Hispolon 對人類肝癌細胞(SK-Hep1)體外抗轉移機制探討;In vitro anti-metastatic effects of hispolon on liver cancer cell line, SK-Hep-1 cell

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Title:	Hispolon 對人類肝癌細胞(SK-Hep1)體外抗轉移機制探討;In vitro anti-metastatic effects of hispolon on liver cancer cell line, SK-Hep-1 cell
Authors:	王曉潔;Hsiao-Chieh wang
Contributors:	中國醫藥大學：中國藥學研究所
Keywords:	桑黃;人類肝癌細胞;轉移;Hispolon;SK-Hep1 cell;Phellinus Linteus
Date:	2008-06-27
Issue Date:	2009-08-10 18:02:15 (UTC+8)
Abstract:	癌症的轉移擴散是許多癌症病人致死的主要原因之ㄧ，而許多癌細胞均具有分泌基質金屬蛋白水解酶 (matrix metalloproteinases, MMPs) 的能力，其中MMP-9以及MMP-2扮演重要的角色。MMP-9和MMP-2是一種可以水解細胞外基質(extracellular matrix, ECM)和基底膜(basement membranes)的酵素，亦為癌細胞在從原位組織侵入遠處組織的重要因子。已有文獻指出桑黃(Phellinus linteus)會抑制腫瘤生長和轉移。而桑黃中Hispolon成分為具有高抗氧化作用的化合物，Hispolon對於人類epidermoid KB Cell具有誘導細胞凋亡的作用，但是目前對於Hispolon在癌症轉移方面的研究仍然不清楚。因此，在本研究中，我們以高轉移活性的人類肝癌細胞SK-Hep-1細胞為實驗模式，探討桑黃成分之Hispolon對SK-Hep-1細胞之移行、侵入、黏附和MMPs活性與表現之影響。以Transwell chamber之方法，來評估Hispolon對於SK-Hep-1細胞黏附能力、移動能力和侵入能力之影響。利用Gelatin zymography分析細胞外之 MMP-9和MMP-2活性。以西方墨點法分析細胞內MMP-9和MMP-2蛋白質表現。結果發現，以 Hispolon (0、1、2.5、5、7.5、10、25和50 μg/ml)共同培養24和48小時後, 對SK-Hep-1細胞在24小時，濃度1、2.5、5、7.5和10 μg/ml並無細胞毒性，因此選用無細胞毒性的濃度和時間點來做之後的實驗研究。當Hispolon 濃度在5 μg/ml時，抑制細胞移行效果會達到49%，在10 μg/ml時抑制率更達到63%。而Hispolon 濃度在5 μg/ml時，抑制細胞侵入效果會達到56%，在10 μg/ml時抑制率更達到62%。而在傷口瘉合試驗中發現，SK-Hep-1細胞經Hispolon (0~10 μg/ml)處理6、12、24和48小時後，會有明顯的抑制SK-Hep-1細胞移行的距離，以10 μg/ml效果最顯著。Hispolon (1~10 μg/ml)均無法顯著的抑制細胞黏附能力。Hispolon(1~10 μg/ml)會顯著抑制SK-Hep-1細胞所分泌之MMP-9和MMP-2的活性，10 μg/ml時對於MMP-2的抑制效果有55%，MMP-9抑制效果會達到53%，且具有劑量依存關係。進一步以西方墨點法研究發現，Hispolon可顯著增加SK-Hep-1細胞內TIMP-1和TIMP-2的蛋白質表現量，以及減少SK-Hep1細胞內FAK和p-FAK的蛋白質表現量，且具有劑量依存性的關係。本研究發現，Hispolon抗轉移機制可能是透過抑制MMP-9和MMP-2活性及增加TIMP-1和2的表現及抑制FAK的表現，進而抑制癌細胞侵入及移行作用，最終達到抗SK-Hep-1細胞轉移的能力。 Metastasis is one of the major causes of cancer-associated mortality, and a large number of cancerous cells have the ability to secret matrix metalloproteinases (MMPs). Among them, MMP 2 and MMP 9, an extracellular matrix enzyme, is a key enzyme for cancerous cells to invade distant tissue from in-situ tissue. Many literatures showed that Phellinus linteus (PI) inhibited the cancer proliferation and metastasis. Hispolon was a highly antioxidant compound in PI and it possessed the effect of apoptosis on human epidermoid KB cells. However, the mechanism is still unclear on metastasis. In this study, we examined the effects of hispolon on invasion、migration、adhesion、MMP activity and protein expression on highly metastastic hepatocarcinoma. The effect of Hispolon was evalvated on invasion、migration and adhesion in SK-Hep-1 by Transwell chamber. The extracellular MMP 2, 9 activity and intracellular protein expression was used by gelatin zymography and Western blotting. We found that hispolon inhibited cell migration and invasion about 49% and 56% at 5 μg/ml, and inhibited about 63% and 62% at 10 μg/ml. Hispolon could not inhibit cell adhesion, significantly. Also, we found that Hispolon inhibited MMP 2, 9 activities after 24hr in a dose-dependent manner. The results demonstrated that the antimetastatic mechanisms of hispolon might be inhibit of MMP-2, 9 activities and enhance the TIMP 1, 2 expressions and inhibit of FAK and p-FAK expressions.
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