| Abstract: | 癌細胞轉移是癌症導致死亡以及治療複雜度提昇的主要原因; 而癌細胞轉移 已知與多種細胞生理改變密切相關，如改變癌細胞與胞外基質間及基底膜的貼附能 力，破壞細胞間交互作用的力量等。癌細胞之所以能轉移主要是透過癌細胞分泌蛋 白分解酵素，如 matrix metalloproteinases (MMPs)來分解胞外基質，最後導致 intercellular matrix 分離和基底膜的分解，進而使癌細胞 migration及 invasion提昇。 而在這些水解酵素中，MMP-2及 MMP-9在癌細胞 migration及 invasion過程中扮 演著最重要的角色。 類黃酮(flavonoids)屬於天然多酚類(polyphenol)廣泛存在於日常生活蔬果之 中。具有抗過敏、抗發炎、抗氧化、強化毛細血管、抗癌化以及調節酵素活性等生 物活性。黃酮化合物(flavones)是類黃酮的主要基本結構，在自然界中具有許多不 同取代基之衍生物，其中以氫氧基(-OH)、甲基(-CH3)及醣苷基(-glycoside, -rutinoside) 的取代最為常見。在抗氧化的研究中，有文獻指出黃酮化合物可有效抑制 LPS 或 LTA 所誘導之 NO 活性，且結構上之氫氧基數目越多其抗氧化能力越強。而在細 胞癌化的化學預防方面，發現其可促使癌細胞的凋亡並抑制其增生能力及抑制正常 細胞的癌化。但對於抑制癌瘤細胞侵襲轉移能力方面的研究，則尚未明瞭。本實驗 室去年國科會計畫送審時，委員建議先完成細胞凋亡部分，所以先給一年期。因此， 本實驗室已完成 7個黃酮類化合物與四株骨癌細胞株其細胞存活率的相關性，發現 2’-Hydroxyflavone可最有效可以降低其細胞存活率，我們也完成其路徑及機制的探 討，而動物實驗也證明其效果。因此，總和以上結論，以 2’-Hydroxyflavone而言， 的確可以誘導骨癌細胞株走向凋亡。但是其他黃酮類化合物在低劑量下可否影響骨 癌的轉移，甚至其轉移相關蛋白如MMPs family或其抑制劑 TIMPs之表現仍有待 釐清。因此，本計畫第一年擬探討不同的黃酮類化合物(Flavone, 3’-Hydroxyflavone, 4’-Hydroxyflavone, 5’-Hydroxyflavone, 6’-Hydroxyflavone及 7’-Hydroxyflavone) 對 不同骨癌細胞株其細胞移動、侵襲、貼附和轉移能力、基質分解相關蛋白酶活性的 影響，並進一步探討不同的黃酮類化合物抑制不同骨癌細胞株其細胞移動、侵襲、 貼附和轉移能力之蛋白酶分泌的相關訊息傳遞路徑的蛋白變化及上述基因的相關 轉錄因子表現。本計畫第二年擬探討不同的黃酮類化合物對不同骨癌細胞株其上皮 -間質轉化(epithelial-mesenchymal transition, EMT)的影響，並進一步觀察 EMT相關 因子及 EMT相關的 microRNA的影響。本計畫第三年擬進行選殖基質分解相關蛋 白酶(MMP-2、MMP-9及 TIMP-1 )的 promoter region，構築 luciferase/reporter gene plasmid，送入不同骨癌細胞株，同時處理對目標基因有抑制效果的黃酮類化合物， 以 luciferase assay分析黃酮類化合物對個別基因 promoter的調控。並以免疫缺陷的 小鼠接種人類不同骨癌細胞株，並處理黃酮類化合物，進一步觀察其腫瘤大小、重 量、血管新生相關基因及其動物存活率，分析黃酮類化合物對於接種不同骨癌細胞 的老鼠其腫瘤大小、重量、動物存活率及轉移到肺臟的影響。
Metastasis of cancer cells, a primary cause of cancer death and a multiple and intricate processes, may complicate the clinical management and lead to a poor prognosis for cancer patients and has tremendous physical or economical impact to patients or communities. Among these involved proteases, MMP-2 and MMP-9 are the most vital ones for degradation of base membrane and therefore deeply involved in cancer invasion and metastasis. Flavonoids are a group of polyphonic compounds that widely distributed in dietary foods of vegetables and fruits. In the anti-cancer potency aspect, flavones might be able to influence processes that are dysregulated during cancer development, and possess the propensity to anti-proliferation and induce apoptosis. Compared to the abovementioned aspects, studies on the inhibitory effect of flavones on cancer cell invasion behavior have been relatively less and warrant a further study. In our last year study, four osteosarcoma cells (MG-63，U2OS，143B，SaOS2) have been treated with flavones and then subjected to assays for cell viability. The results showed that 2’-Hydroxyflavone significantly induce apoptosis. However, the involvement mechanisms of other flavone and their analogues in osteosarcoma cell migration ability have not been studied. Therefore, in the first year of this study, we propose to analyze and compare the effects of different flavones on cell migration ability of different osteosarcoma cells and demonstrate the mechanism involved in reduced invasion of different flavones. Then, in the second year of this study, we aim to analyze and compare epithelial-mesenchymal transition (EMT) related gene, after different flavones treatment in different osteosarcoma cells and to determine the transactivity of these transcription factors and binding activity with its target binding sequence. Finally, in the third year of this study, we aim to clone the promoter region of MMP-2, -9, TIMP-1 and create promoter constructs driving luciferase to detect the effect of different flavones treatment on regulation of selected gene expression via luciferase assay. Finally, investigate tumor growth and angiogenesis of different osteosarcoma cells in vivo via cancer cell xenografted nude mice model. |
