钾通道MaxiK经NF-κB/MGMT途径参与恶性胶质瘤细胞替莫唑胺耐药的机制研究

Temozolomide (TMZ) is a widely used chemotherapeutic agent in the treatment of glioblastoma multiforme (GBM), however the resistance of GBM to TMZ results in the dismal treatment outcomes. By far, the mechanism underlying the TMZ resistance of GBM still remains unclear. Growing evidence has demonstrated that the O6-methylguanine-DNA methyltransferase (MGMT), the DNA repair protein whose activity was regulated by NF-κB signaling pathway, played an important role in the resistance of GBM to TMZ. Findings from our work have revealed that MaxiK, a cellular potassium channel, was capable of regulating NF-κB signaling cascades in murine macrophages. Moreover, the involvement of MaxiK in the resistance of GBM to TMZ was also found in our previous work. Base on the findings above, the hypothesis was proposed that NF-κB mediated expression of MGMT was responsible for the resistance to TMZ in GBM cells, which was probably modulated by MaxiK channel. In this study, the roles of MaxiK in the chemo-resistance of GBM cells to TMZ will be verified in vitro and in vivo using MaxiK knockdown cell lines and xenograft tumor model, respectively. We will further disclose the function and the mechanism of caspase-1 in the NF-κB activation regulated by MaxiK using RNA interference. This study will bring new insight into the understanding of mechanism of TMZ induced chemo-resistance in GBM.

替莫唑胺（TMZ）是治疗多形性胶质母细胞瘤(GBM)的常用化疗药物，但其耐药性影响了治疗效果，且耐药机制尚未完全阐明。研究表明，DNA修复酶MGMT活性上调是GBM细胞对TMZ耐药的主要诱因，该过程可被NF-κB调控。我们发现在巨噬细胞中MaxiK通道可调控NF-κB的激活，而在GBM中MaxiK参与了TMZ耐药的形成。因此我们推测：在GBM细胞中MaxiK通过调控NF-κB活性来影响MGMT表达，介导TMZ耐药。本课题拟使用干扰沉默和抑制剂等手段在细胞及裸鼠成瘤模型上明确MaxiK在GBM细胞TMZ耐药形成中的作用；利用基因沉默等手段考察caspase-1在MaxiK调控NF-κB中的作用，进一步通过沉默RIP2或/和MAL来研究该调控作用的实现途径，从而揭示MaxiK调控NF-κB/MGMT并介导TMZ耐药的机制。本项目的实施将为GBM细胞TMZ耐药机制及其对抗策略的研究提供新的思路。

替莫唑胺（TMZ）是治疗人神经胶质瘤的常用化疗药物，但其耐药性影响了治疗效果，且耐药机制尚未完全阐明。本项目证明TMZ可引起胶质瘤细胞中中MaxiK通道基因及蛋白水平上调，同时，组织芯片结果指出，胶质瘤患者Maxi K蛋白表达增加。采用特异性抑制Maxi K或干扰其表达后，可增强TMZ细胞毒性。另一方面，裸鼠荷瘤模型结果显示，TMZ与Maxi K特异性抑制剂paxilline联合治疗效果优于TMZ单独治疗。并且，由于paxilline水溶性差，本项目成功制备paxilline脂质体，为其真正应用于临床做出准备。机制研究方面，采用基因芯片技术，课题组首次发现Maxi K与HMGB1密切相关。抑制Maxi K可抑制HMGB1表达。同时，基于HMGB1与肿瘤细胞自噬的密切关系，本项目对Maxi K与自噬的关系进行研究，研究证明抑制Maxi K可抑制自噬，过表达HMGB1可逆转该过程，该结果说明HMGB1是Maxi K参与自噬的重要环节。但是，过表达HMGB1不影响Maxi K对TMZ的增敏作用，该结果说明，除自噬外，Maxi K还通过其他方式参与胶质瘤耐药，该结果为课题的进一步研究提供理论基础。