【作者】 伍俊；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 呼吸病学， 2010， 博士

【摘要】 研究背景肺癌是全球最常见的恶性肿瘤之一,严重危害人类健康,其中大部分为非小细胞肺癌,约占80%。早期手术治疗是首选的治疗方法,但是由于多数病人在早期不易诊断,就诊时已是中晚期以及术后存在高的复发率,化疗仍是主要的治疗方法。以铂类药物为基础的联合化疗是非小细胞肺癌目前一线化疗方案,然而由于铂类药物的毒副作用和肺癌耐药的出现,限制了铂类药物的广泛应用。因此,寻找有效的方法和药物逆转肺癌耐药具有十分重要的意义。研究表明,顺铂的耐药性是由多因素,多因子参与的,存在多种机制,包括药物的蓄积降低、巯基分子如谷胱甘肽(GSH)的过度流入及其钝化作用、DNA修复能力增强以及细胞凋亡的抑制等。肺癌对顺铂的化疗敏感性改变可能与细胞周期管理基因、凋亡相关基因的表达改变有关。表观遗传修饰的分子机制主要包括DNA甲基化及组蛋白乙酰化,它们在基因表达调控起着重要的作用。表观遗传修饰的改变与肿瘤的发生发展密切相关,越来越多的证据表明,它们在肿瘤耐药中起着重要的作用。组蛋白去乙酰化酶抑制剂能抑制组蛋白乙酰化酶的活性,促进基因转录能诱导肿瘤细胞生长抑制、分化、凋亡,具有较广泛的应用前景。曲古抑素A(Trichostatin A,曲古抑素A)是组蛋白去乙酰化酶抑制剂的经典化合物之一,最初是作为一种抗真菌药物,能抑制Ⅰ、Ⅱ类组蛋白去乙酰化酶,研究发现曲古抑素A诱导多种肿瘤细胞分化促进肿瘤细胞凋亡,并且对正常细胞毒性较低,因而被认为是一种有希望的新的抗癌药物。新近研究发现组蛋白去乙酰化酶抑制剂能增强化疗或放疗的敏感性,具体机制仍待进一步研究。本实验拟以体外培养人肺腺癌A549细胞及其耐顺铂细胞株A549/CDDP细胞为研究对象,探讨曲古抑素A能否促进A549/CDDP细胞凋亡及提高其对顺铂的敏感性,并对其作用机制进一步研究,为临床肺癌的治疗寻找新途径和提供实验基础。目的研究曲古抑素A能否诱导人肺腺癌耐顺铂细胞株凋亡并增强对顺铂的敏感性。方法1.常规培养A549、A549/CDDP细胞株,用中性红方法检测耐药指数。2曲古抑素A处理A549/CDDP细胞株3.荧光染色、流式细胞仪检测细胞凋亡,流式细胞仪分析细胞周期及线粒体膜电位的变化。4.曲古抑素A联合顺铂处理A549/CDDP细胞株。结果1.A549/CDDP细胞耐药指数为12.8。2.TSA对A549/CDDP细胞的IC50是446.59±27.32 nmol/L,随着曲古抑素A浓度升高,A549/CDDP细胞生长率明显下降。3.曲古抑素A处理A549/CDDP细胞株后,荧光染色主要表现为核染色质固缩,荧光染色增强。流式细胞仪检测亚二倍峰出现。流式细胞仪检测发现曲古抑素A阻滞细胞于S期,线粒体膜电位降低。4.低浓度(31.25、62.5 nmol/L)的曲古抑素A联合顺铂处理后,逆转倍数分别为1.52,1.70。结论1.人肺腺癌耐顺铂细胞株A549/CDDP耐药性良好,耐药指数为12.8。2.曲古抑素A能诱导A549/CDDP细胞株凋亡。3.曲古抑素A阻滞A549/CDDP细胞株细胞周期于S期。4.曲古抑素A增加A549/CDDP细胞株对顺铂的敏感性。目的探讨曲古抑素A是否通过上调死亡相关蛋白激酶(Death-associated protein kinase,DAPK)表达增强人肺腺癌耐顺铂细胞株对顺铂敏感性。方法为了明确死亡相关蛋白激酶在曲古抑素A增加A549/CDDP细胞对顺铂敏感性的作用,将pcDNA3.1(+)-DAPK转染入A549/CDDP细胞,使其表达较高的DAPK水平。将对DAPK有负调控作用的DAPK-C末端导入A549/CDDP细胞,并应用RNA干扰技术抑制DAPK的表达,用曲古抑素A及顺铂进行处理,采用Western blot检测蛋白表达,中性红方法检测细胞抑制率。结果死亡相关蛋白激酶蛋白水平在A549/CDDP比在A549细胞中表达教低。当死亡相关蛋白激酶过表达时,A549/CDDP细胞对顺铂敏感性增加,曲古抑素A的作用增强。当死亡相关蛋白激酶活性被DAPK-C末端抑制及RNA干扰下调死亡相关蛋白激酶表达水平时,曲古抑素A的作用减低。结论死亡相关蛋白激酶的表达可能参与顺铂耐药,它的表达上调可能是曲古抑素A提高人肺腺癌耐顺铂细胞株A549/CDDP对顺铂敏感性机制之一目的探讨曲古抑素A诱导人肺腺癌耐顺铂细胞株凋亡的作用及机制。方法Western blot分析蛋白表达的变化,比色法测定Capase-8活性。结果Western blot结果显示,曲古抑素A处理细胞后,cFLIP蛋白水平下降,procaspase-8减少,提示Caspase-8被激活,促使Bid蛋白切割，使全长Bid蛋白水平减少，截断Bid条带增加。procaspase-3减少,提示此Caspase-3被激活。聚腺苷二磷酸核糖聚合酶(PARP)蛋白减少,PARP切割的条带逐渐增加,提示PARP活性增加,加促切割DNA,促进细胞凋亡。Western blot结果显示,曲古抑素A处理细胞后,cFLIP蛋白水平下降,同时观察到Caspase-8, Caspase-3, PARP被激活,Bid切割增加,曲古抑素A对Caspase-8活性和cFLIP的影响具有浓度和时间依赖性。结论曲古抑素A可以通过降低cFLIP水平激活Caspase-8诱导人肺腺癌耐顺铂细胞株A549/CDDP凋亡。目的探讨曲古抑素A诱导人肺腺癌耐顺铂细胞株凋亡的作用机制。方法Western blot分析凋亡相关蛋白的变化。转染Bcl-2表达质粒过表达Bcl-2和利用siRNA下调Bcl-2表达水平。结果Westernblot结果显示,曲古抑素A处理细胞后,Bcl-2蛋白水平下降,而Bax表达上调,同时观察到Caspase-3被激活。转染Bcl-2表达质粒可以抑制曲古抑素A诱导的细胞凋亡,而沉默Bcl-2表达可以增加细胞对曲古抑素A的敏感性。结论曲古抑素A可能通过线粒体途径诱导人肺腺癌耐顺铂细胞株A549/CDDP凋亡。更多还原

【Abstract】 BackgroundLung cancer is one of the most common cancers worldwide. There are about 80% of lung cancers are defined as non-small cell lung cancer (NSCLC). While surgery remains to be the primary treatment at the early stage, many stageⅡandⅢpatients will progress within few months after tumor resection. For most NSCLC patients, they are diagnosed at local or more often distant advanced stage, chemotherapy is the leading way to lung cancer.The use of platinum-based combination chemotherpy remains the standard treatment for non-small lung cancer,However, the resistance to platinum limits further treatment clinically. For this reason, it is important to search for an appropriate and effective way to reverse cisplatin resistance in lung cancer.The mechanism of cisplatin-resistance has been associated with multi-factors including different cellular accumulation and detoxification of the drug, inhibition of apoptosis and DNA repairs. In treating lung cancers, the chemo-sensitivity of cisplatin is affected by the changes of gene expression, including those known to be associated with cell cycle regulation and apoptosis.Epigenetic alterations, such as the histone acetylation and DNA methylation in the promoter of genes, contribute to the changes of gene expression. The dys-regulation in the epigenetics can lead the onset and progression of cancer, increasingly more reports have demonstrated that it also plays an important role in drug resistance. Histone deacetylase (HDAC) inhibitors have been known to promote transcription of genes required for cell differentiation and apoptosis; therefore,they emerged as a class of cancer therapeutic agents. Trichostatin A (TSA) is one of such HDAC inhibitors. It was originally isolated from Streptomyces and identified as a fungicidic antibiotic and inhibited all class I and II HDACs. TSA can act as a chemo-sensitizer in ovarian cancer, gastric cancer, erythroleukemia cells, the molecular mechanisms of TSA-sensitized cytotoxicity of chemotherapeutic drugs remain largely unknown.In this study, we investigate the apoptosis-inducing effect of TSA in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP cell line) and examine whether TSA can enhance the sensitivity to cisplatin treatment and the underlying molecular mechanisms.Objective To investigate the apoptosis-inducing effect of TSA in the human lung adenocarcinoma cisplatin-resistant cell line A549/CDDP and to examine whether TSA can enhance the sensitivity to cisplatin treatment. Methods 1.A549 and A549/CDDP Cell lines were cultured with DMEM medium. Neutral Red assay Cell viability was performed to evaluate the resistance of A549/CDDP cells. 2. The A549/CDDP cells were treated with different concentrations of TSA.3. Apoptosis was studied using Hoechst 33258 staining and flow cytometry analysis. cell cycle and mitochondrial membrane potential were analyzed by flow cytometry.4. The A549/CDDP cells were treated with the combined treatment of cisplatin and TSA. Results 1.The resistant index of A549/CDDP cell was 12.8.2. TSA was 446.59±27.32 nmol/L,The growth curve showed the ratio of growth decreased with the increase of concentration of TSA.3.TSA induced apoptosis in A549/CDDP cells. morphologic changes including nuclear chromatin condensation fluorescence strength was observed with fluorescence microscope. Apoptosis was measured as the percentage of sub-G1 DNA content by flow cytometry.Treated by TSA, mitochondrial membrane potential was descreased and cells were arrested at S phase.4. Treated by the combined treatment of cisplatin and TSA(31.25 nmol/L and 62.5 nmol/L), the reverse multiple was 1.52,1.70. Conclusions 1.Compared with A549 cells, A549/CDDP cells have good resistance.2. TSA induced apoptosis in A549/CDDP cells.3. Treated by TSA, mitochondrial membrane potential was descreased and cells were arrested at S phase.4 TSA enhances the sensitivity of CDDP-resistant A549 cells to cisplatin.Objective To investigate whether DAP kinase contributes to cisplatin resistance and the underlying molecular mechanisms. Methods Different proteins was detected by Western blot method. To confirm the role of Death-associated protein kinase (DAPK) in the TSA-induced apoptosis in A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has higher expression level of DAPK expression,and the DAPK activity was inhibited by C-terminal fragment of DAPK and RNA interference.Results TSA induced apoptosis in A549/CDDP cells, along with the concomitant DAPK up-regulation. When DAPK is over-expressed, CDDP-resistant A549 cells become sensitive to both TSA and cisplatin. Moreover, the cytotoxicity of TSA can be alleviated when DAPK activity is inhibited by the expression of a recombinant C-terminal fragment of DAPK or RNA interference. Conclusions DAPK might involve in cisplatin-resistance of the A549/CDDP cells,the up-regulation of DAPK is one of the mechanisms mediating TSA action to sensitize cisplatin-resistant cells to apoptosis.Objective To investigate the mechanism of TSA induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Different proteins were analyzed by Western blot method, Colorimetric method was used to measure Caspase-8 activities. Results Treated by TSA,Western blotting analyses showed that the levels of cFLIP decreased, procaspase-8 decreased, and it resulted in the activation of Caspase-8 and Bid cleavage. The levels of procaspase-3 and PARP decreased, the cleavage of the activated PARP molecules would undergo more rapid apoptosis. Caspase-8 activity increase and cFLIP level decrease were regulated by TSA in a dose and time-dependent manner. Conclusion TSA induce A549/CDDP cell apoptosis by decreasing cFLIP level, activating caspase-8.Objective To investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Different proteins were analyzed by Western blot method, A549/CDDP cells were transfected by Bcl-2 expression Vector or endogenous Bcl-2 was downregulated by siRNA. Results Western blot analyses showed that the levels of Bcl-2 decreased, while expression of Bax increased. Simultaneously Caspase-3 was activated.Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis, while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA.Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.更多还原