基于氯通道差异性表达的Disulfiram-Cu靶向抗肿瘤作用及其机制

Recently studies including our own showed that chloride channel may be considered as a potential tumor marker and therapeutic target for human carcinoma. Emerging evidence indicates that Disulfiram (DSF), a deterrent to ethanol abuse in the management of alcoholism, when chelated with metal ions such as copper,zinc, has a highly selective anti-tumor effect. The underlying mechanisms,however, are unclear. Our previous studies found that, compared those in normal nasopharyngeal epithelial cells, the functional activities and protein expression of chloride channel were up-regulated in nasopharyngeal carcinoma cells. Functional blockages of chloride channel or silencing of the expression of chloride channel can significantly inhibit tumor cell proliferation. These results demonstrated that proliferation of cancerous was more dependent on the activities of chloride channel. Our preliminary studies for this project found that disulfiram chelate copper (DSF-Cu)can induce cell apoptosis and activate the chloride current in nasopharyngeal carcinoma cells, but with little effect on the normal nasopharyngeal epithelial cells. These results suggest that the chloride channel plays important roles in the process of cell apoptosis and may mediate the highly selective anti-tumor effect of DSF-Cu. This project will be performed on the molecular, cellular and whole animal levels, to explore: 1) Whether the highly selective toxicities of DSF-Cu in carcinoma cells is related to the different expression of the chloride channel in normal and carcinoma cells? 2) What are the molecular mechanisms of chloride channel involving in the anti-tumor activities of the DSF-Cu? 3) What are the signal pathways of DSF-Cu in activating of chloride channel. 4) What are the mechanisms that activation of chloride channels by DSF-Cu which induce the irreversible apoptosis? Investigation of the roles of the chloride channel in cell apoptosis induced by DSF-Cu and its molecular mechanisms will benefit for the development of the anti-tumor drugs which targets on the chloride channel.

我们前期研究发现氯通道在相同组织起源的人鼻咽上皮细胞(NP69-SV40T)和人低分化鼻咽癌细胞(CNE-2Z)呈现蛋白和功能的差异性表达、提示氯通道可作为肿瘤诊断和干预的潜在新靶标。近年来发现双硫仑(DSF)与铜、锌等金属离子螯合后具有高选择性杀伤肿瘤细胞的抗肿瘤活性，然其作用机制未明。预实验发现DSF螯合铜(DSF-Cu)可诱导CNE-2Z细胞凋亡、激活氯电流，而对NP69-SV40T细胞的影响甚微，这些结果提示氯通道很可能是DSF-Cu的高选择性抗肿瘤作用的未知靶标。本项目将从分子、细胞和整体水平研究：1）氯通道是否为DSF-Cu抗肿瘤作用的靶向分子？2）参与DSF-Cu抗肿瘤作用的氯通道的分子本质；3）DSF-Cu激活氯通道的途径；4）DSF-Cu经由氯通道介导凋亡的机制。从氯通道角度探索DSF-Cu高选择性抗肿瘤作用的潜在新靶点，为发展基于氯通道为靶标的抗肿瘤药物提供实验依据。

我们前期发现氯通道在相同组织起源的人鼻咽上皮细胞(NP69-SV40T)和人低分化鼻咽癌细胞(CNE-2Z)呈现蛋白和功能的差异性表达、提示氯通道可作为肿瘤诊断和干预的潜在新靶标。近年来发现双硫仑(DSF)与铜、锌等金属离子螯合后具有高选择性杀伤肿瘤细胞的抗肿瘤活性，然作用机制未明。本项目经过4年的研究工作，按计划顺利完成本课题的主要的研究内容。研究分3个部分来进行：1）氯通道及其亚型CLC-3蛋白是否为DSF-Cu靶向抗肿瘤效应的潜在靶点。在鼻咽癌（CNE-2Z）及其对应的正常鼻咽上皮细胞（NP69-SV40T）、乳腺癌（MCF-7，MDA-MB-231）及其对应的正常乳腺上皮（MCF-10A）细胞，我们发现CLC-3蛋白在肿瘤细胞高表达，DSF-Cu可以选择性激活氯电流、开放氯通道、使胞内氯离子外流从而带动水外流，产生凋亡的早期事件——凋亡性细胞容积回缩（Apoptotic Volume Decrease），并诱导后期凋亡事件——磷脂酰丝氨酸外翻及细胞坏死。但是在对应的正常细胞，DSF-Cu这些作用并不明显。在整体效应中，DSF-Cu可以显著抑制裸鼠移植瘤的生长，氯通道阻断剂NPPB和DIDS则抑制其作用；2）DSF-Cu靶向抗肿瘤的途径及3）机制。细胞膜结构完整性是细胞赖以生存的前提，DSF-Cu可选择性破坏肿瘤细胞膜超微结构；影响生物力学特性；破坏F-actin、FLNa、Tubulin等骨架蛋白的完整性及黏附蛋白的表达，继而抑制肿瘤细胞的迁移和运动能力，该作用与Rho家族调节骨架蛋白相关。CLC-3氯通道使DSF-Cu作用的早期靶点，药物作用5-10分钟内即可激活氯电流，使氯通道开放。在药物处理1.5小时左右，CLC-3氯通道蛋白表达明显增加，到6小时达高峰，随后逐渐减少。研究较为透彻的NF-kB、钙超载、ROS产生、Caspase-3的活化等均发生在CLC-3氯通道开放之后，且干扰CLC-3后，氯通道激活、AVD、凋亡及钙超载等均被抑制。说明后续的凋亡信号受CLC-3通道功能和表达的调控。本项目的完成拓展和加深了CLC-3在肿瘤发生发展及作为抗肿瘤药物潜在新靶点的认识，为发展基于氯通道为靶标的抗肿瘤药物提供可靠的依据。