指導教授：謝銘鈞臺灣大學：醫學工程學研究所魏名峰Wei, Ming-FengMing-FengWei2014-11-302018-06-292014-11-302018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/264478最近的研究指出，癌症幹細胞 (CSCs) 是導致對目前臨床上的抗癌治療模式產生拮抗的主要來源，也包括對光動力療法（PDT）的拮抗。並更進一步影響大腸直腸癌（CRC）的復發。因此，將癌症幹細胞視為標的，發展更有效的抗癌策略，將是未來治療癌症的關鍵。在本研究中，我們利用流式細胞儀從大腸直腸癌細胞中篩選出具CD133表面抗原的癌症幹細胞，同時驗證是否具備幹細胞相關的特性。首先，我們證明在PDT治療過程中會誘發CD133細胞產生較高的細胞自噬能力，這可能是對PDT產生抗性的主因。藉由相關的藥物與shRNA抑制細胞自噬能促進CD133細胞凋亡並降低的形成colonosphere和致瘤的能力。第二，我們抑制CD133 細胞內硫氧還蛋白1（Thioredoxin 1; Trx1）的表現，提升PDT誘導的ROS產量，增強毒殺CD133細胞的能力。除此之外，我們發現Trx1的抑制降低了CD133細胞形成colonosphere能力，也使常規化療藥物更易於毒殺CD133細胞。第三，我們採用EMT (Epithelial–mesenchymal transition)的特性去增加癌症細胞內CD133 細胞所占比例，並建立癌症幹細胞的動物模式。經由EMT過程產生的癌細胞會更有效的誘發腫瘤生成。綜合上述結果，期盼我們的發現有助於發展有效針對癌症幹細胞的新型療法，達到癌症治癒的目的。Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). Therefore, more effective anticancer strategies targeting CSCs will be critical for developing a cure for cancer. In this study, CSCs were isolated from colorectal cancer cells using CD133 expression and analyzed it’s stemness-associated properties. We demonstrated first that autophagy may be the main cause of resistance to PpIX-mediated PDT in CD133+ cells. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of autophagy-related gene substantially triggered apoptosis of CD133+ cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. Second, we elevated ROS levels to improve the cytotoxicity of PDT to CSCs by inhibition of thioredoxin 1 (Trx1). In addition, we found that silencing of Trx1 reduced the ability of sphere formation of CD133+ cells and sensitized CD133+ cells to conventional chemotherapy. Third, we increased the percentage of CD133+ cells in cancer population and established an in vivo CSCs model by inducing EMT in colorectal cancer cells. Cells underwent an EMT are more efficient in tumor-initiating potential and increase stemness-associated properties. Based on the results, these findings would aid in the development of novel therapeutic approaches for CSC treatment.口試委員會審定書……………………………………………………………………I 誌謝……………………………………………………………………………………II 中文摘要……………………………………………………………………………III Abstract………………………………………………………………………………IV Chapter 1. Introduction……………………………………………………………1 1.1 Colorectal cancer stem-like cells……………………………………………1 1.2 The resistance to photodynamic therapy in CSCs…………………2 1.3 The role played by autophagy in cytotoxicity of PDT………………………4 1.4 The functions of thioredoxin…………………………………………………6 1.5 The establishment of stable colorectal CSCs and their animal models……………8 Chapter 2. Materials and Methods……………………………………………11 Chapter 3. Results……………………………………………………………………21 Part 1. The resistance to photodynamic therapy in CSCs……………………21 3.1.1 CD133+ cells of colorectal cancer exhibit the characteristics of CSCs……………………………………………………………………21 3.1.2 CD133+ cells take up more protoporphyrin IX………………22 3.1.3 CD133+ cells of colorectal cancer exhibit the resistance to PDT……………………………………………………………………23 3.1.4 CD133+ cells of colorectal cancer can inhibit generation of ROS produced by PDT…………………………………………………………24 Part 2. Autophagy promotes resistance to photodynamic therapy-induced apoptosis in colorectal cancer stem-like cells…………………………………26 3.2.1 PDT induces autophagy in CD133+ cells…………………………………26 3.2.2 PDT-induced autophagy protects CD133+ cells from PDT-induced cell death……………………………………………………………………27 3.2.3 Silencing of autophagy-related genes sensitized CD133+ cells to PDT…………………………………………………………………28 3.2.4 Inhibition of PDT-induced autophagy enhances the apoptotic effect of PDT in CD133+ cells…………………………………………30 3.2.5 Blocking autophagy enhances the antitumorigenicity of PDT in a xenograft mode……………………………………………………………31 3.2.6 Blocking the Akt-mTOR pathway through the application of PDT also promoted the activation of autophagy in CSCs………………………33 Part 3. Thioredoxin 1 plays a critical role to sustain CSCs properties………34 3.3.1 Inhibition of Trx1 enhances the cytotoxicity of PDT in CD133+ cells………………………………………………………………………34 3.3.2 Trx1 contributes to the stemness-associated properties in CD133+ cells………………………………………………………………………35 Part 4. Elevation of tumorigenicity in colorectal cancer cells and establishment of CSCs animal model by EMT…………………………………………………37 3.4.1 An increase in the percentage of CD133+ cells by genetic silencing of E-cadherin……………………37 3.4.2 Knockdown of E-cadherin enhances the tumor-initiating potential of colorectal cancer cells in a xenograft model………38 Chapter 4. Discussion………………………………………………………………40 References……………………………………………………………………………46 Figure legends………………………………………………………………………64 Figures………………………………………………………………………………80 APPENIX…………………………………………………………………………107 Publication list………………………………………………………………………1089621792 bytesapplication/pdf論文公開時間：2017/08/04論文使用權限：同意有償授權(權利金給回饋學校)癌症幹細胞光動力療法大腸直腸癌CD133細胞自噬硫氧還蛋白1EMT[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264478/1/ntu-103-D95548004-1.pdf