逆转肿瘤细胞耐药性的潜在新靶标-氯通道在跨细胞膜药物转运中的作用及其与耐药的关系

The normal transport of drugs across the cell membrane is a key step for maintaining intracellular drug concentration and preventing drug resistance. However, the underlying mechanism has not been clarified. It was found recently by us that the activation of chloride channels in the cell membrane promoted cellular uptake of the anticancer drugs, polypyridyl ruthenium complexes, and fluorescent dyes; these actions could be inhibited by chloride channel blockers and by down-regulation of the expression of chloride channel proteins. These data suggest that chloride channels may be involved in the regulation of drug transport across the cell membrane and affect the sensitivity of cells to drugs by regulating intracellular drug concentration. By focusing on the roles of chloride channel in the regulation of transmembrane drug transport and drug resistance, the aims of this project are to investigate or clarify: 1) whether chloride channels can mediate, or regulate transmembrane drug transport; 2) the action mechanism of chloride channels in regulating transmembrane drug transport; 3) the molecular identity of the chloride channels involved in transmembrane drug transport;; 4) the differences in chloride channel expression and transmembrane drug transport between the original drug-sensitive tumour cell lines and their counterpart, the drug-insensitive cell lines; and the relationships between chloride channels, transmembrane drug transport and drug sensitivity. This study may result in the finding of new roles of chloride channels in regulation of transmembrane drug transport, which may benefit the development of ion channel-targeting drugs for reversing drug resistance.

细胞膜对药物的正常转运是维持细胞内药物浓度、防止耐药性发生的关键环节，但其作用机制远未明确。我们前期研究发现，激活细胞膜氯通道可促进肿瘤细胞摄取抗肿瘤药钌多吡啶配合物和荧光染料，该作用被氯通道阻断剂和下调氯通道蛋白表达所抑制，提示氯通道可能参与调节药物跨细胞膜转运，从而影响细胞内药物浓度和细胞对药物的敏感性。本课题以氯通道在药物转运和逆转耐药性中的作用为切入点，重点探讨：1）氯通道在调节细胞膜药物转运中的作用；2）氯通道调节膜药物转运的机制；3）参与细胞膜药物转运的氯通道分子本质；4）肿瘤亲本细胞和耐药细胞的氯通道表达和细胞膜药物转运功能的差异，分析氯通道-药物转运-药物敏感性之间的相关性。从氯通道的角度来探索和发现调控细胞膜药物转运、逆转耐药性的潜在新靶点，有望对离子通道在细胞膜药物转运中的作用提出新的观点，为研发以氯通道为靶标的促进药物摄取和逆转耐药性的药物提供基础理论依据。

细胞膜对药物的正常转运是维持细胞内药物浓度、防止耐药性发生的关键环节，但其机制远未明确。我们前期研究发现，氯通道可能参与调节药物跨细胞膜转运，从而影响细胞内药物浓度和细胞对药物的敏感性。本课题以氯通道在药物转运和逆转耐药性中的作用为切入点，重点探讨：氯通道在调节细胞膜药物转运中的作用及机制；参与细胞膜药物转运的氯通道分子本质；肿瘤耐药细胞的氯通道表达和细胞膜药物转运功能的变化。主要研究结果如下：..1)氯通道参与调节细胞对钌多吡啶配合物（RuPOP）、阿霉素跨细胞膜转运，氯通道开放有助于细胞摄取药物, 提高胞内药物浓度，阻断氯通道作用相反；动力学研究表明，上述药物转运过程具有非饱和特征；2）与正常鼻咽上皮细胞相比，鼻咽癌细胞内源性高表达ClC-3氯通道蛋白，RuPOP和阿霉素激活的氯电流较大，对RuPOP、阿霉素的跨膜转运增强，细胞内药物浓度提高；3）下调ClC-3表达，显著削弱RuPOP、阿霉素诱导的氯电流及药物跨膜转运，降低胞内药物浓度；上调ClC-3表达，促进药物进入细胞，提示ClC-3 氯通道可能是参与调节药物跨膜转运的主要氯通道亚型；4）与乳腺癌阿霉素敏感细胞株（MCF-7）相比，乳腺癌耐阿霉素细胞株（MCF-7/Adr）ClC-3氯通道表达下调，阿霉素的摄取和胞内浓度明显减少，且此种药物转运可被氯通道阻断剂阻断，提示耐药细胞氯通道下调与阿霉素跨膜转运减弱及胞内药物浓度降低密切相关。进一步实验所表明，上调ClC-3表达后耐药细胞对阿霉素的摄取能力增强，胞内药物浓度提高，细胞对阿霉素敏感性恢复。5）耐药细胞脱耐药后ClC-3表达上调，细胞膜对药物转运功能增强，胞内药物浓度提高，下调ClC-3的表达，细胞对阿霉素的摄取能力以及对阿霉素敏感性均减弱。. . 本研究表明，氯通道参与调节药物的跨细胞膜转运，是逆转耐药性的潜在作用靶点。本项目的完成拓展了我们对离子通道调节细胞跨膜药物转运的认识，对离子通道在细胞膜药物转运中的作用提出新的观点，为研发以氯通道为靶标的促进药物摄取和逆转耐药性的药物提供基础实验依据。