Intracellular β-Tubulin/Chaperonin Containing TCP1-β Complex Serves as a Novel Chemotherapeutic Target against Drug-Resistant Tumors
Intracellular β-Tubulin/Chaperonin Containing TCP1-β Complex Serves as a Novel Chemotherapeutic Target against Drug-Resistant Tumors
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DOI：
10.1158/0008-5472.can-08-4700
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发表时间：
2009-09-01
期刊：
影响因子：
11.2
通讯作者：
Liang, Po-Huang
中科院分区：
文献类型：
--
作者：
Lin, Yuan-Feng;Tsai, Wen-Ping;Liang, Po-Huang
在本研究中，用亚微摩尔浓度的合成小分子N-碘乙酰基-色氨酸（I-Trp）处理HEK-293细胞有效地诱导细胞凋亡，这从亚Go细胞的积累和细胞内DNA片段化来判断。在I-Trp处理的细胞中检测到除caspase-1外的所有细胞内caspase的激活。蛋白质组学分析表明，P-微管蛋白作为一个特定的细胞内目标的I-Trp。蛋白质指纹分析表明，β-微管蛋白的肽片段TAVCDIPPR中的Cys（354）残基，其位于与含有TCP 1-β（CCT-β）的伴侣蛋白的结合界面处，被I-Trp烷基化。此外，Cys（354）（Cys-Ala）的定点突变消除了I-Trp掺入β-微管蛋白，表明Cys（354）确实是I-Trp的靶向位点。免疫沉淀显示β-微管蛋白/CCT-β复合物组成型形成，但在用I-Trp处理后被破坏。截短的β-微管蛋白（T351-S364）的过表达或用I-Trp或合成肽Myr-TAVCDIPPRG的处理在多药耐药MES-SA/Dx 5癌细胞中引起更严重的细胞凋亡，这是由于相对于野生型MES-SA癌细胞的更高水平的CCT-β。沉默CCT-β的表达使MES-SA/Dx 5细胞对I-Trp诱导的凋亡性细胞死亡不太敏感。这些发现表明β-微管蛋白/CCT-β复合物可作为治疗临床微管蛋白结合剂耐药或CCT-β过表达肿瘤的有效化疗靶点。[Cancer Res 2009;69（17）：6879-88]
In the present study, treatment of HEK-293 cells with the synthetic small molecule N-iodoacetyl-tryptophan (I-Trp) at submicromolar concentrations efficiently induced cell apoptosis as judged from the accumulation of sub-Go cells and intracellular DNA fragmentation. Activation of all intracellular caspases, except caspase-1, was detected in I-Trp-treated cells. Proteomic analysis revealed that P-tubulin acted as a specific intracellular target of I-Trp. Protein fingerprinting analysis indicated that the Cys(354) residue in the peptide fragment TAVCDIPPR of beta-tubulin, which is located at the binding interface with chaperonin containing TCP1-beta (CCT-beta), was alkylated by I-Trp. Moreover, site-directed mutagenesis of Cys(354) (Cys-Ala) abolished the incorporation of I-Trp into beta-tubulin, suggesting Cys(354) is indeed the targeting site of I-Trp. Immunoprecipitation showed that the beta-tubulin/CCT-beta complex was constitutively formed but disrupted after treatment with I-Trp. Overexpression of the truncated beta-tubulin (T351-S364) or treatment with I-Trp or the synthetic peptide Myr-TAVCDIPPRG caused more severe cell apoptosis in multidrug-resistant MES-SA/Dx5 cancer cells due to higher levels of CCT-beta relative to wild-type MES-SA cancer cells. Silencing the expression of CCT-beta rendered MES-SA/Dx5 cells less sensitive to I-Trp-induced apoptotic cell death. These findings suggest that the beta-tubulin/CCT-beta complex may serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-beta-overexpressing tumors. [Cancer Res 2009;69(17):6879-88]