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Aurora激酶抑制剂VX-680对人膀胱癌T24细胞增殖和凋亡影响的实验研究

Studies on the Influence of Aurora Kinase Inhibitor VX-680 on the Proliferation and Apoptosis in Human Bladder Carcinoma Cell Line T24

【作者】 肖宁

【导师】 赵晓昆;

【作者基本信息】 中南大学 , 外科学, 2010, 博士

【摘要】 研究背景:膀胱癌在全球恶性肿瘤中位居第九位,其中在男性恶性肿瘤中居第七位,而女性中居第十七位,其中移行细胞癌(BTCC)占90%以上,给患者家庭和社会带来沉重的心理压力和经济负担。虽然在我国膀胱癌发病率远低于西方发达国家,但是近年来,我国部分城市膀胱癌发病率有增高趋势,且城市居民膀胱癌死亡率明显高于农村。非肌层浸润性膀胱癌行经尿道膀胱肿瘤电切术(TUR-BT)后并行膀胱内灌注化疗或免疫治疗后仍容易复发,易发展为肌层浸润性膀胱癌。而肌层浸润性膀胱癌,根治性手术难度高、并发症多以及辅助性化疗和放疗效果不佳。所以探索膀胱癌有效治疗的新方法一直是肿瘤治疗研究的热点。细胞增殖是高度有组织的时序调控过程。细胞进行有丝分裂必须依靠纺锤体检查点(spindle checkpoint),又称有丝分裂检查点(mitotic checkpoint)监控纺锤体形态、染色体着丝点与微管连接及其产生的张力、染色体排列,确保姐妹染色单体精确分离完成有丝分裂。纺锤体检查点功能缺陷将导致染色单体错误分配,产生非整倍体子代细胞,致使最终肿瘤发生。有丝分裂缺陷产生非整倍体导致的肿瘤在众多肿瘤发生中已得到证实。Aurora蛋白家族是一种丝/苏氨酸蛋白激酶,在细胞有丝分裂的进程中起重要作用,它包括Aurora A, Aurora B, Aurora C 3种,业已证明许多实体肿瘤都存在Aurora A的过度表达。目前正在开发的3个小分子Aurora激酶抑制剂(ZM447439,Hesperadin, VX-680)均非针对某一种Aurora激酶的特异性抑制剂。研究提示,VX-680对激酶的抑制作用更有临床应用作用。肿瘤细胞水平的实验证实VX-680可诱导多种肿瘤细胞凋亡,能抑制包括乳腺癌、淋巴瘤、前列腺癌、胰腺癌、黑色素瘤、颈部肿瘤等众多肿瘤细胞增殖。所以Aurora激酶抑制剂将有可能成为肿瘤治疗的有力武器。Aurora激酶抑制剂在治疗肿瘤中具有广阔前景,我们通过体外实验研究VX-680对人膀胱癌T24细胞的抑瘤作用,并从诱导细胞凋亡、调控细胞分裂和增殖等方面对其作用机理进行较深入的研究,为BTCC的治疗提供新的思路。第一章人膀胱移行细胞癌中Aurora A, CyclinD1, CyclinB1和Bcl-2的表达及其相关性的研究目的:探讨Aurora A, CyclinD1, CyclinB1和Bcl-2在BTCC中的表达及其意义方法:用免疫组化的方法检测Aurora A, CyclinD1, CyclinB1和Bcl-2在56例BTCC和15例正常膀胱组织中的表达情况,并分析其与BTCC临床病理因素的关系。结果:膀胱癌组织Aurora A阳性率为51.8%(29/56),高于正常膀胱组织的13.3%(2/15), (P<0.01); CyclinD1阳性率为46.4%(26/56),高于正常膀胱组织的6.7%(1/15), (P<0.01); CyclinB1阳性率为75.0%(42/56),高于正常膀胱组织的33.3%(5/15),(P<0.01);Bcl-2的表达为62.5%(35/56),高于正常膀胱组织的6.7%(1/15), (P<0.01)。Aurora A, CyclinB1和Bcl-2阳性率与BTCC的病理分级呈正相关,但是CyclinD1随病理分级增加而阳性率下降。Aurora A阳性率与临床分期呈正相关,CyclinD1则呈负相关;CyclinB1和Bcl-2阳性率与临床分期无关。Aurora A阳性率与CyclinD1 (R=0.508, P<0.01)、CyclinB1 (R=0.594, P<0.01)和Bcl-2 (R=-0.535, P<0.01)阳性率明显相关。结论:1.BTCC组织中Aurora A、CyclinD1、CyclinB1和Bcl-2的阳性率高于正常膀胱组织。2. BTCC组织中Aurora A、CyclinD1、CyclinB1和Bcl-2的阳性率高低与BTCC的肿瘤病理分级明显相关,病理分级与Aurora A、CyclinB1和Bcl-2阳性率呈正相关,与CyclinD1阳性率呈负相关。3. BTCC的肿瘤临床分期与Aurora A阳性率呈正相关,与CyclinD1阳性率呈负相关,与CyclinB1和Bcl-2阳性率与临床分期无关。第二章Aurora激酶抑制物VX-680对人膀胱癌T24细胞增殖和凋亡影响目的:探讨Aurora激酶抑制物VX-680对人膀胱癌T24细胞生物学行为的影响,即细胞增殖和细胞凋亡的影响。方法:设定不同浓度的Aurora激酶抑制物VX-680作用不同时间于T24细胞,利用MTT法检测细胞增殖,流式细胞术检测T24细胞的凋亡指数,Hochest染色观察细胞形态学变化。结果:Aurora激酶抑制物VX-680不同浓度(0nM,100nM,300nM,500nM)作用T24细胞于四个不同时间(12h,24h,48h,72h),MTT法检测细胞的生长抑制率,流式细胞术检测其凋亡率,Hochest染色观察细胞形态学变化,均发现呈明显的剂量效应关系和时间效应关系(P<0.05),分别在72h和500nM时其效应最明显,在72h时VX-680的IC50=761.222nM。MTT法检测72h时细胞存活率,VX-680浓度100nM:0.790±0.038,300nM:0.737±0.033,500nM:0.532±0.013,各浓度间均有差异(P<0.05)。流式细胞术检测其凋亡率,72h时VX-680浓度为0nM:(0.493±0.033)%,100nM:(6.608±0.550)%,300nM:(12.225±1.232)%,500nM:(18.275±4.986)%,各浓度组间两两相比差异性显著(P<0.01)。Hochest染色可见到不同程度的凋亡细胞,低剂量VX-680(100nM)处理细胞后,细胞凋亡较少,增加VX-680剂量(500nM),细胞凋亡明显增多,并且可见凋亡小体形成。结论:1.VX-680作用于人膀胱癌T24细胞后,对T24细胞的增殖有明显抑制作用,并且呈显著的剂量效应关系和时间效应关系。2.VX-680作用于人膀胱癌T24细胞后,明显诱导细胞凋亡,凋亡的诱导作用呈显著剂量效应关系和时间效应关系。第三章Aurora激酶抑制物VX-680抑制人膀胱癌T24细胞分子生物学机制的研究目的:探讨VX-680抑制人膀胱癌T24细胞的分子生物学机制。方法:设定不同浓度的VX-680作用不同时间于T24细胞,半定量RT-PCR检测T24细胞的Aurora A, CyclinD1, CyclinB1和Bcl-2mRNA的变化,Western blot检测Aurora A, CyclinD1, CyclinB1和Bcl-2蛋白的表达。结果:半定量RT-PCR检测,不同浓度(0nM,100nM,300nM,500nM) VX-680作用不同时间(12h,24h,48h,72h)于T24细胞后,降低Aurora A、CyclinD1、CyclinB1和Bcl-2 mRNA表达比值和其蛋白表达比值,并呈明显的时间效应关系和剂量效应关系。不同VX-680浓度作用72h时Aurora A mRNA表达比值(Aurora A/ GAPDH)分别为,0nM组:0.749±0.012,100nM组:0.638±0.014,300nM组:0.272±0.011,500nM组:0.253±0.003,各组间差异均有统计学意义(P<0.05); CyclinD1、CyclinB1和Bcl-2 mRNA表达比值在72h时也有类似趋势。Western blot检测,Aurora A、CyclinD1、CyclinB1和Bcl-2基因在蛋白表达水平。72h时各浓度组细胞Aurora A蛋白表达比值(Aurora A/GAPDH)分别为,0nM组:0.766±0.016,1O0nM:0.637±0.013,300nM:0.478±0.009,500nM:0.335±0.004,各组间均差异明显(P<0.01); CyclinD1、CyclinB1和Bcl-2 mRNA基因在蛋白表达比值在72h时也有类似趋势。但是短时间(12h)作用于T24细胞时,不同浓度VX-680(0 nM,100 nM,300 nM,500 nM)对Aurora A、CyclinD1、CyclinB1和Bcl-2mRNA表达比值下降不明显,各浓度组间均无统计学意义(P>0.05)。结论:1.VX-680引起的Aurora A、CyclinD1、CyclinB1和Bcl-2的表达下调可以从多个通路抑制细胞增殖和诱导凋亡,并呈剂量效应关系和时间效应关系。2. Aurora A与CyclinD1、CyclinB1和Bcl-2基因及蛋白的表达下调呈现明显相关性,他们之间可能存在相互调控。

【Abstract】 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.

  • 【网络出版投稿人】 中南大学
  • 【网络出版年期】2010年 11期
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