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研究生: 呂奕靜
LU, I-Ching
論文名稱: 植化素Withaferin A 引發人類非小細胞肺癌鐵依賴型細胞死亡的影響
Effect of Phytochemical Withaferin A-Induced Ferroptosis in Human Non-Small Cell Lung Cancer Cells
指導教授: 蘇純立
Su, Chun-Li
口試委員: 蕭寧馨
Shaw, Ning-Sing
黃奇英
Huang, Chi-Ying
口試日期: 2021/08/23
學位類別: 碩士
Master
系所名稱: 營養科學碩士學位學程
Graduate Program of Nutrition Science
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 122
中文關鍵詞: 鐵依賴性細胞死亡肺癌基因分析Withaferin A臨床化療藥物
英文關鍵詞: iron-dependent cell death, lung cancer, genetic analysis, Withaferin A, clinical chemotherapy drugs
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202101153
論文種類: 學術論文
相關次數: 點閱:154下載:0
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  • 鐵依賴型細胞死亡(ferroptosis)為一調節性細胞死亡形式,其導致細胞死亡的特徵為:還原活化態的鐵存在、氧化含多元不飽和脂肪酸的磷脂質及脂質過氧化修復能力缺失,被認為是對抗惡性癌細胞的新契機。肺癌是全球死亡人數最多的癌症,預後不佳且死亡率逐年攀升,除傳統治療方法外,臨床也針對EGFR突變型(mutant-type)的肺癌細胞株開發標靶藥物,但對於無突變的EGFR 原生型(wild-type)肺癌細胞效果不彰。本研究透過基因分析發現肺癌細胞內鐵相關基因FTH1表現量不僅與ferroptosis發生有關,亦影響南非醉茄之萃取物Withaferin A (WA)作用於肺癌細胞的活性。經實驗證實儲鐵蛋白FTH1表現量較低的惡性非小細胞肺癌細胞CL1-5對ferroptosis inducer敏感性高,且WA經由提高進鐵蛋白TFRC提升細胞內鐵含量、增加生成細胞lipid ROS與抑制細胞抗氧化能力,並透過自噬體包裹、降解FTH1,促使容易產生氧化還原作用的游離鐵釋出,誘導CL1-5細胞發生ferroptosis。另外,WA合併肺癌臨床化療藥物能藉由引發ferroptosis造成肺癌細胞死亡。綜合以上結果,透過天然植化素WA誘導癌細胞產生ferroptosis,以改善EGFR 原生型之非小細胞肺癌細胞對化療藥物及標靶藥物療效有限的治療困境,是對抗惡性肺癌腫瘤相當具有潛力的新策略。

    Ferroptosis is a regulated cell death characterized by the availability of redox active iron, oxidation of polyunsaturated fatty acid containing phospholipids and loss of lipid peroxide repair capacity. It is considered as a new hope for fighting malignant cancer cells. Lung cancer has been the leading cause of cancer death worldwide. The prognosis of lung cancer is poor and the mortality rate is increasing year by year. In addition to traditional treatment methods, targeted therapy has also been developed for EGFR mutant lung cancer cell lines, but the efficacy of EGFR wild-type lung cancer cells is limited. In the present study, database analysis results show that iron-related gene, FTH1 expression in lung cancer was not only associated with ferroptosis, but also the drug activity of withaferin A (WA), a kind of phytochemical extracted from Ashwagandha. CL1-5, a malignant non-small cell lung cancer (NSCLC) cell line, with a lower protein expression of FTH1 was sensitive to ferroptosis inducer, and WA-induced ferroptosis in CL1-5 proceeded through elevating the intracellular iron level by upregulating the protein expression of TFRC, increasing the production of lipid ROS. The antioxidant capacity was also inhibited. Besides, WA caused the degradation of FTH1 by formation of autophagosomes to release free iron which triggers ferroptosis, and the cell death effect of the combination of WA and lung cancer chemotherapeutic drugs on NSCLC is related to WA-induced ferroptosis. Overall, our results suggest that WA may increase the chance to fight against EGFR wild-type NSCLC and improve the quality of medical treatment.

    第一章 緒論 1 第一節 鐵依賴型細胞死亡(ferroptosis) 1 一、Ferroptosis的特徵 2 二、Ferroptosis作用機制 4 三、Ferroptosis的調控 5 四、Ferroptosis於癌症治療之應用 6 第二節 腫瘤與鐵(tumor and iron) 7 第三節 肺癌(lung cancer) 9 一、肺癌的流行與發生 9 二、肺癌的類型及治療 10 三、CL1肺腺癌細胞株 12 第四節 臨床化療藥物 13 一、順鉑(cisplatin)13 二、Alimta®愛寧達(pemetrexed) 14 第五節 Withaferin A (WA) 16 第二章 研究目的 19 第三章 材料與方法 21 第一節 藥品與試劑 21 第二節 儀器與實驗耗材 25 第三節 實驗方法 29 一、細胞培養、繼代、解凍與冷凍保存 29 二、藥品配製 33 三、基因資料庫分析(database analysis) 36 四、細胞存活率分析(cell viability analysis) 38 五、脂質過氧化分析(lipid ROS analysis) 42 六、西方墨點法(Western blotting) 44 七、免疫螢光染色(immunofluorescence) 55 八、免疫沉澱法(immunoprecipitation) 58 九、統計分析(statistical analysis) 62 第四章 結果 63 第一節 FTH1基因表現量與ferroptosis敏感度具有相關性 63 第二節 惡性非小細胞肺癌細胞CL1-5較容易誘導ferroptosis發生 67 第三節 低濃度WA造成惡性非小細胞肺癌細胞CL1-5的生長抑制 70 第四節 WA透過ferroptosis引發惡性非小細胞肺癌細胞CL1-5死亡 73 第五節 WA改變惡性非小細胞肺癌細胞CL1-5中鐵相關蛋白表現量,且透過調節儲鐵蛋白影響細胞內游離鐵分布 76 第六節 WA增加惡性非小細胞肺癌細胞CL1-5產生lipid ROS 79 第七節 WA合併化療藥物會誘導非小細胞肺癌細胞發生ferroptosis 82 第五章 討論 85 第六章 結論 97 參考文獻 98 附錄 108 Supplementary data 110

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