以肺癌細胞株與動物模式探討新穎的吲哚結構合成化合物1,1,3-tri(3-indolyl)cyclohexane抑制腫瘤細胞生長機制

A novel two-step synthetic indole compound 1,1,3-tri(3-indolyl)cyclohexane inhibits cancer cell growth in lung cancer cells and xenograft models

李慶孝(Ching-Hsiao Lee)

指導教授 ： 王憶卿 ； 李桂楨

國立臺灣師範大學/理學院/生命科學研究所/博士(2008年)

摘要

目的：肺癌在世界各地無論男性或女性都是發病率、死亡率名列前茅的惡性腫瘤。因此，發現與合成新穎的肺癌治療抗癌藥物是刻不容緩的工作。材料與方法：本研究發展了一種新穎的吲哚結構合成化合物1,1,3-tri(3-indolyl)cyclohexane (3-indole)，設計使用二步法合成，該技術方法縮短製備過程，產品質量和產量也獲得提高，並藉由人類肺癌細胞株 (A549, H1299, H1435, CL1-1, and H1437) 來探討新穎抗癌藥物對於肺癌細胞的毒殺作用及其機制，同時進行前臨床動物實驗測試。結果：新穎的抗癌藥物3-indole經由不同濃度處理，可以誘導人類肺癌細胞株 (A549, H1299, H1435, CL1-1, and H1437) 進行細胞週期休止 (cell cycle arrest) 及細胞凋亡 (apoptosis)。細胞週期研究初步實驗結果顯示調控細胞週期休止的蛋白p53與p21表現增加，顯示p53/p21相關訊息傳遞路徑重要性。目前已知有兩個機轉可以調控細胞凋亡現象，第一個作用機轉是經由caspases (cysteine-dependent aspartate-specific proteases) 相關性機轉活化而引起細胞凋亡，目前已被認定有粒線體參與訊息傳遞的內在路徑與細胞外死亡訊息接受器作用的外在路徑；第二個機轉是經由caspases非相關性機轉。西方墨點法實驗結果顯示，調控細胞凋亡進行的促進凋亡蛋白Bax、Bad表現增加，抗凋亡蛋白Bcl-2表現下降，而粒線體細胞色素C釋放至細胞質情形也有增加，另外一方面，透過caspases活性分析實驗結果顯示，3-indole主要是藉由caspases-9、caspases-3參與粒線體訊息傳遞的內在路徑以誘發細胞凋亡發生。此外，3-indole誘導A549人類肺癌細胞株粒線體膜電位下降、活性氧分子 (reactive oxygen species, ROSs) 產量增加，與細胞生長調節相關MAPK (Mitogen-activated protein kinase) 家族分子c-Jun N端蛋白質激酶 (JNK) 表現增加，同時顯示有DNA損傷情形。進一步活性氧分子抑制劑實驗結果顯示，JNK表現與DNA損傷可部分減少。3-indole誘導細胞凋亡情形受到活性氧分子抑制劑或JNK訊息抑制劑阻斷，顯示活性氧分子與JNK壓力相關訊息傳遞路徑重要性。此外，初步實驗結果，其他生長調節相關訊息傳遞蛋白 (如Akt與p38/COX-2) 表現也受到3-indole抑制，顯示PI3K/Akt與p38/COX-2訊息傳遞路徑重要性。同時前臨床動物實驗測試結果顯示3-indole抑制A549及H1435肺癌細胞株生長。結論：3-indole在細胞模式與動物模式呈現具有抑制肺癌細胞株生長的作用，其誘導細胞死亡是透過ROS與JNK路徑之粒線體訊息傳遞的內在細胞凋亡，同時可誘導細胞週期休止以及抑制肺癌細胞株Akt與p38/COX-2的表現，顯示使用二步法合成，具有高質量和產量的3-indole具有發展作為新穎的抗癌症用藥的價值。

關鍵字

吲哚結構化合物 ； 肺癌 ； 微管聚合作用 ； 抗微管作用 ； 粒線體訊息傳遞相關細胞凋亡 ； 活性氧分子 ； c-Jun N端蛋白質激酶

並列摘要

BACKGROUND. Lung cancer is the most common malignancies in both men and women worldwide. Thus, the development of more effective anti-cancer drugs for lung cancer is urgently needed
METHODS. This study generated a 2-step novel synthetic compound, referred to as 1,1,3-tri(3-indolyl)cyclohexane (3-indole), in high purity and yield. 3-indole was tested for its biological activity in A549, H1299, H1435, CL1-1, and H1437 lung cancer cells. Animal studies were also performed.
RESULTS： The data indicated that 3-indole induced cell cycle arrest and apoptosis in various lung cancer cells. Increased expression of p53 and p21 protein suggested the importance of p53/p21 pathway in 3-indole-induced cell cycle arrest. Increased cytochrome c release from mitochondria to cytosol, decreased expression of anti-apoptotic Bcl-2, and increased expression of pro-apoptotic Bax and Bad were observed. In addition, 3-indole stimulated caspases-3, -9 and to a lesser extent caspase-8 activities in cancer cells, suggesting that the intrinsic mitochondria pathway was the potential mechanism involved in 3-indole-induced apoptosis. 3-indole-induced a concentration-dependent mitochondrial membrane potential dissipation, and increase in reactive oxygen species (ROSs) production. Activating c-Jun N-terminal kinase (JNK) and triggering DNA damage were also apparent. Note that 3-indole-induced JNK activation and DNA damage can be partially suppressed by ROS inhibitor. Apoptosis induced by 3-indole could be abrogated by ROS or JNK inhibitors, suggesting the importance of ROS and JNK stress-related pathways in 3-indole-induced apoptosis. Preliminary data of decrease expression of Akt, p38, and COX-2 proteins suggested the importance of PI3K/Akt and p38/COX-2 pathways in 3-indole induced cell apoptosis. Moreover, 3-indole showed in vivo anti-tumor activities against human xenografts in murine models.
CONCLUSIONS. The result from the present study suggest that 3-indole inhibited the growth of various human lung cancer cells in cell and animal models and induced intrinsic apoptosis by ROS production and activation of the JNK signaling pathways. Together, these data confirmed that the 2-step synthetic 3-indole compound of high purity and yield is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment.

並列關鍵字

indole compound ； lung cancer ； tubule polymerization ； antimicrotubule ； mitochondria-mediated apoptosis ； reactive oxygen species ； c-Jun N-terminal kinase

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