米尔贝类抗生素逆转胶质瘤多药耐药的作用及机制研究

How to reverse multidrug resistance ( MDR ) in glioma chemotherapy is key to effective chemotherapy, and development of novel MDR reversal agents is crucial. Early in our study we found that milbemycin compounds could reverse the MDR of the human breast cancer cell line (MCF-7/adr) through inhibiting the expression of P-gp and its pumping function. The project on this basis, the effects and the mechanisms of milbemycin compounds on reversing MDR of glioma are further investigated. First, adriamycin-resistant glioma cells ( C6/adr ) in vitro are established by exposing the parent C6 cell line to increasing doses of adriamycin. Then we evaluate the effects of milbemycin compounds ( nemadectin, moxidectin, milbemycin β1 and secomilbemycin D ) on reversing MDR of C6/adr cells in vitro. The molecular mechanisms of milbemycin compounds reversing multidrug resistance are researched via real-time fluorescence quantitative PCR and flow cytometric analysis, and it is tested that the milbemycin compounds inhibiting the main protein pump transport function. At the same time, the blood-brain barrier ( BBB ) model is established in vitro, and the effect of milbemycin compounds on enhancing BBB permeability is detected to develop the effects of milbemycin compounds on reversing MDR. Furthermore, we establish C6/adr xenograft in nude mice to investigate the effects of milbemycin compounds on reversing MDR of glioma and enhancing BBB permeability, and the reversal mechanisms in vivo. In this study, we clarify the effects and the mechanisms of milbemycin compounds on reversing MDR of glioma, and provide theoretical evidences for adjuvant therapy of tumor chemotherapy.

如何逆转胶质瘤化疗过程中多药耐药（MDR）现象是对其有效化疗的关键，开发新型的MDR逆转剂至关重要。前期我们研究发现米尔贝类抗生素通过抑制P-gp的表达及泵出功能有效地逆转了人乳腺癌(MCF-7/adr)的MDR。本项目在此基础上，进一步探讨其逆转胶质瘤MDR的作用及机制。首先在体外构建胶质瘤耐阿霉素细胞系C6/adr，检测米尔贝类抗生素在体外逆转胶质瘤细胞MDR的作用，并利用实时荧光定量PCR及流式细胞仪分析其逆转胶质瘤MDR的分子机制，及对主要蛋白泵功能抑制的作用机制；同时，建立体外血脑屏障（BBB）模型，检测米尔贝类抗生素增强BBB通透性而辅助其逆转MDR的作用。进一步在体内建立荷耐药胶质瘤Balb/c裸鼠模型，探讨米尔贝类抗生素在体内逆转MDR作用、主要逆转机制，以及增强BBB通透性的作用。拟阐述米尔贝类抗生素逆转胶质瘤MDR的作用及机制，对肿瘤化疗的辅助治疗提供重要理论依据。

由于在临床化疗过程中肿瘤细胞产生的多药耐药(multidrug resistance, MDR)，使化疗药物难以起效，甚至无效。可见，成功逆转肿瘤细胞MDR直接关系到肿瘤患者的化疗效果。本研究成功诱导胶质瘤耐ADR的细胞株C6/adr，其耐药指数大约为41倍。以体外耐药细胞为研究模型，确定伊维菌素及莫西克汀具有明显的逆转肿瘤细胞的MDR及增加BBB通透性。0.1μmol/L、1μmol/L、5μmol/L的伊维菌素及莫西克汀可显著降低C6/adr细胞的ADR IC50值，并呈剂量依赖关系，使耐药倍数明显下降。我们明确了伊维菌素及莫西克汀对体外培养的人成纤维细胞和人外周血单个核细胞的毒性较小，安全剂量可达4μmol/L左右。成功建立荷耐药肿瘤裸鼠模型，明确伊维菌素及莫西克汀具有体内增加BBB通透性促进其逆转胶质瘤MDR的作用；阐明伊维菌素及莫西克汀体内外逆转MDR作用的主要分子机制：一是与P-gp竞争性结合，抑制其泵出功能；二是降低MDR 1基因和P-gp的表达，以及MRP 1基因的表达。于2013年、2014年发表中文核心期刊两篇，目前一篇SCI文章在投，2个发明专利申请中。本研究从逆转胶质瘤MDR的特点出发，结合文献报道及前期研究基础，将作为杀虫药物的伊维菌素及莫西克汀用于医学逆转肿瘤多药耐药的基础研究，扩展了该类抗生素的应用领域。同时，也为肿瘤多药耐药的逆转提供了更为广泛的药物来源。另外，我们进一步阐述伊维菌素及莫西克汀增强BBB通透性而实现逆转胶质瘤MDR的新作用及机制，对肿瘤化疗的辅助治疗提供重要理论依据。