Silibinin抑制骨癌細胞侵入，貼附能力及細胞型態之機制探討

Study of the inhibitory effect and mechanism of silibinin on osteosarcoma invasion, adhesion, and cell morphology

劉育銓(Yu-Chuan Liu)

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(2005年)

https://doi.org/10.6834/CSMU.2005.00037

摘要

癌細胞的轉移往往是惡性腫瘤的重要特徵之一，並且也是在臨床治療上最難以根治的。因此，在很多研究中，大都是以針對癌細胞的轉移過程，去開發新藥或者是利用天然藥物去觀察是否會影響細胞的轉移。而本篇所選用的silibinin是由奶薊子(milk thistle)所萃取出的類黃酮(flavonoid)，早期用於治療肝臟的解毒劑和當作抗氧化物。但近年來，許多研究都用來治療癌症，它會促使癌細胞的自我凋亡，但對於癌細胞的轉移作用及機制尚未明瞭。因此，我們選用一株具有高度轉移能力的人類骨肉瘤細胞：MG-63，分別處理不同濃度的silibinin來探討此藥物對於癌細胞侵入能力的影響。而當癌細胞轉移時常常會伴隨著細胞外基質的分解、細胞貼附能力以及細胞移動能力的改變。所以，先藉由modified Boyden chamber invasion assay，我們發現silibinin具有抑制MG-63侵入的能力，另外，在gelatin zymography與casein zymography assay中也發現到silibinin可以抑制MG-63 骨肉癌細胞的MMP-2及u-PA的表現。此外，利用cell-matrix adhesion assay 發現MG-63在處理 silibinin之後，其細胞與基質的貼附能力也有明顯的下降。接著進一步了解其調控的機制，利用西方點墨法，發現在訊息傳導路徑中的p-ERK1/2會明顯受到silibinin的抑制，並且我們也用了ERK1/2的抑制劑：U0126，證實silibinin是透過ERK1/2路徑來影響細胞的侵入能力以及相關因子。同時，當細胞受到silibinin的處理，也發現其細胞型態上會有改變，而且利用西方點墨法觀察與細胞骨架相關的蛋白。結果發現細胞在FAK的表現量確實會受到藥物的抑制，此外也使用免疫螢光染色觀察細胞中的組成，觀察細胞中actin的組成，發現細胞確實有變得萎縮細長的現象。最後，推測silibinin可能是透過改變細胞中細胞骨架的排列和抑制FAK進而影響細胞的貼附以及侵入能力。

關鍵字

骨癌 ； 侵入

並列摘要

The metastasis of cancer is a vital trait in malignance with extreme difficulties in early diagnosis and therapeutic management. Therefore, the development of new remedies or the utilization of natural medicines targeted at metastasis has been interested and studied extensively. In this study, silibinin, a flavonoid antioxidant extracted from milk thistle, was studied for this purpose. Recently, silibinin has been reported to have various anti-carcinogenesis properties, including inducing cell apoptosis. However, the in vitro effect of silibinin on metastasis of cancer cells was still unclear. Hence, a highly metastatic human osteosarcoma cancer cell line, MG-63, was chosen to be treated with various concentrations of silibinin to investigate its potential for inhibiting cancer cells invasion based on that tumor metastasis are accompanied with proteolytic degradation of the extracellular matrix, changes in cell-matrix adhesion, and regulated cell motility. For a start, via modified Boyden chamber invasion assay, we found that silibinin inhibited MG-63 cells invasion without toxicity while both of MMP-2 and u-PA activity were also inhibited by silibinin via gelatin zymography and casein zymography assay. In addition, the adhesion ability of cells was also suppressed by a treatment with silibinin. To further explore the detailed molecular mechanisms, we found that the expression of phospho-ERK1/2 was dramatically suppressed by silibinin through a western blotting. We also demonstrated that silibinin could inhibit cell invasion and suppress the expression of related molecules via ERK1/2 pathway by using ERK1/2 inhibitor U0126. After cell being treated with silibinin, cell morphology was altered to become shrank and threadlike. Immunofluorescence assay also revealed that silibinin influenced the cell cytoskeletal arrangement. Then, the effect of silibinin on cellular morphology-related proteins was also examined by western blotting to reveal that silibinin could repress FAK expression. Finally, it was concluded that silibinin may influence cell morphology and inhibit FAK expression, and further suppress cell adhesion and cell invasion.

並列關鍵字

silibinin ； invasion

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