透過活化p53引起人類非小細胞肺癌與肝癌細胞凋亡的吲哚喹嚀基藥物與其作用機制的探討

Study of a novel indolylquinoline compound that induces apoptosis in human non-small cell lung cancer and hepatocellular carcinoma cells through p53 activation

劉俊彥(Liu Chun-Yen)

指導教授 ： 方剛

國立臺灣師範大學/理學院/生命科學系/博士(2016年)

https://doi.org/10.6345/NTNU202203664

摘要

肺癌是全世界癌症中死亡率第一的癌症。非小型細胞肺癌（NSCLC）佔肺癌病患的比例約75％至80％。化療所造成的副作用與抗藥性在肺癌治療中是有待處理的棘手問題。因此發展出新的抗癌藥物對肺癌的病患是有必要的。此篇論文旨在篩選可抑制肺癌細胞生長新穎的合成化合物。透過MTT的分析方式鑑定EMMQ對非小型肺癌細胞的IC50的濃度。數據顯示低濃度的EMMQ即可減少A549和H460此兩種非小型肺癌細胞的生長速率。研究也證明，EMMQ可誘導具有正常p53基因的非小細胞肺癌的細胞凋亡，而且該藥物對p53-null的非小細胞肺癌則無明顯的效果。研究顯示EMMQ會誘導細胞DNA損傷，活化p53蛋白，干擾粒線體的膜電位而釋放細胞色素c，造成Bcl-2的下降，活化caspase-3， 讓PARP裂解而造成的內生性細胞凋亡。活體的實驗證明，EMMQ可以抑制裸鼠異種移植腫瘤的生長。最後，此研究證實EMMQ可在低濃度時活化非小細胞肺癌細胞的p53而造成細胞凋亡。此外，本研究還發現EMMQ可抑制具有正常 p53基因之非小細胞肺癌細胞的細胞轉移。因此本論文的數據顯示EMMQ可能成為一種新穎且有潛力的人類肺癌治療藥物。

肝癌是世界排名第五大常見的癌症。臨床顯示治療這些肝癌腫瘤中的重要的限制是治療過程中化療藥物長久投藥後的失效且肝癌對這些藥物產生抗藥性的問題。因此，開發肝癌治療中所產生化療失效與抗藥性的抗癌藥物是迫切需要的。之前我們已經確認EMMQ於肺癌細胞與活體的模式中是有效治療的效果。在此研究中證明EMMQ可抑制肝癌細胞的細胞生長進而誘導細胞凋亡。 EMMQ誘導的細胞凋亡為wild type p53的肝細胞癌（HCC）細胞，但是對mutant p53和p53-null細胞不敏感。數據顯示此化合物以內源性的途徑方式誘導細胞死亡。研究證明了EMMQ通過兩個主要途徑誘導細胞凋亡。此化合物使HepG2細胞的DNA損傷進而活化p53和γ-H2AX，降低cyclin D1和CDK 2的表現，導致細胞週期於G1期停滯。其次，此化合物使腫瘤抑制基因p53活化，干擾粒線體膜電位，使得ROS產生，Akt與 Bcl-2表現降低，Bax和細胞色素c的釋放，讓caspase-3和PARP裂解。細胞實驗的結果證明，在肝癌的治療上，EMMQ是一個有潛力的抗癌藥物。

關鍵字

肺癌 ； p53 ； EMMQ ； 細胞凋亡 ； 肝癌

並列摘要

Lung cancer is the leading cause of cancer mortality worldwide. NSCLC accounts for about 75% to 80% of lung cancer cases. Chemotherapy adverse side effects and resistance to current anticancer agents have been the pressing problems in the success of lung cancer therapy. Anticancer drugs are in urgent need especially for lung cancer. A program to develop a new anti-lung cancer agent by screening novel synthetic compounds was initiated.
EMMQ was selected by MTT cell viability assay. The cell growth rate of A549 and H460 NSCLC cells was reduced by a low concentration of EMMQ treatment. Our study revealed that EMMQ induced apoptosis in NSCLC cells with wild-type p53, while the drug is less potent to against p53-null cells. The study elucidated that EMMQ-induced apoptosis is was mediated through the intrinsic pathway involving DNA damage, activation of p53, interference of mitochondrial ΔΨm that led to release of cytochrome c and down regulation Bcl-2, activation of caspase family proteins, and finally cleavage of PARP polymerase cleavage.
In vivo study showed that EMMQ reduced tumorigenesis and significantly suppressed growth rate of xenograft tumors in nude mice. In addition, metastasis studies demonstrated that EMMQ may inhibit wild-type p53 cells migration at low concentration. In conclusion, EMMQ was demonstrated as an effective p53 regulator in NSCLC cells. Our findings indicate that EMMQ may serve as a promising new and potential therapeutic agent for human lung cancer.

Human liver cancer is the fifth most frequently diagnosed cancer worldwide. The important limitation in the clinical battle against this tumor is its marked intrinsic and acquired refractivity to the available chemotherapies. Anticancer agents effective against chemo-resistant cells are greatly needed for liver cancer treatment. Previously, our study have identified EMMQ as an effective drug in the treatment of lung cancer cells in vitro and in animal models. In this work, results show EMMQ treatment may inhibit cell growth and induce apoptosis in HCC. EMMQ induced apoptosis in HCC cells with wild-typed p53, and is less potent in cells with mutant p53 and in p53-null. The study also demonstrated that EMMQ induces apoptosis through two major pathways. First, the compound induced cell death through the intrinsic pathway by first damaging DNA increasing expression of p53 and γ-H2AX and decreasing cyclin D1 and CDK 2, finally leading to G1 arrest in HepG2 cells as studied cell cycle. Second, the tumor suppressor gene p53 was activated following a reduction of ΔΨm, ROS generation and down-regulating Akt, Bcl-2, Bax, cytochrome c, caspase-3 and cleavage of PARP, the critical events leading to cell death in HepG2 cells treated with EMMQ. The in vitro findings indicate that EMMQ is a promising candidate for the treatment of liver cancer.

並列關鍵字

none

參考文獻

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