異丁香酚抑制不同時期結腸癌細胞的生長、遷移和侵襲

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劉子瑄(Zih-Shuan Liou) 查詢紙本館藏

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論文名稱

異丁香酚抑制不同時期結腸癌細胞的生長、遷移和侵襲
(Isoeugenol inhibits the growth, migration and invasion in different stages of colorectal cancer cells)

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至系統瀏覽論文 (2028-1-30以後開放)

摘要(中)

摘要
　　結腸癌（colorectal cancer，CRC）是世界上第二大癌症死亡原因，根據侵犯腸壁、淋巴結和轉移的程度，可分為Dukes A至D四個階段，儘管 CRC 的發展可以通過食物營養素和草藥來調節，但是關於荖藤異丁香酚在 CRC 不同階段的控制的訊息傳導路徑的研究相對較少。因此，本論文的總體目標旨在研究異丁香酚調節 CRC 細胞不同階段的生長、遷移和侵襲的訊息路徑。
　　使用 SW480（Dukes B 期）和 SW620（Dukes C 期）CRC 細胞，我們發現異丁香酚抑制細胞生長，細胞存活率、細胞數量和集落形成減少就是證明。在較小程度上，異丁香酚影響正常 FHC 結腸細胞的生長。進一步的以傷口癒合試驗表示異丁香酚抑制 SW480 和 SW620 細胞的遷移。此外，Boyden chamber assay證實異丁香酚抑制了這兩種 CRC 細胞的侵襲。當檢查訊息路徑時，觀察到異丁香酚會降低 pAkt、CDK2、CDK4、CDK6、細胞週期蛋白 D1、N-鈣粘蛋白和 Snail 蛋白的水平，並增加 pAMPK、pERK、pJNK、pp38 MAPK、ATF3、LC3β 和 E -鈣粘蛋白的表現，也觀察到異丁香酚對細胞週期蛋白 D3、p21 和 p27 蛋白水平的階段依賴性影響，酶譜分析表明異丁香酚抑制 MMP-2 和 MMP-9 蛋白的活性。
　　使用 AMPK（Compound C）和 p38（SB203580）抑製劑治療可拮抗異丁香酚對 CRC 細胞生長、遷移和侵襲以及 SW480 和 SW620 細胞中大多數訊息蛋白的影響。有趣的是，用 ERK (U0126) 和 JNK (SP600125) 抑製劑治療會增強異丁香酚對 CRC 細胞生長、遷移和侵襲以及大多數訊息蛋白的影響。
　　這些數據表明，異丁香酚可能通過活化 AMPK 和 p38 MAPK 來抑制 CRC 細胞的生長、遷移和侵襲，並使 CRC 細胞能夠抑制表皮特徵轉變為具有 MMP 依賴性的間質特徵。本論文關於異丁香酚調節 CRC 細胞各個階段活動的訊息機制的結果，將突出其在預防和治療 CRC 方面的可能用途。

摘要(英)

ABSTRACT
Colorectal cancer (CRC) is the second common cause of cancer death in the world and can be divided into four stages from Dukes A to D according to the degrees of invasion of intestinal wall, lymphatic node involvement, and metastasis. Although development of CRC can be regulated by food nutrients and herbal medicines, relatively little information is known about the control of Piper betle isoeugenol in different stages of CRC. Thus, the overall objective of this thesis was designed to study the signal pathways involved in isoeugenol modulations of growth, migration, and invasion in different stages of CRC cells.
Using SW480 (Dukes B stage) and SW620 (Dukes C stage) CRC cells, we found that isoeugenol inhibited cell growth, as evidenced by decreased cell viability, cell number and colony formation. To a much lesser extent, isoeugenol affected the growth of normal FHC colon cells. Further wound healing assay indicated that isoeugenol inhibited the migration of SW480 and SW620 cells. In addition, the Boyden chamber assay confirmed that isoeugenol inhibited the invasion of these two CRC cells. When the signaling pathways were examined, isoeugenol was generally observed to decrease levels of pAkt, CDK2, CDK4, CDK6, cyclin D1, N-cadherin and Snail proteins and increase levels of pAMPK, pERK, pJNK, pp38 MAPK, ATF3, LC3β and E-cadherin proteins. The stage-dependent effects of isoeugenol on levels of cyclin D3, p21, and p27 proteins were also observed. Zymography assay indicated that isoeugenol inhibited the activities of MMP-2 and MMP-9 proteins.
Treatment with the inhibitors of AMPK (compound C) and p38 (SB203580) antagonized the effects of isoeugenol on CRC cell growth, migration, and invasion and most of those signaling proteins in SW480 and SW620 cells. Interestingly, treatment with the inhibitors of ERK (U0126) and JNK (SP600125) generally enhanced the effects of isoeugenol on CRC cell growth, migration, and invasion, and most of those signaling proteins.
These data suggest that isoeugenol may inhibit CRC cell growth, migration and invasion through activations of the AMPK and particular p38 MAPK member, as well as enabling CRC cells to suppress the epithelial features changing to mesenchymal ones with MMP dependency. Results of this thesis for signaling mechanisms of isoeugenol to coordinate the activity of various stages of CRC cells would highlight its possible uses for prevention and cure of CRC.

關鍵字(中)

★ 結腸癌

關鍵字(英)

★ Colorectal cancer

論文目次

目錄
摘要 I
ABSTRACT II
致謝 IV
目錄 V
圖目錄 IX
表目錄 XIII
英文縮寫對照表 XIV
壹、緒論 1
1-1結腸癌 1
1-1-1介紹 1
1-1-2結腸癌的癌化 1
1-1-3轉移階段 2
1-1-4結腸癌治療 3
1-2 藥物 3
1-2-1 Piper betle與Isoeugenol 3
1-2-2 Isoeugenol與抗氧化的活性 4
1-2-3 丹參酮IIA（Tanshinone IIA）在結腸癌中的作用 5
1-3結腸癌的調節與訊息傳導 6
1-3-1生長訊息傳導 6
1-3-2結腸癌與細胞週期 10
1-3-3 結腸癌EMT與轉移 12
1-3-4基質金屬蛋白酶 (Matrix metalloproteinases, MMPs) 13
1-4研究動機 14
貳、材料與方法 16
2-1實驗材料(Experimental materials) 16
2-2細胞培養(Cell culture) 16
2-3細胞存活率試驗(MTT assay) 17
2-4細胞計數試驗(Cell number assay) 17
2-5軟瓊脂集落形成測定(Soft agar colony formation assay) 18
2-6傷痕癒合爬行測試(Wound healing assay) 19
2-7細胞侵襲分析(Boyden chamber invasion assay) 20
2-8酶譜法(Zymography) 20
2-9西方墨點法(Western blot) 22
2-10 抑制劑(Inhibitors) 24
2-11 統計(Statistics) 24
參、結果 25
3-1 Isoeugenol對正常人類結腸上皮細胞和人類結腸癌上皮細胞的生長及存活率的影響 25
3-2 Isoeugenol對人類結腸癌上皮細胞群落生長之抑制效果 26
3-3 Isoeugenol對人類結腸癌上皮細胞之細胞週期蛋白的影響 27
3-4 Isoeugenol對人類結腸癌上皮細胞生長訊息蛋白表現量的影響 27
3-5 Isoeugenol對人類結腸癌上皮細胞爬行能力之抑制效果 28
3-6 Isoeugenol對人類結腸癌上皮細胞侵襲能力之抑制效果 29
3-7 Isoeugenol對人類結腸癌上皮細胞上皮間質轉化(EMT)訊息蛋白質的表現量的影響 29
3-8 Isoeugenol對人類結腸癌上皮細胞MMP-2和MMP-9活性之影響 30
3-9 Isoeugenol透過AMPK對人類結腸癌上皮細胞的生長及存活率的影響 30
3-10 Isoeugenol透過AMPK對人類結腸癌上皮細胞之細胞週期蛋白的影響 31
3-11 Isoeugenol透過AMPK對人類結腸癌上皮細胞生長訊息蛋白表現量的影響 35
3-12 Isoeugenol透過AMPK對人類結腸癌上皮細胞上爬行和侵襲的影響 40
3-13 Isoeugenol透過AMPK對人類結腸癌上皮細胞上皮間質轉化(EMT)訊息蛋白質的表現量的影響 41
3-14 Isoeugenol透過AMPK對人類結腸癌上皮細胞上MMP-2和MMP-9活性之影響 43
3-15 Isoeugenol透過ERK對人類結腸癌上皮細胞的生長及存活率的影響 43
3-15 Isoeugenol透過ERK對人類結腸癌上皮細胞之細胞週期蛋白的影響 44
3-16 Isoeugenol透過ERK對人類結腸癌上皮細胞生長訊息蛋白表現量的影響 47
3-17 Isoeugenol透過ERK對人類結腸癌上皮細胞爬行和侵襲的影響 53
3-18 Isoeugenol透過ERK對人類結腸癌上皮細胞上皮間質轉化(EMT)訊息蛋白質的表現量的影響 53
3-19 Isoeugenol透過ERK對人類結腸癌上皮細胞MMP-2和MMP-9活性的影響 55
3-20 Isoeugenol透過JNK對人類結腸癌上皮細胞的生長及存活率的影響 56
3-21 Isoeugenol透過JNK對人類結腸癌上皮細胞之細胞週期蛋白的影響 57
3-22 Isoeugenol透過JNK對人類結腸癌上皮細胞生長訊息蛋白表現量的影響 60
3-23 Isoeugenol透過JNK對人類結腸癌上皮細胞爬行和侵襲的影響 65
3-24 Isoeugenol透過JNK對人類結腸癌上皮細胞上皮間質轉化(EMT)訊息蛋白質的表現量的影響 66
3-25 Isoeugenol透過JNK對人類結腸癌上皮細胞MMP-2和MMP-9活性的影響 68
3-26 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞的生長及存活率的影響 68
3-27 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞之細胞週期蛋白的影響 69
3-28 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞生長訊息蛋白表現量的影響 72
3-29 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞爬行和侵襲的影響 78
3-30 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞上皮間質轉化(EMT)訊息蛋白質的表現量的影響 79
3-31 Isoeugenol透過p38 MAPK對人類結腸癌上皮細胞MMP-2和MMP-9活性的影響 80
肆、討論 82
伍、結論 95
陸、參考文獻 97
柒、附錄 182

參考文獻

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指導教授

高永旭(Yung-Hsi Kao)

審核日期

2023-1-17

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