藉由非共價性破壞β-tubulin:CCT-β蛋白質複合體以引發CL1-5細胞凋亡並抑制其轉移能力的小分子藥物

A non-covalent small inhibitor blocking β-tubulin:CCT-β complex induces apoptosis and suppresses migration and invasion in CL1-5 cells

郭瑾融(Chin-Jung Kuo)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902879

摘要

在先前的研究中，我們發現破壞β-tubulin與CCT-β的蛋白質複合體可以做為新的抗癌方針。並且，經由虛擬篩選，我們從Sigma-Aldrich compound bank中找到一個非共價性的小分子藥物，3112210，可逆地結合於β-tubulin與CCT-β接觸面上的熱點。
本實驗中，我們將3112210測試於具有高度轉移性的非小細胞肺癌細胞株CL1-5。首先，在免疫共沈澱實驗結果顯示，經3112210處理後的細胞中，β- tubulin與CCT-β的蛋白質複合體會被破壞。另外，3112210藉由內質網壓力與細胞凋亡的機制使CL1-5肺小癌細胞死亡。除了探討3112210的癌細胞毒性，我們也以其EC20濃度進行細胞遷移與侵入實驗。實驗結果顯示，3112210可以抑制CL1-5細胞遷移與侵入的能力，並降低基質金屬蛋白酶MMP-2, -9的表現量與活性。在西方點墨法實驗與電泳酵素分析法中，我們推測此藥效是由整合素(Integrin)及其下游訊息傳遞帶動EMT調節因子的表現量改變所造成。
總結而論，3112210是一個新的非共價性地破壞CL1-5肺癌細胞內β-tubulin與CCT-β蛋白質複合體的小分子藥物，它對CL1-5細胞具有毒性並可以抑制其轉移能力。

關鍵字

抗癌症小分子藥物 ； β-tubulin:CCT-β蛋白質複合體 ； 細胞凋亡 ； 抗癌症轉移

並列摘要

Previously, we reported the protein-protein interaction (PPI) between β-tubulin and CCT-β complex as a potential anti-cancer chemotherapeutic target. Through virtual screening, a compound 3112210 from Sigma-Aldrich compound bank was identified to be a reversible inhibitor of the PPI by docking into hot spots on this PPI interface of β- tubulin.
In this study, 3112210 was tested on a highly metastatic non-small cell lung cancer (NSCLC) cell line, CL1-5. The co-IP experiments showed that, in 3112210-treated cancer cells, β-tubulin and CCT-β complex was disrupted. Furthermore, 3112210 caused CL1-5 cell death through ER stress and apoptosis. In addition to verifying its toxicity toward CL1-5, we performed migration and invasion assays using dosage at about IC20. The results indicated that 3112210 also inhibited cancer cell migration and invasion, and MMP-2, -9 were also inhibited. These anti-metastatic effects were endowed via integrin- related pathways and EMT transcriptional factors, as demonstrated by western blot experiments.
To sum, 3112210 is a novel non-covalent inhibitor for β-tubulin:CCT-β complex in CL1-5 lung adenocarcinoma cells to induce cancer cell death and impeded cell metastasis.

並列關鍵字

anti-cancer small molecule ； β-tubulin:CCT-β complex ； apoptosis ； anti-metastasis

參考文獻

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