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系統識別號 U0026-1601201717280800
論文名稱(中文) 探討半乳糖凝集素-3在大腸癌抗藥性中的角色及分子機制
論文名稱(英文) Investigating the molecular roles and mechanisms of Galectin-3 on drug resistance in colon cancer
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 105
學期 1
出版年 105
研究生(中文) 李永國
研究生(英文) Yung-Kuo Lee
學號 S58991081
學位類別 博士
語文別 英文
論文頁數 88頁
口試委員 指導教授-張權發
口試委員-王憶卿
口試委員-呂增宏
口試委員-李政昌
口試委員-林秋烽
口試委員-許昺奇
中文關鍵字 大腸直腸癌  抗藥性  癌症起始細胞  半乳糖凝集素-3  塑化劑 
英文關鍵字 Colorectal cancer  drug resistance  tumor-initiating cells  galectin-3  plasticizing agents 
學科別分類
中文摘要 大腸直腸癌是全球十大癌症死亡原因之一,也是近十年來台灣腫瘤發生率與發生人數最多的一種癌症。由於手術後的高復發率以及化療無效反應(抗藥性)的結果,每年的死亡率和死亡人數也是居高不下。許多研究發現,癌症的高復發率以及高轉移率與兩個因素有高度相關性,其一是多重要抗藥性相關蛋白的表現量上升,另一則是腫瘤中存在的癌症起始細胞(cancer initiating cells, CICs or cancer stem cells, CSCs)。腫瘤幹細胞除了具有分化成癌細胞以及自我修復等特性之外,也具備較高的抵抗藥物能力。因此,研究大腸癌細胞抗藥性的分子機制,進而探討細胞抗藥性在癌症起始細胞中的角色,為本論文主要的目標。我們首先發現半乳糖凝集素3 (galectin-3) 同時參與在大腸癌抗藥能力的調控,以及癌症起始細胞特性的維持。大腸癌細胞會透過大量表現galectin-3,經由β-catenin/GSK-3β路徑會促進細胞內多重抗藥蛋白的表現上升。我們接著發現大腸癌細胞腫瘤懸浮球體(spheres)中的 galectin-3表現量有明顯增加;透過RNAi系統抑制大腸癌細胞中的galectin-3表現,則會減少腫瘤懸浮球體的大小、數量、以及相關蛋白的表現,而外加galectin-3抑制劑也可獲得類似的效果。根據以上的研究結果顯示,大腸癌細胞在抗癌藥物或是塑化劑的刺激之下,可以透過不同的分子機制促進多重抗藥蛋白的表現,以及表現出癌症起始細胞的相關基因與能力;而galectin-3在大腸癌細胞的多重抗藥性與癌症起始細胞特性上,也扮演相當關鍵的角色。基於這些結果,我們建議針對galectin-3可能是改善結腸癌治療的有效方法。
英文摘要 Colorectal cancer (CRC) is among the top ten causes of cancer-related death worldwide, and it also accounts for the highest incidence of cancer in Taiwan over the past decade. The prognosis of CRC remains poor because of the high recurrence rates and low response to chemotherapy (drug resistance). Many studies have demonstrated two factors, namely the upregulation of multidrug resistance (MDR)-associated proteins and presence of tumor-initiating cells (cancer-initiating cells, CICs, or cancer stem cells, CSCs), that are correlated with a high rate of recurrence and high metastatic ability. CSCs are capable of self-renewal and differentiation into various types of cancer. Moreover, CSCs are proposed to be responsible for resistance to chemotherapy. Therefore, this study investigated the roles and molecular mechanisms of drug resistance in CRC, in addition to elucidating the drug resistance mechanisms in colon CSCs. We determined that galectin-3 (Gal-3) participated in drug resistance modulation in CRC and in maintaining stemness in CSCs. Increased Gal-3 expression in colon cancer cells involves the upregulation of MDR-related proteins through the induction of β-catenin/GSK-3β signaling. Furthermore, the expression of Gal-3 increased significantly in colon tumor spheres. The suppression of Gal-3 through RNA interference in colon cancer cells decreased Gal-3 expression and reduced the size, number, and stemness-related proteins in tumor spheres. In addition, these phenomena could be rescued by Gal-3 inhibitor treatment. Thus, the exposure of colon cancer cells to anticancer drug stimulation or plasticizing agents may promote the expression of multidrug-resistant proteins through different molecular mechanisms and exhibit CIC-related gene expression and activity. Taken together, Gal-3 plays key roles in MDR and tumor-initiating cell properties. Accordingly, we suggest that targeting galectin-3 may be a potent approach for improving colon cancer therapy.
論文目次 Abstract in Chinese I
Abstract II
致謝 IV
Table of contents V
List of Abbreviations VIII
Introduction 1
Colorectal cancer and multidrug resistance 1
Galectin-3 in cancer 3
Gal-3 and MDR 4
Colon cancer-initiating cells 6
Colon CICs and MDR 7
Specific aims 9
Results 11
Combined epirubicin and shGal-3 treatment significantly intensified epirubicin cytotoxicity 11
Combined epirubicin and shGal-3 treatment induced apoptosis-mediated morphological changes in Caco-2 cells and increased sub-G1 accumulation in the Caco-2 cell cycle 11
shGal-3 significantly increased intracellular epirubicin retention in Caco-2 cells 12
Combined shGal-3 and epirubicin treatment significantly inhibited the mRNA expression of Gal-3 and β-catenin but increased the expression of GSK-3β 12
Combined shGal-3 and epirubicin treatment inhibited the mRNA expression levels of the cyclin D1 and c-myc that were induced by the epirubicin treatment 12
Combined shGal-3 and epirubicin treatment significantly inhibited the mRNA expression levels of MDR1, MRP1, and MRP2 12
Confirmation of the protein expression level of the Wnt/β-catenin signaling pathway and apoptosis-associated pathway components after epirubicin treatment in wild-type or Gal-3 knockdown Caco-2 cells 13
Knockdown of Gal-3 in Caco-2 cells inhibited P-gp and cyclin-D1 protein expression after epirubicin treatment 13
Identification of a new monoclonal antibody recognizing CD133 14
Gal-3 attenuation reduced sphere formation and stemness-related gene expression 15
Cancer sphere formation indeed requires Gal-3 15
Lactose suppressed colon cancer sphere formation with Oct4 downregulation in the cancer sphere formation process 16
Gal-3 induces the formation of Gal-3/Oct4 complexes for cancer sphere formation 16
Nuclear interaction of Gal-3 with Oct4 in cancer spheres 17
TD-139 inhibited colon cancer sphere formation in a dose-dependent manner 17
Use of efflux pump inhibitors significantly reduces cell viability in DEHP/MEHP-treated HCT116 colon cancer cells 17
DEHP/MEHP-induced drug resistance and antiapoptosis but without Gal-3 increase in HCT116 colon cancer cells receiving anticancer drug treatment 18
Long-term DEHP/MEHP exposure can induce sphere formation, which was inhibited by Gal-3 knockdown in HCT116 colon cancer cells 19
Discussion 20
Conclusion 24
Materials and Methods 25
Materials 25
Antibodies 25
Reagents 26
Plasmid 27
Consumables and devices 27
Methods 28
Cell culture and experimental conditions 28
Construction of galectin-3 shRNA 28
Cytotoxicity assay 28
Apoptosis detection assay 29
Observation of chromatin condensation using a fluorescence microscope 29
Cell cycle analysis 30
Intracellular epirubicin accumulation 30
Construction and overexpression of galectin-3 30
Real-time PCR of P-gp, MRP1, MRP2, Bax, and Bcl-2 31
Western blotting 32
Statistical analysis 32
CD133 antigen design and generation 33
Immunization and monoclonal antibody production 33
Cell culture and experimental conditions 33
Immunofluorescence 34
FACS staining 34
Lentivirus-based RNAi knockdown 35
Construction and overexpression of galectin-3 35
Western blotting 35
Total RNA preparation and RT-PCR 36
Test of Lactose (Lac) effect on spheres formation 36
Co-mmunoprecipitation Assays 37
Test of TD-139 effect on spheres formation 37
Preparation of cell nuclear and cytosol fractions 38
Statistical analysis 38
Cell culture and experimental conditions 39
Western blotting 39
P-glycoprotein activity assay 40
Cell proliferation analysis 40
Sphere formation analysis 41
Statistical analysis 41
Tables 42
Table 1. The sequences of shRNA and primer sequences used for knock-down and real-time PCR. 42
Figures 43
Appendix 82
Reference 84
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