Bcl-2家族蛋白Noxa和Bim在喜树碱诱导细胞凋亡中的功能与机制

Camptothecin (CPT) is a promising therapeutic drug which has been shown to induce apoptosis through DNA damage in a wide variety of cancer cells. However, the mechanism of DNA damage induced apoptosis is not fully understood. In our preliminary studies, we have identified the Bcl-2 family proteins critically involved in this process. We hypothesize that CPT induces apoptosis through multiple pathways that activate select BH3-only proteins, including Noxa and Bim, which cause the inactivation of Mcl-1 and Bcl-xL, and eventually cause mitochondrial damage. Research in this proposal will first examine the mechanisms of transcriptional upregulation of Noxa, analyze the promoter region to locate the cis element(s) as well as the binding factors that mediate the response to CPT.; the second aim is to determine Noxa C-terminal tail involvement in Noxa regulation; and the third aim is to determine the role of Bim dephosphorylation in CPT-induced apoptosis. By using Bid/Bim/Puma TKO cell line from preliminary study, we will exam the necessity of Bid/Bim/Puma direct activating Bax/Bak during CPT-induced apoptosis. The goal of this application is to systematically study the involvement and regulation of the Bcl-2 family proteins during DNA damage-induced apoptosis. Results from this research will provide invaluable knowledge on the mechanisms of apoptosis in cancer cells as well as critical insights into the mechanisms of potential therapeutic strategy.

喜树碱是一种通过DNA损伤诱导肿瘤细胞凋亡的化疗药物，其诱导凋亡的分子机制尚不明确，尤其是相关的Bcl-2家族蛋白分子参与调控的机制需要进一步阐明。我们前期研究发现Bcl-2家族蛋白中参与该过程的关键蛋白包括Noxa和Bim，这两个BH3-only蛋白可能直接与抗凋亡蛋白Mcl-1和Bcl-xl的失活相关。因此本课题将明确Noxa转录上调的关键作用因子，对Noxa启动子序列进行分析，定位顺式作用元件及其效应分子；通过测定重组蛋白水平及半衰期明确NoxaC端降解决定子序列是否通过稳定Noxa蛋白的方式参与调控；定位Bim蛋白的磷酸化位点，明确Bim去磷酸化反应的作用机制，并利用前期研究构建的Bid/Bim/PumaTKO基因敲除细胞系验证喜树碱诱导的细胞凋亡是否依赖Bid/Bim/Puma对Bax/Bak的直接激活作用。从而为发现潜在的肿瘤治疗策略以及利用DNA损伤治疗肿瘤提供新的机制依据。

喜树碱（CPT）是一种高度特异性的拓扑异构酶Ⅰ抑制剂。抑制拓扑异构酶Ⅰ能产生DNA 单链的缺口损伤，并由此转变为双链断裂，DNA 损伤诱导的细胞凋亡被用作治疗肿瘤的一种主要手段。本课题对喜树碱诱导凋亡的分子机制进行了研究，发现喜树碱造成的DNA损伤通过线粒体途径诱导，而ROS激活可能不参与喜树碱诱导的凋亡激活过程；阻断BH3-only的小分子蛋白能够有效的抑制凋亡发生；mcl-1和bcl-xl抗凋亡蛋白能够保护细胞阻止凋亡发生，过表达其中任何一个蛋白能够保护细胞，而低表达其中任何一个蛋白会增加细胞对CPT的敏感性，并且bcl-xl的保护作用更强；PED3A介导的凋亡信号通路能够使抗凋亡mcl-1和bcl-xl表达降低，诱导凋亡发生，但该通路并不参与CPT诱导凋亡的过程；最后我们新发现两个mcl-1的isoform，其具体意义尚不清楚，需要进一步研究探索。本课题的研究结果为利用DNA 损伤治疗肿瘤提供了一些新的机制依据，包括重要的阴性结果，同时也为发现潜在的新的治疗策略提供理论支持。