新化合物TMZ-POH在非小细胞肺癌中的抗肿瘤作用及其对DNA修复蛋白MGMT的调控

发布时间：2018-06-28 09:17

本文选题：新型化合物TMZ-POH + 抗肿瘤作用　；参考：《济南大学》2017年硕士论文

【摘要】：研究背景非小细胞肺癌(NSCLC)在肺癌中占80%,化疗是临床上常用的治疗方法之一,但是严重的毒副作用及化疗耐药的发生在很大程度上影响了其治疗效果。替莫唑胺(Temozolomide,TMZ)是烷化剂类药物氮烯唑胺的衍生物,紫苏醇(Perillyl alcohol,POH)在多种类型的肿瘤中对TMZ具有治疗增敏作用。TMZ-POH是将TMZ和POH以共价键连接形成的新型烷化剂,在三阴性乳腺癌、非小细胞肺癌、人类鼻咽癌,甚至是TMZ耐药的神经胶质瘤中,TMZ-POH比TMZ、POH单独用药或二者联合应用具有更强的抗肿瘤作用。MGMT是一种进化保守的酶,在多种癌症中的高表达往往提示预后较差,且表达水平受到多重作用机制的调控。MGMT能够移除TMZ的细胞毒性产物O6-meG,并使其失活,这是MGMT诱导TMZ化疗耐药发生的决定性因素。在不同肿瘤细胞中的研究发现,TMZ-POH与TMZ等烷化剂不同,前者即使在MGMT高表达的细胞系中同样能发挥较强的细胞毒性作用,这意味着TMZ-POH可能通过调控MGMT来克服在其他烷化剂中发生的耐药。目的研究TMZ-POH对非小细胞肺癌的抗肿瘤作用,并探索其作用机制是否与ROS累积相关;研究TMZ-POH对MGMT的调控作用以及MGMT对TMZ-POH细胞毒性的影响。方法将TMZ和POH以共价键连接形成新型烷化剂TMZ-POH,将其作用于所培养的肿瘤细胞系,通过细胞活性测定、克隆形成实验、活性氧ROS测定、线粒体膜电位定位测定和蛋白印迹等体外实验以及体内实验研究TMZ-POH的抗肿瘤作用及机制。结果TMZ-POH对NSCLC细胞系A549、H460和H520均具有细胞毒性作用,上调磷酸化的ATM、CHEK1、CHEK2、H2AFX蛋白表达,将细胞周期阻滞在G2/M期,并且导致细胞凋亡、线粒体膜电位降低和活性氧累积;TMZ-POH在A549和SPC-A1(NSCLC来源)、A2780(卵巢癌来源系)以及CNE2(人鼻咽癌来源)等多种细胞系中,均能下调MGMT蛋白表达,在A549和SPC-A1细胞系中发现,该调控作用不引起Wnt/β-catenin通路上的β-catenin以及GSK-3β表达水平的差异,用蛋白酶体抑制剂MG132进行预处理之后,TMZ-POH诱导了更高水平的泛素化。结论新型化合物TMZ-POH在NSCLC中具有抗肿瘤作用,这种抗肿瘤作用是通过介导ROS累积完成的,能导致线粒体膜电位降低、线粒体凋亡、DNA损伤和细胞周期阻滞,进而抑制肿瘤生长。TMZ-POH能通过独立的蛋白酶体途径抑制MGMT的表达,避免耐药性的发生,并且在多种癌症细胞中(包括TMZ耐药的细胞),这种抑制作用在TMZ-POH发挥细胞毒性时是一个重要的影响因素。
[Abstract]:Background Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancer. Chemotherapy is one of the commonly used treatment methods in clinic. Temozolomide (TMZ) is a derivative of alkylating agent azenazole amine. Perillyl alcohol POH has the effect of sensitizing TMZ to TMZ in many kinds of tumors. TMZ-POH is a new alkant that connects TMZ and POH by covalent bond, and it is a new alkant in tri-negative breast cancer. Among non-small cell lung cancer, human nasopharyngeal carcinoma, and even TMZ resistant gliomas, TMZ-POH has a stronger antitumor effect than TMZ-POH alone or a combination of the two. MGMT is an evolutionarily conserved enzyme. High expression in multiple cancers often suggests poor prognosis, and the level of expression is regulated by multiple mechanisms. MGMT can remove and inactivate the cytotoxic product O6-meGfrom TMZ, which is the decisive factor in inducing TMZ chemoresistance. Studies in different tumor cells showed that TMZ-POH was different from TMZ, and the former could play a strong cytotoxic effect even in a cell line with high expression of MGMT. Objective to study the antitumor effect of TMZ-POH on non-small cell lung cancer and whether its mechanism is related to Ros accumulation, and to study the regulatory effect of TMZ-POH on MGMT and the effect of MGMT on the cytotoxicity of TMZ-POH. Methods TMZ and POH were covalently bonded to form a novel alkylator TMZ-POH, which acted on the cultured tumor cell lines. The antitumor effect and mechanism of TMZ-POH were studied in vitro and in vivo by mitochondrial membrane potential localization and Western blotting. Results TMZ-POH had cytotoxic effects on NSCLC cell lines A549H 460 and H 520, upregulated the expression of phosphorylated ATMN CHEK1CHEK2H2AFX protein, blocked the cell cycle at G 2 / M phase, and led to apoptosis. The expression of MGMT protein was down-regulated in A549, SPC-A1 (NSCLC) -derived cell line and CNE2 (human nasopharyngeal carcinoma) cell line, as well as in A549 and SPC-A1 cell lines. This regulation did not cause the difference of 尾 -catenin and GSK-3 尾 expression in Wnt- 尾 -catenin pathway. After pretreatment with proteasome inhibitor MG132, TMZ-POH induced a higher level of ubiquitin. Conclusion the new compound TMZ-POH has anti-tumor effect in NSCLC, which is mediated by Ros accumulation and can lead to mitochondrial membrane potential decrease, mitochondrial apoptosis and DNA damage and cell cycle arrest. TMZ-POH can inhibit the expression of MGMT through independent proteasome pathway and avoid the occurrence of drug resistance. And this inhibition is an important factor in the cytotoxicity of TMZ-POH in a variety of cancer cells (including those resistant to TMZ).
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【相似文献】