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研究生:劉懿真
研究生(外文):Yi-Zhen Liu
論文名稱:α-胡蘿蔔素在活體內外抑制小鼠肺癌細胞轉移之探討
論文名稱(外文):α-Carotene inhibits metastasis of Lewis lung carcinoma both in vitro and in vivo
指導教授:胡淼琳胡淼琳引用關係
指導教授(外文):Miao-Lin Hu
口試委員:莊正宏黃晉修
口試日期:2013-07-18
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:99
中文關鍵詞:α-胡蘿蔔素轉移太平洋紫杉醇小鼠肺癌細胞株C57BL/6小鼠
外文關鍵詞:α-CaroteneMetastasisTaxolLLC1C57BL/6 mice
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α-胡蘿蔔素為人類血漿中主要類胡蘿蔔素之一,富含於深綠色水果和蔬菜中,例如:胡蘿蔔、菠菜、南瓜和番茄。流行病學研究指出在血漿中含有較高的α-胡蘿蔔素濃度,可以降低慢性疾病發生的風險,如:癌症。在我們過去的研究發現,α-胡蘿蔔素具有抑制人類肝癌細胞株SK-Hep-1轉移的效果。然而,α-胡蘿蔔素對於肺癌是否也具有同樣效果仍不清楚,而肺癌為台灣因癌症死亡的主要原因之一。因此本論文第一部分的研究目的在於利用具有高度轉移能力的小鼠肺癌細胞株(Lewis lung carcinoma, LLC1)作為細胞模式,探討α-胡蘿蔔素抑制癌轉移的作用及其可能機制,並與β-胡蘿蔔素比較。結果發現α-胡蘿蔔素可以顯著抑制LLC1的侵襲、移動及黏附作用,而在相同濃度下(2.5 μM)其抑制效果也較β-胡蘿蔔素好。我們也發現α-胡蘿蔔素可降低細胞膜上整合素β1 (Integrin β1)所調控的黏著斑激酶(Focal Adhesion Kinase, FAK)表現,並進一步調節有絲分裂活化蛋白質激酶(Mitogen-activated protein kinase, MAPKs)的磷酸化作用,而影響到下游Rho small GTPase蛋白的表現。此外,α-胡蘿蔔素會顯著減少尿素激酶型胞漿素原活化子(Urokinase plasminogen activator, uPA)和基質金屬蛋白酶(Matrix metalloproteinases, MMPs)-2和-9的酵素活性,並提升胞漿素原活化子抑制物(Plasminogen activator inhibitor-1, PAI-1)、基質金屬蛋白酶內生性抑制劑(Tissue inhibitor of matrix etalloproteinase, TIMP)-1及-2和抗轉移蛋白23(Non-metastatic protein 23 H1, Nm23-H1)的蛋白質表現。
在本論文的第二部份,我們更進一步利用異體移植LLC1的B57CL/6小鼠模式探討α-胡蘿蔔素合併太平洋紫杉醇在活體內抑制癌轉移的作用。以皮下注射方式將LLC1接種至C57BL/6小鼠的背部,九天後依小鼠腫瘤大小和體重分成五組:(1)腫瘤控制組;(2) α-胡蘿蔔素(5 mg/kg);(3)太平洋紫杉醇(6 mg/kg);(4) α-胡蘿蔔素(1 mg/kg)+太平洋紫杉醇(6 mg/kg);(5) α-胡蘿蔔素(5 mg/kg)+太平洋紫杉醇(6 mg/kg)。以一週三次的頻率經由管餵給予α-胡蘿蔔素並由腹腔注射給予太平洋紫杉醇,持續二十一天。結果發現單獨給予α-胡蘿蔔素(5 mg/kg)和太平洋紫杉醇(6 mg/kg)可顯著:(1)抑制肺臟轉移的作用;(2)降低肺臟組織中整合素β1、p-FAK和Rho small GTPase的蛋白質表現;(3)增加肺臟組織中TIMP-1、TIMP-2、PAI-1和nm23-H1的蛋白質表現;(4)抑制血漿中uPA、MMP-2和-9的酵素活性。此外單獨給予太平洋紫杉醇可顯著抑制腫瘤的生長。合併給予α-胡蘿蔔素(5 mg/kg)及太平洋紫杉醇(6 mg/kg)對於腫瘤生長及肺臟轉移具有相加性抑制作用;在機制方面,合併給予α-胡蘿蔔素(5 mg/kg)及太平洋紫杉醇(6 mg/kg)對於肺臟組織中Rho和Rac的蛋白質表現具有相加性抑制作用,但對於TIMP-2蛋白質表現則具有加乘性促進作用。這些活體外和活體內試驗的結果顯示:α-胡蘿蔔素本身除了具有癌症化學預防的能力之外,亦具有作為抗癌藥物佐劑之潛力。


α-Carotene, one of the major carotenoids in human plasma, can be found in yellow-orange and dark-green fruits and vegetables, such as carrots, spinach, pumpkin and tomatoes. Epidemiological studies have indicated that higher serum levels of α-carotene are associated with lower risk of chronic diseases, including cancer. Our previous studies have demonstrated that α-carotene exhibits anti-metastatic activity in human hepatocarcinoma SK-Hep-1 cells. However, it is unclear whether α-carotene has similar effects in lung cancer, the major leading cause of death in cancer patients in Taiwan. Therefore, the aim of the first part of this thesis was to determine the anti-metastatic effects and possible mechanisms of α-carotene in comparison with β-carotene in highly invasive Lewis lung carcinoma (LLC1). Results reveal that α-carotene significantly inhibited cell invasion, migration and adhesion in LLC, and that these inhibitory effects of α-carotene are similar to β-carotene at the same concentration (2.5 μM). We also found that α-carotene significantly inhibited the phosphorylation of integrin β1 receptor-mediated focal adhesion kinase (FAK), which in turn reduced the phosphorylation of mitogen-activated protein kinase (MAPKs). Such an inhibition was concomitant to reduction of Rho small GTPase expression, including Rho and Rac. In addition, α-carotene significantly decreased activities of urokinase plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and -9), but increased protein expression of plasminogen activator inhibitor (PAI)-1 and tissue inhibitor of MMP (TIMP)-1 and -2 as well as nm23H1.
In the second part of this thesis, we further examined the anti-metastatic effects of α-carotene in combination with taxol in C57BL/6 mice xenografted with LLC model. After implantation (s.c.) with LLC for 9 d, mice were divided into five groups: group 1, tumor control; group 2, α-carotene (5 mg/kg); group 3, taxol (6 mg/kg); group 4, α-carotene (1 mg/kg) + taxol (6 mg/kg); group 5, α-carotene (5 mg/kg) + taxol (6 mg/kg). Mice were orally supplied with α-carotene and administrated (i.p.) with taxol three times a week for an additional 21 d. Results reveal that α-carotene (5 mg/kg) and Taxol (6 mg/kg) treatment alone resulted in: 1) significant inhibition of lung metastasis; 2) significantly decreased protein expression of integrin β1, p-FAK and Rho small GTPase in lung tissues; 3) significantly increased protein expression of TIMP-1、TIMP-2、PAI-1 and nm23-H1 in lung tissues; and 4) significantly decreased activities of uPA, MMP-2 and MMP-9 in plasma. Taxol (6 mg/kg) treatment alone also significantly inhibited tumor growth, as evidence by inhibition of tumor volume and tumor weight. α-Carotene (5 mg/kg) in combination with Taxol (6 mg/kg) exhibited additive inhibition on tumor growth and lung metastasis. Mechanistically, α-carotene (5 mg/kg) in combination with Taxol (6 mg/kg) additively inhibited Rho and Rac protein expression but synergistically increased TIMP-2 protein expression in lung tissues. Overall, the present results suggest that α-carotene could be used as an adjuvant for anti-cancer drugs or as a chemopreventive agent.


第一章文獻整理.......................................................................................................... 1
1. 肺癌 (Lung cancer) 2
2. 癌症轉移 (Cancer metastasis) 2
3. 癌轉移相關之生化指標 (Metastatic-related biomarkers) 3
3.1 基質金屬蛋白酶 (Matrix metalloproteinases; MMPs) 3
3.2 基質金屬蛋白酶抑制劑 (Tissue inhibitor of matrix metalloproteinases; TIMPs) ………………………………………………………………………………..3
3.3 尿素激酶型胞漿素原活化子系統 (Urokinase plasminogen activator system) ……………………………………………………………………………………...4
3.3.1 尿素激酶型胞漿素原活化子 (Urokinase plasminogen activator; uPA) 4
3.3.2 胞漿素原活化子抑制物 (Plasminogen activator inhibitor-1; PAI-1) 4
3.4 抗轉移蛋白23 (Non-metastatic protein 23 H1; Nm23-H1) 4
3.5 Rho small GTPase 5
3.6 整合素β1 (Integrin β1) 5
3.7 黏著斑激酶 (Focal Adhesion Kinase; FAK) 6
3.8 有絲分裂活化蛋白質激酶 (Mitogen-activated protein kinase; MAPKs) 6
4. 類胡蘿蔔素 (Carotenoids) 8
5. α-胡蘿蔔素 (α-Carotene) 9
5.1 結構和特點 9
5.2 α-胡蘿蔔素的生理功能 9
5.2.1 細胞試驗: 抗增生與抗轉移情形 9
5.2.2 細胞試驗:細胞間隙連接通道 10
5.2.3 動物試驗:抑制腫瘤生長 11
5.2.4 人體試驗 11
5.2.5 流行病學 12
6. 目的與假說 12
7. 實驗架構 14
8. 參考文獻 15

第二章α-胡蘿蔔素抑制小鼠肺癌細胞轉移作用及其相關分子機制之探討 23
摘要........................................................................................................................... 24
1. 前言 25
2. 材料與方法 26
2.1 實驗試劑 26
2.2 α-與β-胡蘿蔔素配製 26
2.3 細胞培養和細胞存活率分析 26
2.4 細胞黏附分析 27
2.5 細胞侵襲和移行分析 27
2.6 酵素圖譜同功分析 28
2.7 西方點墨法 29
2.7.1 蛋白質萃取 29
2.7.2 蛋白質定量 29
2.7.3 聚丙烯醯胺膠體之製備 29
2.7.4 電泳 30
2.7.5 Transfer和Blocking………………………………………………………….30
2.8 統計分析 30
3. 結果………………………………………………………...…………………31
3.1 α-胡蘿蔔素抑制小鼠肺癌細胞株(Lewis lung carcinoma,LLC1)的轉移 31
3.1.1 α-胡蘿蔔素和β-胡蘿蔔素抑制LLC1侵襲作用 31
3.1.2 α-胡蘿蔔素和β-胡蘿蔔素抑制LLC1移行作用 31
3.1.3 α-胡蘿蔔素和β-胡蘿蔔素抑制肺癌細胞黏附作用 32
3.1.4 α-胡蘿蔔素和β-胡蘿蔔素對於LLC1存活率之影響 32
3.2 α-胡蘿蔔素和β-胡蘿蔔素抑制MMP-2、MMP-9和uPA的酵素活性 33
3.3 α-胡蘿蔔素促進TIMP-1、TIMP-2、nm23-H1和PAI-1蛋白質表現 33
3.4 α-胡蘿蔔素降低Rho和Rac 蛋白質表現 34
3.5 α-胡蘿蔔素抑制整合素β1蛋白質表現、FAK和MAPKs磷酸化作用 34
4. 討論 35
4.1 α-胡蘿蔔素抑制LLC1轉移的分子機制 35
4.1.1 α-胡蘿蔔素造成MMPs和TIMPs比例失衡 35
4.1.2 α-胡蘿蔔素影響尿素激酶型胞漿素原活化子系統(urokinase plasminogen activator system, uPA system) 35
4.1.3 α-胡蘿蔔素增加抗轉移蛋白nm23-H1的表現 36
4.1.4 α-胡蘿蔔素弱化整合素β1所調控的訊號傳遞路徑 36
4.2 α-胡蘿蔔素與β-胡蘿蔔素在抑制LLC1轉移作用的分子機制是不相同的 …………………………………………………………………………………….37
4.3 α-胡蘿蔔素抑制LLC1轉移的作用與其轉換為維生素A的能力無關α-胡蘿蔔素………………………………………………………………………….……….37
4.4 α-胡蘿蔔素與β-胡蘿蔔素的生理濃度 38
4.5 α-胡蘿蔔素所產生的U型或鐘型效應 39
4.6 結論 39
5. 參考文獻 40

第三章α-胡蘿蔔素合併太平洋紫杉醇在異體移植小鼠肺癌細胞株(LLC1)模式中抑制轉移作用和原位腫瘤生長之研究 55
摘要 56
1. 前言 58
2. 材料與方法 60
2.1 實驗試劑與α-胡蘿蔔素及太平洋紫杉醇(Taxol)配製 60
2.2 細胞培養 60
2.3 動物與分組 61
2.4 計數肺臟轉移數目和測量腫瘤體積 61
2.5 酵素圖譜同功分析 61
2.6 西方點墨法 62
2.6.1 蛋白質萃取 62
2.6.2 蛋白質濃度測定 62
2.6.3 聚丙烯醯胺膠體之製備 63
2.6.4 電泳 ………………………………………………………………………...63
2.6.5 Transfer和Blocking 63
2.7 相加效應 64
2.8 統計分析 64
3. 結果…..………….……………………………………………………………64
3.1 單獨α-胡蘿蔔素或合併Taxol對於小鼠體重的影響 64
3.2 單獨α-胡蘿蔔素或合併Taxol對於小鼠腫瘤體積及重量的影響 65
3.3 單獨α-胡蘿蔔素或合併Taxol對於小鼠肺臟腫瘤轉移數的影響 66
3.4 單獨α-胡蘿蔔素或合併Taxol對於小鼠血漿中MMP-2、MMP-9和uPA的酵素活性之影響 66
3.5 單獨α-胡蘿蔔素或合併Taxol對於小鼠肺臟組織中TIMP-1、TIMP-2 nm23-H1和 PAI-1蛋白質表現之影響 67
3.6 單獨α-胡蘿蔔素或合併Taxol對於小鼠肺臟組織中Rho和Rac蛋白質表現之影響 ………………………………………………………………………………68
3.7 單獨α-胡蘿蔔素或合併Taxol對於小鼠肺臟組織中整合素β1和p-FAK蛋白質表現之影響 69
4. 討論.... 70
4.1 α-胡蘿蔔素與Taxol在體內抗轉移機制 70
4.1.1 α-胡蘿蔔素與Taxol降低血漿中MMP-2、MMP-9和uPA的酵素表現 70
4.1.2 α-胡蘿蔔素與Taxol增加肺臟組織中TIMPs、PAI-1和nm23-H1蛋白質表現…...……………………………….…………………………………………………………………………...……..71
4.1.3 α-胡蘿蔔素與Taxol抑制肺臟組織中Rho、Rac、p-FAK和整合素β1的蛋白質表現…………………………………………………………………………………………….……………..72
4.2 加成效應 72
4.3 將α-胡蘿蔔素給予小鼠劑量換算為人體劑量 73
4.4 結論.. 73
5. 參考文獻 74
總結..............................................................................................................................98



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