臺灣博碩士論文加值系統

乳癌為全球女性發生率及死亡率最高的癌症，其中三陰性乳癌佔15%。三陰性乳癌因荷爾蒙治療及標靶治療效果不佳且腫瘤生長速度快，成為較難治療的類型。乳癌具有早期轉移的特性，雖然藥物能有效得治療，但其抗藥性、腫瘤復發及轉移仍是乳癌患者死亡的主因。臺灣蜂膠是由蜜蜂從樹芽中收集的天然樹脂與蜂蠟混合而成，其活性成分蜂膠素G已被證實具有多種抗腫瘤的效果。本研究旨在探討蜂膠素G抑制三陰性乳癌細胞之移行、侵襲能力及其機制。結果顯示，蜂膠素G能夠抑制細胞移行及侵襲的數量，上調癌細胞的表皮-間質轉化 (Epithelial-mesenchymal transition, EMT) 上皮細胞指標蛋白E-cadherin，並抑制間質細胞指標Vimentin及轉錄因子Snail的表現。進一步發現蜂膠素G活化GSK-3β，使Snail被磷酸化並抑制其進入細胞核中，最後使Snail在細胞質中被降解。另外蜂膠素G能抑制HDAC6活性，使Hsp90過度乙醯化，造成其與Vimentin的結合能力下降，使Vimentin受到降解。由以上結果顯示，蜂膠素G調控GSK-3β來降低Snail的蛋白穩定性，同時抑制HDAC6而促使Vimentin蛋白穩定性下降，造成三陰性乳癌細胞移行及侵襲的能力降低。

Breast cancer is the highest incidence and mortality rate of cancer in female. About 15% of breast cancers are diagnosed as triple-negative breast cancer (TNBC), which responds poorly for endocrine therapy and human epidermal growth factor receptor 2 (HER-2) -targeted therapy. Highly metastasis activity is the main causes of poor prognosis and high mortality rate in TNBC patients. It is urgent to search the strategy for improving the TNBC therapeutic effect. Taiwanese propolis is produced by honeybee, and its extract propolin G has been demonstrated to possess the anti-cancer effects. However, the anti-cancer activity and molecular mechanisms of propolin G on TNBC was still a mystery. The aim of this study was to investigate the anti-metastatic ability of propolin G on TNBC. The results showed that propolin G suppressed the migration and invasion activities of TNBC cells. Up-regulation of E-cadherin, an epithelial marker of epithelial-mesenchymal transition (EMT), and down-regulation of Vimentin and Snail, the mesenchymal marker of EMT, was observed in propolin G-treated cells. Furthermore, down-regulation of snail expression and blockage it translocating to the nucleus was through GSK-3β-mediated Snail phosphorylation via propolin G. The Snail degradation was promoted by ubiquitin-proteasome degradation system and the E-cadherin expression was up-regulated in propolin G treatment. Meanwhile, the degradation of Vimentin expression was through disruption heat shock protein 90 (Hsp90)/vimentin association, which resulted from the accumulation of hyper-acetylation of Hsp90 via HDAC6 activity suppression in propolin G-added cells. Accordingly, propolin G disrupted the stability of Snail through GSK-3β-mediated phosphorylation and nucleus translocation inhibition and HDAC6-mediated Hsp90/vimentin stability repression, which restrained the migration and invasion activity of TNBC cells.

目錄
第一章、文獻回顧 1
一、乳癌病理發展與風險因子 1
二、乳癌型態與治療方式 2
三、乳癌的惡性轉移與機制 5
四、組蛋白去乙醯化對癌細胞惡性轉移的影響 9
五、臺灣綠蜂膠 11
第二章、研究目的與設計 13
一、研究目的 13
二、實驗設計 13
三、實驗流程圖 14
第三章、實驗材料與方法 15
一、實驗材料 15
二、實驗儀器 18
三、實驗耗材 19
四、常用溶液配置 20
五、實驗方法 25
第四章、實驗結果 34
一、三陰性乳癌細胞株轉移能力之比較 34
二、蜂膠素G對三陰性乳癌細胞產生的細胞毒性 34
三、蜂膠素G抑制三陰性乳癌細胞轉移及侵襲的能力 35
四、蜂膠素G調控三陰性乳癌細胞的EMT marker 36
五、蜂膠素G透過GSK-3β調控Snail蛋白穩定性 38
六、蜂膠素G抑制HDAC6活性，並促進Vimentin蛋白穩定性下降 39
第五章、討論 41
第六章、圖表 47
第七章、參考文獻 73 
圖目錄
圖一、四株三陰性乳癌細胞的移行及侵襲能力之比較 47
圖二、四株三陰性乳癌細胞的EMT marker蛋白表現之比較 49
圖三、PPG對三陰性乳癌細胞MDA-MB-231、Hs578T及人類乳腺上皮細胞MCF-10A的細胞毒性影響 50
圖四、PPG對MDA-MB-231細胞株的細胞週期影響 51
圖五、PPG對 MDA-MB-231、Hs578T細胞株的轉移能力及侵襲能力之影響 52
圖六、PPG對MDA-MB-231細胞株EMT蛋白表現之濃度及時間依賴效應 54
圖七、PPG對MDA-MB-231細胞株EMT mRNA表現之影響 55
圖八、在抑制蛋白酶體的情況下，PPG對細胞EMT 蛋白表現之影響 56
圖九、PPG降低了Snail蛋白在細胞核內的含量，使其在細胞核外堆積並被降解 57
圖十、PPG能夠降低GSK-3β的磷酸化情形 58
圖十一、在抑制GSK-3β的情況下，PPG調控細胞轉移及侵襲的能力有所逆轉 59
圖十二、在抑制GSK-3β的情況下，PPG調控細胞EMT marker蛋白的能力有所逆轉 61
圖十三、PPG能促進Snail與GSK-3β的結合能力，使其大量被磷酸化並誘導泛素化，最終導致Snail蛋白降解 63
圖十四、PPG能夠誘導α-tubulin的乙醯化 65
圖十五、在HDAC6大量表現的情況下，由PPG抑制細胞侵襲的能力有所逆轉 66
圖十六、在HDAC6大量表現的情況下，由PPG誘導α-tubulin的乙醯化及抑制Vimentin蛋白情形都有所逆轉 68
圖十七、PPG能抑制HDAC6與Hsp90結合的能力，使Hsp90乙醯化增加，並降低Hsp90及Vimentin結合狀況 69
圖十八、PPG抑制Hsp90與Vimentin的結合後，促使Vimentin被泛素化，導致Vimentin蛋白受到降解 70
圖十九、PPG抑制TNBC cell進行EMT作用的機制圖 72

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