680 down-regulated expression of Aurora A mRNA in Semi-quantitive RT-PCR tests (P<0.05). The expression ratio of Aurora A protein(Aurora A/GAPDH) in 72h was 0.766±0.016(0nM), 0.637±0.013(100nM),0.478±0.009(300nM),0.335±0.004(500nM), VX-680 significantly down-regulated expression of Aurora A protein in Western blot tests (P<0.01). It is also found that VX-680 down-regulated the expression of CyclinD1,CyclinB1 and Bcl-2 in similar to it of Aurora A in both tests, Semi-quantitive RT-PCR and Western blot(P<0.05). However, no significant decreasing expression of Aurora A,CyclinD1,CyclinB1 and Bcl-2mRNA, respectively, in treatment time of 12h in Semi-quantitive RT-PCR tests.Conclusion1. VX-680 can inhibit proliferation and induce apoptosis of T24 cell, by means of down-regulating Aurora A,CyclinD1,CyclinB1 and Bcl-2 through multiple pathway.2. The significant correlation found between down-regulated expression of Aurora A and it of CyclinD1,CyclinB1,Bcl-2 respectively, which may be regulated each other.