覆盆子酒精萃取物抑制人類肺癌細胞之MMP及u-PA表現及降低腫瘤之生長能力

Rubus idaeus L. ethanolic extract inhibits MMP and u-PA of Human Lung Cancer A549 Cells and Reduces Tumor Growth in Vivo

王敏雄(Min-hsiung Wang)

指導教授 ： 謝易修

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2015年)

https://doi.org/10.6834/CSMU.2015.00177

摘要

覆盆子拉丁學名為 Rubus idaeus ，來源為薔薇科落葉灌木植物華東覆盆子的未成熟果實。根據明代李時珍於《本草綱目》記載，覆盆子的命名是由於其「子似覆盆之形，故名之。」亦有文獻記載覆盆子："益腎臟，縮小便，服之當覆其溺器(即服用後可將小便的器皿覆轉收起不需再用)，如此取名也。
本實驗使用藥物為覆盆子萃取物 (Rubus idaeus extract: RIE)，RIE 為50％酒精溶液萃取物利用高效能液相層析分析(High-performance liquid chromatography: HPLC)覆盆子之萃取物，覆盆子萃取物在 HPLC 的分析下有gallic acid、EGC(epigallocatechin)、catechin、p-coumaric acid幾種主要成分。我們進一步分析覆盆子萃取物 (RIE) 在MTT assay ( 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) 中覆盆子萃取物 (RIE)對肺癌細胞A549 和 H1299並沒有毒殺細胞的作用，接著用Western blot 分析方法，觀察 RIE 對 A549 的影響後蛋白質的表現量，結果也顯示加藥處理 RIE的A549肺癌細胞會減少MMP-2(matrix metalloproteinases-2)和u-PA(urokinase type plasminogen activator)的蛋白分泌量，而使TIMP-1(Tissue inhibitors of etalloproteinases) 和PAI-1( Plasminogen activator inhibitor-1)的蛋白分泌量有所上升 。MMP-2和u-PA為基質金屬蛋白水解酵素利於細胞的移動，TIMP-1和PAI-1則為MMPs的內生性抑制劑會抑制細胞的移動。接著利用Boyden chamber 的分析方法來分析細胞移動性。分析結果在使用 RIE的加藥處理下A549和 H1299 細胞株的Invasion 能力會隨著RIE的濃度增加而降低，和GM6001 (galardin, ilomastat) MMP抑制劑濃度5μM對照組相比較。以上這幾個實驗我們得知 RIE 可能具抑制癌細胞的移能力。同時，在小鼠皮下注射癌細胞的動物模式下，由腫瘤重量觀察、螢光強度的觀察，可以看出有餵食RIE藥物的組別其腫瘤生長的情形較Control組別有較低的趨勢，也說明了 RIE 具有減緩 A549 腫瘤變大的情形。另外再用組織切片使用免疫染色的方法觀察腫瘤，Ki-67為臨床上判斷腫瘤惡性程度的標誌。利用免疫組織染色的方法對腫瘤切片進行染色。在免疫組織染色的分析上也顯示出餵食 RIE後老鼠腫瘤Ki-67表現量較Control組有減少的趨勢。癌症中E – cadherin為上皮細胞的標誌，上皮細胞可塑性差細胞不易轉移，vimentin為間質細胞的標誌，間質細胞的可塑性很強，可以在不同的環境中轉換成不同的細胞型態。在免疫組織染色的分析上也顯示出餵食 RIE後老鼠腫瘤E – cadherin較Control組有減增加的趨勢，而餵食RIE的組別其vimentin較Control組別相比有減少的趨勢。
由以上種種的實驗我們可以得知，在抑制肺癌細胞的侵襲上，RIE 很可能具備潛在的治療效果。絕大部分的癌症是死於轉移而非腫瘤本身，越惡化的癌症其轉轉移能力越強，若是能有效抑制其轉移能力則能控制癌正的惡化。

關鍵字

覆盆子 ； 基質金屬蛋白水解酵素 ； 肺癌

並列摘要

Raspberry, Rubus idaeus L., is a plant belonging to the Rosaceae family, genus Rubus. The purpose of this article is to evaluate anti-tumor effect of Rubus idaeus L. fruit. Rubus idaeus L. fruit was extracted by 50% ethanol(50% ethanol and 50% water) and analyzed by HPLC. The major compositions of Rubus idaeus extract(RIE) are gallic acid, epigallocatechin (EGC), catechin, p-coumaric acid. RIE has no cytotoxicity to human lung cancer cell A549 and H1299 under MTT assay. On Western blot analysis, RIE group has downregulation of MMP-2 /u-PA secretion and upregulation of TIMP-1/PAI-1 compared to control group. Furthermore, we used Boyden chamber to measure its cell invasion. Invasion ability in RIE group reduced both in A549 and H1299 cell line. The reduced invasion ability is positively correlation to concentration of RIE. In view of above mentioned study, RIE may inhibit tumor invasion. In mice animal study, less tumor weight was noted 40 days later. Immunohistochemistry revealed less Ki-67 performance in RIE group, indicating less cell proliferation .E-cadherin is a transmembrane glycoprotein, involved in cell-cell adhesion and epithelial-mesenchymal transition (EMT). Vimentin is highly expressed in mesenchymal cells and is positively correlated with increased metastasis. Cancer metastasis is the leading cause of death in all cancer patients. Immunohistochemistry also showed upregulation of E-cadherin and downregulation of vimentin in RIE group compared to control group. In conclusion, RIE has potential benefit in lung cancer treatment owing to reducing tumor proliferation and metastasis. Furthermore, it needs further detail study for involving signaling pathway.

並列關鍵字

Rubus idaeus ； MMP ； lung cancer

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