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研究生:葉名威
研究生(外文):YEH, MING-WEI
論文名稱:分析生醫資料庫預測並驗證可促進5-FU毒殺LoVo大腸癌細胞株之潛力藥物
論文名稱(外文):In silico predication and validation of the potential compound to promote 5-FU mediated cytotoxic effect in LoVo colorectal cancer cells
指導教授:賴金美
指導教授(外文):LAI, JIN-MEI
口試委員:黃奇英楊小青
口試委員(外文):HUANG, CHI-YINGYANG, HSIAO CHING
口試日期:2022-09-05
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:36
中文關鍵詞:大腸癌老藥新用
外文關鍵詞:colon cancerdrug repurposing
相關次數:
  • 被引用被引用:0
  • 點閱點閱:170
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  • 下載下載:16
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大腸癌(CRC)是台灣癌症死亡的第三大主要原因。一般而言大腸癌的主要治療
方式包含手術、化療和標靶藥物治療。然而這些藥物治療,往往會遇到抗藥性
的問題造成治療上困境,以至於大腸癌患者的存活率無法有效提高。近年來,
老藥新用(Drug Repurposing)-即原先已經核准臨床使用的藥物賦予新用途,已常見的癌症被用作尋找新抗癌藥物的新策略。基於大腸癌的高基因異質性,目前已有研究利用大腸癌組織的基因表徵(gene signature)進行Consensus molecular subtypes(CMS)分型,分別為CMS1-4。其中CMS4分型的大腸癌細胞具有較強的轉移能力、病患治療後復發率較高及較差之整體生存率。實驗室先前已利用
DepMap資料庫(含多種癌細胞之藥物敏感性資料庫),預測數個可對抗CMS4
大腸癌的候選藥物,其中,Disulfiram 為本論文主要探討的藥物。Disulfiram在數個對於傳統化療藥物 5-FU 敏感性較不佳的大腸癌細胞株,如 LoVo (5-FU作24小時之IC50>400 µM),皆具毒殺效果(Disulfiram作用 48小時之IC50約為13.1 µM);而Disulfiram在有銅離子(Cu2+)合併處理的情況下,細胞毒殺效果更加提升使得IC50下降至52.5 nM。利用Western blot探討藥物作用機制得知Disulfiram可促進細胞之HO-1, γ-H2AX, p62 等蛋白質的表現量增加,由於ROS抑制劑可明顯抑制Disulfiram/Cu2+所誘導之細胞死亡及上述蛋白質之增加,目前將繼續探討 ROS/UPR (unfolded protein response)及細胞凋亡相關之訊息傳遞路徑在其中扮演的角色,並測試合併使用Disulfiram/Cu2+是否可藉上述之機制以增強5-FU對大腸癌細胞株的細胞毒殺效果。

Colorectal cancer (CRC) is the third leading cause of cancer death in Taiwan. In general, the main treatment methods for colorectal cancer include surgery, chemotherapy and targeted drug therapy. However, these drug treatments often encounter drug resistance, which makes the treatment difficult and reduces the survival rate of colorectal cancer patients. Recently, repurposing drugs by identifying new uses for approved or investigational drugs, has been used as a strategy to identify new anti-cancer drugs. Based on the high genetic heterogeneity of colorectal cancer, there have been studies using the gene signature to classify colorectal cancer tissue by Consensus molecular subtypes (CMS), which are CMS1-4. Among CMS, CMS4-typed colorectal cancer cells have strong metastatic ability, high recurrence rate after treatment, and poor overall survival rate. We used the DepMap database (a database with drug sensitivity of various cancer cells) to predict several candidate drugs that are effective in treating CMS type 4 colorectal cancer. In these drugs, Disulfiram is the main focus of this paper. Disulfiram has cytotoxicity effect on several colorectal cancer cell lines that are less sensitive to traditional chemotherapeutic drugs 5-FU, such as LoVo (IC50 of 5-FU for 24 hours>400 µM) (IC50 of Disulfiram for 48 hours is about 13.1 µM); and Disulfiram in the case of combined treatment with copper ions (Cu2+), the cytotoxic effect was further improved to IC50 of 52.5 nM. The mechanism action was evaluated by Western blot which showed Disulfiram promoted the expression of HO-1, γ-H2AX, p62 and other proteins in cells. Since ROS inhibitors can significantly attenuate Disulfiram/Cu2+-induced cytotoxicity and up-regulated protein expression, we will continue to explore the role of ROS, UPR (unfolded protein response) and apoptosis-related signaling pathways, and test whether the combined treatment of Disulfiram/Cu2+ and 5-FU can enhance the cytotoxicity of 5-FU in colorectal cancer cell lines through the above mechanism.
摘要 i
Abstract ii
壹、序論 1
1.1、大腸癌的介紹 2
1.2、大腸癌治療現況 2
1.3、使用於CRC的化療藥物的介紹 2
1.4、癌症抗藥性 5
1.5、大腸癌的分類與共識分子亞型 5
1.6、DepMap資料庫 6
1.7、Profiling relative inhibition simultaneously in mixtures (PRISM) 7
1.8、精準醫療 8
1.9、老藥新用 8
1.10、候選藥物資訊 8
1.11、研究動機及目的 9
貳、實驗材料 10
一、細胞株 10
二、化學藥品試劑 10
三、抗體 11
參、實驗方法 12
一、大腸癌LoVo細胞之繼代與培養 12
二、以Sulforhodamine B (SRB) assay進行細胞活性測試 12
三、以MTT assay進行細胞活性測試 12
四、細胞藥物處理與蛋白質萃取 13
五、蛋白質濃度定量 13
六、Western blot analysis 13
肆、實驗結果 16
4.1、尋找具有抑制大腸癌細胞之潛力藥物 16
4.2、測試傳統化療藥物5-FU藥物對LoVo大腸癌細胞株存活率的抑制能力 16
4.3、測試候選藥物Bromhexine及Disulfiram對LoVo大腸癌細胞株存活率的影響力 16
4.4、測試候選藥物 Disulfiram/Cu2+ 對LoVo大腸癌細胞株存活率的變化 17
4.5、以Western blot分析Disulfiram/Cu2+ 對LoVo大腸癌細胞株的作用機制 17
4.6、以添加抑制劑的方式探討 Disulfiram/Cu2+ 影響LoVo大腸癌細胞株存活率的主要機制 17
伍、討論 19
一、Disulfiram與Disulfiram/Cu2+對癌細胞的效果 19
二、Disulfiram和5-FU對大腸癌細胞誘發死亡的藥效差異 19
三、Disulfiram/Cu2+ 誘發大腸癌細胞死亡的機制探討 19
四、對於Disulfiram /Cu2+ 所引發的細胞死亡機制或其他細胞毒性的後續探討 20
五、銅離子錯合物Elesclomol/Cu2+ 與本實驗結果Disulfiram/Cu2+ 的關聯性 21
六、關於 Disulfiram/Cu2+ 是否透過銅死亡的方式產生細胞毒性的後續探討方式 23
陸、圖表 24
Figure 1. Figure 1. Drug sensitivity score of 5-FU and Disulfiram in CRC cells identified in Depmap/PRISM database 25
Figure 2. The cytotoxic effect of 5-FU in LoVo cells 27
Figure 3. The cytotoxic effect of Bromhexine and Disulfiram in LoVo cells 28
Figure 4. The cytotoxic effect of Disulfiram/Cu2+ in LoVo cells 29
Figure 5. To investigate the mechanism of how Disulfiram/Cu2+ inhibit the cell viability of LoVo cells 30
Figure 6. To investigate the mechanism of how Disulfiram/Cu2+ inhibit the cell viability of LoVo cells by adding various inhibitor 32
柒、附錄 31
Supplementary Table 1. Previous clinical drug screening. 33
捌、參考文獻 34

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