臺灣博碩士論文加值系統

結腸癌病人常伴有葉酸營養不良狀態，此不良葉酸營養微細環境是否調轉Warburg能量代謝誘導結腸癌幹細胞之惡性發展及其分子作用機轉，尚不清楚，故本研究探討不良葉酸營養微細環境調轉生物能量代謝誘導結腸癌幹細胞惡性特質之蛋白質體指標檢定。以人類結腸腺癌細胞株SW480，模擬腸癌病人低葉酸血清濃度 (3-6 ng/ml) 培養4天，以LC-MS/MS與MaxQuant進行定量性蛋白質體及磷酸化蛋白質體表現，以gene ontology生物資訊軟體及STRING功能關聯網絡，分析低葉酸微細環境所調控能量代謝與癌幹細胞惡性發展的總體生物代謝路徑、訊息網絡與標的指印分子。結果指出低葉酸微細環境顯著影響83個蛋白分子表現，包括細胞外基質組織、細胞內胞器、黏附因子和分子伴侶蛋白中與細胞結構及細胞骨架相關之DYNLL1、AKAP12、PPIF、HSP90β蛋白表現顯著上調，CD9、STMN2、AKAP8L蛋白表現顯著下調，以及能量代謝調轉因子相關之EBP、UQCRQ、PIH1D1、PDP1、mTOR蛋白表現顯著上調，PFDN1、LCLAT1、SLC16A1蛋白表現顯著下調。磷酸化蛋白質體分析結果中，同樣發現顯著上調與細胞結構及細胞骨架相關之磷酸化蛋白質DYNLL1、AKAP12、PKA、PKC、HSPβ1表現，以及能量代謝調轉因子相關之磷酸化蛋白質Akt、p70S6K、PDK、MAPK1表現。以Western blot驗證與細胞結構及細胞骨架相關蛋白質體特徵表現，並分析Warburg能量代謝相關的蛋白質表現，發現在低葉酸組中pAkt、mTOR、HIF-1α蛋白顯著表現，PDH蛋白表現量有增加的趨勢；下調Akt、FoxO3a、CK1-α、ACLY、LDH蛋白表現。於低葉酸微環境 (10 nM) 中培養結腸癌細胞SW480誘發低葉酸代謝壓力，進行無貼附性腫瘤球形成實驗 (anchorage-independent oncospheroid formation)，觀察癌細胞自我更新之幹性表現 (self-renewal)，發現低葉酸組形成的腫瘤球數量顯著高於對照組。分析乳酸堆積量，發現低葉酸組培養液乳酸堆積濃度顯著低於正常葉酸組。檢測細胞內NADP＋及NADPH濃度，結果顯示NADP＋/NADPH ratio在低葉酸營養狀態下有較高的趨勢。mTOR抑制劑rapamycin (5 μM) 處理後能夠反轉低葉酸誘發能量代謝壓力訊息傳遞與癌幹細胞特性的表現。本研究檢定不良葉酸營養微細環境調轉生物能量代謝誘導結腸癌幹細胞惡性特質之蛋白質體標的指印，提供降低結腸癌惡性轉移的營養介入防治策略之檢測蛋白分子參考效標。

Colon cancer patients are often accompanied with folate malnutrition. Folate malnutrition microenvironment may alter Warburg energy metabolism to induce malignant development, but the mechanisms are unknown. Therefore, the aims of the study were to investigate that proteomic signatures of low folate metabolic stress-induced metastasis potentials in human colonic adenocarcinoma cells: reprogramming bioenergetics and cancer stem cell features. SW480 cells were cultured in medium containing 2.2 μM folate (control (C) group) and 10 nM folate (low folate (LF) group) for 4 days, which is simulated colorectal cancer patients with low folic acid serum concentration (3-6 ng/ml). Quantitative proteomics and phosphorylated proteomics expression was conducted by LC-MS/MS and MaxQuant. The overall biological metabolic pathways, signaling networks and target fingerprints of low folate microenvironment regulated energy metabolism and malignant development of cancer stem cells were analyzed by gene ontology bioinformatics software and STRING functional association network. The data indicate that low folate microenvironment significantly affected the expression of 83 protein molecules including extracellular matrix origanization, intracellular organelles, cell adhesions, chaperones and energy metabolism regulatory factors. The expression of cell structure and cytoskeleton related proteins DYNLL1, AKAP12, PPIF and HSP90β were significantly up-regulated, and CD9, STMN2 and AKAP8L were significantly down-regulated. The expression of energy metabolism regulatory factors related proteins EBP, UQCRQ, PIH1D1, PDP1, mTOR were significantly up-regulated, and PFDN1, LCLAT1, SLC16A1 were significantly down-regulated. The results of phosphorylated proteomics were also found up-regulated cell structure and cytoskeleton related proteins, DYNLL1, AKAP12, PKA, PKC, HSPβ1, and energy metabolic regulatory factors related proteins, Akt, p70S6K, PDK, MAPK1. Western blot was used to verify the proteomic signiatures of cell structure and cytoskeleton and analyze the energy-metabolism related proteins. The data showed that mTOR, HIF-1α protein were significantly up-regulation and Akt, FoxO3a, ACLY, LDH protein were down-regulation in LF-group. SW480 cells were cultured low folate microenvironment (10 nM) to induce low folate metabolic stress. Cells were conducted anchorage-independent oncospheroid formation to observe stemness expression of self-renewal and found that the number of onco-speroids in LF-group were significant higher than the other groups. The concentration of lactic acid was significant lower in LF-group medium. The NADP+/NADPH ratio was higher in LF-group. mTOR inhibitor, rapamycin (5 μM) treatment could reverse the expression of low folate induced energy metabolism stress signaling pathways and the expression of cancer stem cell characteristics. In this study, proteomics fingerprints of folate malnutrition microenvironment altered bio-energy metabolism to induce malignant characteristics in colon cancer stem cells, to reduce the colon cancer malignant metastasis of the nutritional intervention strategy for the detection of protein molecules reference effect.

摘要 I
Abstract IV
致謝 VII
目錄 IX
圖目錄 XIV
表目錄 XVI
縮寫表 (Abbreviations) XVII
第一章 前言 1
第二章 文獻回顧 3
一、 結腸惡性腫瘤進展的相關因子 3
二、 結腸惡性腫瘤進展與細胞結構骨架相關蛋白之相關性 3
三、 癌幹細胞與結腸惡性腫瘤進展之相關性 4
四、 結腸惡性腫瘤能量代謝相關分子機轉 7
(一) 腫瘤細胞能量代謝機轉 7
(二) PI3K/AKT/mTOR訊息傳遞分子機制 10
五、 葉酸營養狀態與結腸癌幹細胞轉移之關係 12
(一) 葉酸營養代謝與罹患結腸癌之風險性 12
(二) 先前葉酸營養狀態不良調控結腸癌研究之發現 13
六、 蛋白質體學分析重要意義 15
七、 研究假說與目的 16
第三章 實驗材料與方法 17
一、 實驗設計 17
二、 實驗材料 19
(一) 細胞株 19
(二) 實驗用試藥 19
(三) 培養基 20
(四) 儀器設備 21
三、 實驗方法 21
(一) 細胞培養 21
(二) 藥劑配置 22
(三) 腫瘤球培養 22
(四) 乳酸濃度分析 22
(五) 氧化還原電位分析 23
(六) 蛋白質體與磷酸化蛋白質體定量分析 23
(七) 西方點墨法 24
四、 統計分析 27
第四章 結果 28
一、 低葉酸微細環境下SW480結腸癌細胞的定量蛋白質體學分析 28
二、 低葉酸微細環境下SW480結腸癌細胞的磷酸化蛋白質體分析 30
三、 低葉酸營養狀態調控結腸癌細胞能量代謝訊息傳遞機轉 33
(一) 低葉酸微細環境下對結腸癌細胞SW480代謝及癌化相關訊息路徑蛋白之影響 33
(二) 低葉酸微細環境下對結腸癌細胞SW480能量代謝相關蛋白之影響 35
(三) 低葉酸微細環境下對結腸癌細胞SW480細胞結構及骨架相關蛋白之影響 36
四、 低葉酸營養狀態誘導結腸癌細胞SW480自我更新之幹性表現 37
五、 結腸癌細胞SW480於低葉酸營養狀態下能量代謝調轉之功能性指標 38
(一) 低葉酸微細環境下結腸癌細胞SW480的腫瘤球細胞培養液乳酸堆積情形 38
(二) 低葉酸微細環境下結腸癌細胞SW480培養液乳酸堆積情形 39
(三) 低葉酸微細環境下結腸癌細胞SW480細胞內氧化還原電位 40
第五章 討論 42
一、 低葉酸微細環境下對結腸癌細胞能量代謝及癌化相關指標之機制探討 42
(一) 低葉酸微細環境下對結腸癌細胞粒線體呼吸能量代謝訊息之影響 42
(二) 低葉酸微細環境下對結腸癌細胞能量代謝訊息傳遞路徑之影響 43
(三) 低葉酸微細環境下對結腸癌細胞代謝與及癌化相關訊息PI3K/Akt/mTOR訊息傳遞路徑之影響 47
(四) 低葉酸微細環境下對結腸癌細胞代謝與及癌化相關訊息Akt/FoxO3a、mTOR/HIF-1α及CK1-α訊息傳遞路徑之影響 49
二、 低葉酸微細環境下對結腸癌細胞SW480細胞骨架及結構相關蛋白之影響 51
三、 低葉酸微細環境下結腸癌細胞SW480受MTOR調控細胞能量代謝訊息傳遞路徑與細胞骨架結構蛋白之影響 54
四、 低葉酸微細環境下結腸癌細胞SW480受MTOR調控細胞能量代謝調轉之功能性指標探討 55
第六章 總結 59
第七章 結論 60
第八章 參考文獻 88

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