Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23145
標題: 綠茶多酚 EGCg 影響人類乳癌細胞株 MCF-7 細胞增生
The anti-proliferative effect of green tea polyphenol (-)-epigallocatechin-3-gallate on human MCF-7 breast cancer cells
作者: 許原彰
Hsu, Yuan-Chang
關鍵字: green tea polyphenol
綠茶多酚
human MCF-7 breast cancer cell
fatty acid synthesis
lipid raft
Ca2+ homeostasis
the MTT assay
western blot
quantitative real time PCR
immune-cellular-fluorescence microscopy
tumor-suppressive gene
pro-apoptotic activity
anti-apoptotic protein
adherens junction proteins
EGFP
laminin receptor
apoptosis
人類乳癌細胞株-MCF-7
脂肪酸
鈣離子細胞溶解
脂質筏
訊息傳遞
細胞存活率
西方墨點法
即時聚合連鎖反應定量儀
免疫螢光染色
細胞凋亡
細胞黏附
基因表現
腫瘤抑制蛋白
促細胞凋亡蛋白
抗凋亡蛋白
綠螢光蛋白
轉質載體
螢光光譜儀
LR接受器
出版社: 生命科學系所
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摘要: 研究報告指出:綠茶多酚具有抑制人類乳癌細胞生長的能力。以人類乳癌細胞株-MCF-7而言,綠茶多酚可分別經由影響乳癌細胞合成脂肪酸的能力、降低胞內鈣離子、以及增加細胞溶解等作用路徑來抑制乳癌細胞的生長能力。另外,研究報告顯示:綠茶多酚也可以經由改變細胞膜上組成脂肪分子移動所造成的脂質筏,而影響許多位在細胞膜上的蛋白接受體的訊息傳遞,因此可抑制許多癌細胞的生長能力。本論文旨在“進一步探討綠茶多酚是否可經由活化細胞膜上的訊息傳遞,而影響細胞內鈣離子濃度的變化以及其他可能的作用機轉”,而產生有效的抑制人類乳癌細胞株-MCF-7細胞的生長。首先,檢視以及量化綠茶多酚影響 MCF-7細胞的存活率;再藉由西方墨點法、即時聚合連鎖反應定量儀、以及免疫螢光染色等方法來“偵測綠茶多酚影響癌細胞內調控細胞凋亡以及細胞黏附相關性蛋白基因表現的變化”。結果發現:綠茶多酚可具有劑量與時間依賴性的抑制 MCF-7細胞的存活率、並會隨著作用濃度提高而活化腫瘤抑制蛋白-Tropomyosin-1基因與蛋白的表現量、增加活化型20kDa 促細胞凋亡蛋白-Caspase-3的表現量、降低抗凋亡蛋白-Gelsolin表現量、以及細胞黏附相關性蛋白 E-cadherin/β-catenin 的表現,並改變其在細胞內的分佈位置;最後也可抑制 MCF-7分泌乳癌細胞標記蛋白 OPN 的能力。另外,利用帶有已建構有綠螢光蛋白基因的轉殖載體轉染 MCF-7細胞,再藉由螢光光譜儀來探討綠茶多酚可能作用在細胞膜上的蛋白接受體的訊息傳遞。結果發現:逐漸提高綠茶多酚劑量會造成螢光強度呈現遞減的結果,而綠茶多酚抑制螢光蛋白活性的能力在加入不同濃度(0.03%-0.1%)Triton X-100破壞細胞膜性構造後會有不同程度的恢復。此外,先破壞細胞膜性構造的實驗中,也發現:綠茶多酚影響綠螢光強度變化反應的敏感性有降低的變化。這些結果暗示:綠茶多酚似乎可經由細胞膜的路徑進而影響細胞內的反應活性。最後,利用抑制 TPK 與 AKT 訊息傳遞的藥物以及 LR 接受器的抗體,來深入探討綠茶多酚如何經由這些訊息傳遞路徑來調控細胞內鈣離子濃度的變化。實驗結果顯示:綠茶多酚可以經由 AKT 與 LR 接受器等作用路徑,來影響細胞內鈣離子濃度的變化。綜合以上實驗結果:綠茶多酚可以經由細胞膜的路徑進入到細胞內,而造成細胞內調控細胞凋亡與細胞黏附相關性蛋白基因的表現,並藉由 AKT 與 LR 接受器的作用路徑共同影響到細胞內鈣離子濃度變化,最後誘導乳癌細胞走向凋亡的路徑。
It has been reported that green tea polyphenol (e.g. (-)-epigallocatechin-3-gallate, EGCg)can inhibit human MCF-7 breast cancer cell growth via the signal transduction pathways to modify the activity of fatty acid synthesis. In addition, EGCg has been shown to cause inhibition of tumor cell growth by altering the lipid compositions on cell membranes such as to induce the lipid raft associated with membrane receptors including human estrogen receptor (HER)and epidermal growth factor receptor(EGFR)in many cancer cells. The main goal of this study was to investigate whether EGCg can inhibit human MCF-7 breast cancer cell proliferation via the membrane-evoked signaling pathway to modulate cytoplasmic Ca2+ homeostasis. Using the MTT assay, EGCg was found to attenuate mitochondria-dependent cell viability of MCF-7 cells. Analyses by using western blot, quantitative real time PCR、and immune-cellular-fluorescence microscopy revealed the chemo-protection of EGCg on inhibition of MCF-7 breast cancer cell proliferation. The anti-tumor effects of EGCg included (1) re-activation of tumor-suppressive gene, tropomyosin-1, (2) increases of pro-apoptotic activity of 20 kD caspase-3, (3) decreases of anti-apoptotic protein-gelsolin gene expression, (4) modulation of gene expression and cellular distribution of adherens junction proteins of E-cadherin,β-catenin, and (5) attenuating extracellular secretion of breast cancer cell marker protein, osteopontin. To examine further whether EGCg acts by the membrane-associated signaling pathway to modulate cytoplasmic Ca2+ homeostasis, we first forced EGFP expression in MCF-7 cell and measured the EGCg-dependent decrease in the EGFP fluorescence. The observation that the EGCg-induced decrease of fluorescence intensity was recovered after cell membrane removal indicates that membrane portion is required for the action of EGCg. Since a multitude of intracellular Ca2+ signaling is sensitive to membrane activation, the dosage effect of EGCg was also determined on changes in intracellular Ca2+. The specific receptor on the membrane was identified by applying a specific antibody to laminin receptor (LR) and genistein, an inhibitor of TPK and AKT receptors. Results obtained demonstrated that EGCg can act through the LR and Akt signaling pathway to influence the intracellular Ca2+. Taken together, EGCg acts by modifying the membrane signal transduction to alter intracellular Ca2+ levels, which might promote apoptosis and attenuate MCF-7 cell viability.
URI: http://hdl.handle.net/11455/23145
其他識別: U0005-2707201014465300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2707201014465300
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