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標題: | 運用熱穩定分析探討中國橄欖萃取物在大腸直腸癌細胞株的蛋白質標的與促進細胞凋亡機制 Identification of protein targets and proapoptotic mechanism of Chinese olives extract in colorectal cancer cell lines with thermal stability analysis |
作者: | GUAN-TING LIAN 練冠霆 |
指導教授: | 謝淑貞(Shu-Chen Hsieh) |
關鍵字: | 大腸直腸癌,中國橄欖,細胞凋亡,蛋白質體學,細胞熱轉變分析, Apoptosis,Canarium album L.,Colorectal cancer,the Cellular thermal shift assay,Label-free quantitative proteomics, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 大腸直腸癌 (colorectal cancer, CRC)的發生率與致死率皆為台灣癌症前三高,富含蔬果且未經加工的飲食模式與高纖維攝取為大腸直腸癌的預防因子,其可能原因為蔬果中的植化素。中國橄欖 (Canarium album L.)過去被用作傳統中藥材,本實驗室先前研究顯示中國橄欖果實水萃物殘渣甲醇萃取乙酸乙酯區分層 (Chinese olive extract, COE)對大腸直腸癌細胞具有抗增生與促進細胞凋亡的功效。然而,詳細作用機制與上游作用標的並不清楚。 本研究指出,相較於正常腸上皮細胞IEC-6 cell line,COE對腸癌細胞株HCT116與CaCO-2有較低的半致死劑量。差異蛋白質體學的結果也顯示COE處理後,除了影響HCT116細胞週期與細胞凋亡途徑外,代謝相關途徑 (如glycolysis process)也有顯著差異。進一步利用細胞熱轉變分析 (cellular thermal shift assay, CETSA)結合蛋白質體學分析配體結合作用標的時所引發的熱穩定性變化,結果發現6-phosphofructokinase (PFK1)熱穩定性受COE影響,並以intact cell與cell lysate的條件下進行CETSA證明溶解曲線改變,推論COE含有PFK1的配體。酵素活性分析發現在in vitro與cell lysate實驗中PFK1的活性受到COE抑制。美國癌症基因體圖譜計畫中的大腸癌資料庫也發現PFKM表現量較高的患者有較低的三年存活率。經實驗結果得知,PFK1的活性下降導致ATP濃度降低與ADP/ATP比例上升,進而活化AMPK促進p53相關細胞凋亡基因 (BAX與BID)表現量提高。最後,我們以分子模擬 (molecular Docking)尋找COE中可能抑制PFK1的功效性成分,發現COE內的amentoflavone相較於PFK1的受質有更高的親和力,最後在細胞存活試驗中也發現對HCT116細胞具有毒殺性。綜合上述,本研究分析蛋白熱穩定性變化證實COE與PFK1的直接交互作用,導致細胞產生能量壓力,進而促進AMPK與p53路徑活化,最終導致細胞凋亡。 The prevalence and motility of colorectal cancer (CRC) has been ranked top 3 in Taiwan. Prudent dietary pattern with high intake of vegetables, fruits and total fiber is preventive factor for CRC, and the phytochemicals rich in vegetables and fruits could be the contributing factor. Canarium album L. is used as traditional Chinese medicine. Previous reports in our lab revealed that the methanol-ethyl acetate partitioned fraction from Chinese olive fruits (COE) exhibited profound anti-proliferative and proapoptotic activities in the human colon cancer cell line, HCT116. However, the proapoptotic mechanism and binding targets of COE are still unclear. In this research, the concentrations of the half of maximal growth inhibition (GI50) in colorectal cancer HCT116 and CaCO-2 cells were much lower comparing to the normal intestine IEC-6 cells. Differential proteomics also indicated that there are significant differences in cell cycle, apoptosis and metabolic pathways in HCT116 cells after COE treatment. We combined cellular thermal shift assay (CETSA) and label-free quantitative proteomics to find out the possible target candidates based on ligand-induced change in protein thermal stability. We found that the thermal stability of 6-phosphofructokinase (PFK1) was affected by COE treatment. We utilized intact cell and cell lysate to conduct CETSA experiments to prove the shift of protein melting curve and we deduced that PFK1 is a possible target for COE. The inhibitory effect of COE on PFK1 was revealed with enzyme activity assay. The Cancer Genome Atlas (TCGA) database analysis revealed that colon cancer patients with higher PFK1 expression have lower survival probability within three years. Our results displayed the correlation of decreased PFK1 activity and dramatic decrease of cellular ATP concentration and increase of ADP:ATP ratio, which induced AMPK activation and the consequent higher p53-related genes (BAX and BID) expression. Finally, we conducted molecular docking to find out possible inhibitors of PFK1 in COE, and amentoflavone turn out to be the candidate that exhibits higher binding affinity and cytotoxicity when compared with PFK1 substrates. In conclusion, we analyzed protein thermal stability changes to confirm the direct interaction between COE and PFK1, and the inhibition of PFK1 resulted in cell energy stress following activation of proapoptotic AMPK/p53 pathway. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55762 |
DOI: | 10.6342/NTU202002008 |
全文授權: | 有償授權 |
顯示於系所單位: | 食品科技研究所 |
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