氯化两面针碱逆转DNA拓扑异构酶介导的肿瘤多药耐药作用研究

Multidrug resistance(MDR) is the main reason for high recurrence rate and poor prognosis of tumor. Topo which mediate atypical multidrug resistance is a key protein in generating MDR. Preliminary studies have shown that nitidine chloride (NC)is safe and has significant antitumor activity in a variety of tumor cell lines, inchluding MDR tumor, which is related to inhibiting Topo. The data have indicated that NC can reverse the human drug-resistant strains of lymphoblastoid expressing DNA topo I. Is there a relationship between reversing MDR of NC and Topo? To solve this problem, we will detect the accumulation of chemotherapeutic drugs in MDR cells and the sensitivity of cells to the agents before and after treatment with NC, and check the tumor volume and the accumulation of chemotherapy drugs in blood and tumor tissue of nude mice, which can define the reversing MDR effect of NC. Catalytic activity, protein and gene expression of Topo will be determined to know the relationship between NC inhibiting antitumor activity and Topo. DNA-Topo complex stability, molecular docking technology and SPR technology will be used to clear the interaction of NC with Topo. The chemotherapeutic drugs sensitivity and Topo expression will be detected in Topo overexpreesion cells to further understand the mechanisms of NC in reversing MDR. The study would provide a basis for clinical applications of NC as an MDR-reversing agent.

多药耐药（MDR）是肿瘤复发率高、预后不良的主要原因。Topo是MDR过程的关键蛋白，介导不典型的多药耐药。前期研究显示NC具有显著的抗肿瘤活性及良好的安全性，对MDR肿瘤细胞亦有显著抑制作用，且该作用与抑制Topo有关。资料表明NC具有逆转TopoⅠ构建的人淋巴母细胞耐药株的作用。NC逆转MDR作用是否与Topo有关？本研究针对该问题，检测NC处理前后MDR细胞内化疗药物聚集浓度变化和细胞对化疗药的敏感性，并测定移植瘤裸鼠的肿瘤体积、血液和瘤内化疗药物聚集浓度，明确NC对MDR的逆转作用；检测Topo蛋白/基因表达和酶催化活性，明确NC与Topo的关系；通过DNA-Topo复合物稳定性实验，分子对接技术和SPR技术预测并验证NC与Topo相互作用；检测不同Topo表达水平细胞对化疗药敏感性及Topo表达量改变，深化对NC逆转MDR机制的理解，为NC以MDR逆转剂用于临床提供依据。

多药耐药（MDR）是肿瘤复发率高、预后不良的主要原因。Topo是MDR过程的关键蛋白，介导不典型的多药耐药。前期研究显示NC具有显著的抗肿瘤活性及良好的安全性，对MDR肿瘤细胞亦有显著抑制作用，且该作用可能与抑制Topo有关。本研究通过NC作用于KB/ADM多药耐药细胞及KB/ADM多药耐药细胞裸鼠移植瘤模型，检测NC对细胞MDR的逆转活性,检测Topo在细胞内表达量的改变，明确NC对MDR的逆转作用及其与Topo的相关性；通过DNA-Topo复合物稳定性实验，分子对接技术验证NC与Topo相互作用；采用慢病毒转染技术检测不同Topo表达水平细胞对化疗药敏感性及Topo表达量改变，为NC以MDR逆转剂用于临床提供依据。结果显示NC对KB/ADM多药耐药细胞有较明显的耐药逆转作用，细胞内Topo表达量下调，细胞凋亡增加；裸鼠移植瘤模型中肿瘤组织减小，肿瘤细胞凋亡增加；NC对DNATopo催化活性和计算机分子对接实验结果显示NC能作用于TopoI及TopoⅡTopo的分子结构；慢病毒转染构建TopoⅡα高表达、低表达细胞系结构显示NC作用后耐药细胞对化疗药敏感性改变，TopoⅡα在细胞中的表达量改变。研究结果表明NC具有逆转耐药细胞MDR的作用，其作用机制可能是通过抑制耐药细胞Topo来实，研究为NC以MDR逆转剂用于临床提供了一定的依据。