- 學位論文
覆盆子抑制肺癌細胞之上皮間質轉化與其 移動及侵入之機制探討
Study of the inhibitory effects and its mechanisms of Rubus idaeus on Epithelial-Mesenchymal Transition, invasion and migration of lung cancer cells
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，開發新藥或是利用天然藥物處理是否會影響癌細胞的轉移。目前已有多種中藥已被確認具有抗發炎，抗過敏及抗癌的活性，且其中具有生物活性的有效成份，部分已被單離並經分析鑑定。中藥對癌症所引起的諸多症狀的治療能有所助益，已是眾所皆知的事實，而中藥的抗癌有效成份之作用機轉，常為有效地促使癌細胞凋亡及抑制癌細胞增生，但對其抑制侵襲及轉移作用和其詳細機制方面的研究還很少。因此，本研究選用一株具有高度轉移能力的人類肺癌細胞：A549，處理覆盆子的萃取物來探討對於癌細胞移動和侵入能力的影響。首先藉由wound healing assay與Boyden chamber assay，發現覆盆子萃取物具有抑制A549移動和侵入的能力，而且不具有細胞毒性。我們藉由cells adhesion assay也發現覆盆子萃取物能抑制細胞的貼附和延伸。而當癌細胞轉移時常常會伴隨著細胞外基質的分解及細胞移動能力的改變，在gelatin zymography與casein zymography assay中也發現到它們可以抑制A549人類肺癌細胞的MMP-2及u-PA的表現。因此我們再利Western blot發現覆盆子萃取物會抑制A549人類肺癌細胞FAK和Erk1/2的磷酸化。此外，當處理覆盆子後會增加上皮細胞的特徵，如:E-cadherin和α-catenin，以及減少間質細胞的特徵，如:Snail-1、vitmentin、fibronectin和N-cadherin，使細胞的入侵和轉移降低。我們也發現覆盆子萃取物具有抑制細胞核內AP-1的表現及DNA binding的活性。綜合以上結果，覆盆子萃取物可能是透過抑制MAPK pathways中蛋白的磷酸化及轉錄因子的活性，與抑制癌細胞蛋白水解酶來達到反轉EMT以及抑制癌細胞轉移的能力。
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. Recently, Chinese medicinal herbs have been reported to have various anti-carcinogenesis properties, including inducing cell apoptosis and suppressing cell proliferation. However, the in vitro effect and detailed mechanism of Rubus idaeus on metastasis of cancer cells was still unclear. Hence, a highly metastatic human lung cancer cell line, A549, was chosen to be treated with Rubus idaeus extracts (RIE) to investigate their potential for inhibiting cancer cell migration and invasion. For a start, via wound healing assay and Boyden chamber assay, we found that RIE can suppress A549 cells migration and invasion, and RIE did not have toxicity to A549 cells by MTT assay. We also found that RIE can suppress A549 cells adhesion and cell spreading by cell adhesion assay. Based on that tumor metastasis are accompanied with proteolytic degradation of the extracellular matrix and changed cell motility. Both of MMP-2 and u-PA activity were also inhibited by RIE via gelatin zymography and casein zymography assay. We performed western blot analysis to find that RIE inhibited phosphorylation of FAK and Erk1/2. Furthermore, Treatment with RIE which was resulted in induction of epithelial markers such as E-cadherin and α-catenin and repression of mesenchymal markers such as Snail-1, vitmentin, fibronectin, and N-cadherin, which reduce cell invasion and metastasis. A treatment with RIE to A549 cells also suppressed the DNA binding activity and the nuclear level of activator protein-1. Finally, it was concluded that RIE may reverse Epithelial-Mesenchymal Transition and inhibit tumor cell metastasis via suppressing the expression of proteases, phosphorylation of Mitogen-activated protein kinase and the activity of transcription factors metastasis-related proteinase.