| 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： serine proteinase、matrix metalloproteinases (MMPs)以及 plasminogen activator (PA)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、 MMP-9及 u-PA在癌細胞 migration及 invasion過程中扮演著最重要的角色。 斑螫素(cantharidin)是萃取自翹翅目、莞青科、斑莞青屬的一種名為斑螫的昆 蟲，而斑螫素與其去甲基斑螫素(norcantharidin)與其之亞胺(imide)衍生物具有抗癌 的作用。而由斑螫素所發展的一系列亞胺結構與其衍生物之抗腫瘤活性亦被發現是 經由誘導癌細胞走向凋亡。但是斑螫素及其衍生物對抑制肝癌細胞侵襲轉移能力及 誘導 apoptosis的詳細路徑方面的研究，則尚未明瞭。本實驗室初步以 norcantharidin 處理二株不同的肝癌細胞株(Huh7及 SK-Hep1)，發現在高濃度(100 μM)會誘導其細 胞凋亡，但進一步以較低濃度(0-10 μM) 的 norcantharidin處理，則發現對這兩株細 胞株的細胞侵襲轉移能力都有明顯的抑制效果，此結果已經以第一作者發表在 2012年的 PLoS ONE期刊中。但針對其他的斑蝥素及其去甲基斑蝥素之亞胺類的 衍生物抑制肝癌細胞侵襲轉移能力及誘導 apoptosis 的效果仍須進一步的研究。因 此，本計畫第一年擬進一步觀察其他不同的斑螫素衍生物對不同肝癌細胞株其存活 率、細胞凋亡及其細胞移動、侵襲、貼附和轉移能力、基質分解相關蛋白酶活性的 影響。本計畫第二年擬探討不同的斑螫素衍生物對不同肝癌細胞株其與細胞週期相 關因子及其 migration/invasion 和 adhesion 相關基因的影響，並進一步探討不同的 斑螫素衍生物抑制不同肝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。本計畫第三年 擬進行選殖基質分解相關蛋白酶 (MMP-2、MMP-9、u-PA、TIMP-1 及 PAI-1)的 promoter region，構築 luciferase/reporter gene plasmid，送入不同肝癌細胞株，同時 處理對目標基因有抑制效果的斑螫素衍生物，以 luciferase assay分析斑螫素衍生物 對個別基因 promoter 的調控。並以免疫缺陷的小鼠接種人類不同肝癌細胞株，並 處理斑螫素衍生物，進一步觀察其腫瘤大小、重量及其動物存活率，分析斑螫素衍 生物對於接種不同肝癌細胞的老鼠其腫瘤大小、重量、動物存活率及轉移到肺臟或 肝臟的影響。
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, Matrix metalloproteinase (MMP)-2, MMP-9 and u-PA are the most vital ones for degradation of base membrane and therefore deeply involved in cancer invasion and metastasis. The anticancer activities of cantharidin, norcantharidin and their analogues have been discussed by suppressing the activity of serine/threonine protein phosphatases. The thiazole derivatives are considered as kinds of antibiotic which can induce apoptosis in human cancer cells. Compared to the abovementioned aspects, studies on the inhibitory effect of cantharidin and their analogues on hepatocellular carcinoma (HCC) cell invasion behavior have been relatively less and warrant a further study. In our preliminary study, two HCC cells (Huh7 and SK-Hep1) have been treated with norcantharidin and then subjected to assays for cell migration. The results showed that norcantharidin significantly inhibit cell migration ability (published in PLoS ONE). However, the involvement mechanism of other cantharidin and their analogues in HCC cell migration ability or apoptosis have not been studied. Therefore, in the first year of this study, we propose to analyze and compare the effects of cantharidin, norcantharidin and their different analogues on cell viability of different HCC and demonstrate the mechanism involved in reduced invasion of cantharidin, norcantharidin and their different analogues. Then, in the second year of this study, we aim to analyze and compare cell cycle related kinases cyclins, after cantharidin, norcantharidin and their different analogues treatment in different HCC 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, u-PA, TIMPs, PAIs and create promoter constructs driving luciferase to detect the effect of cantharidin, norcantharidin and their different analogues treatment on regulation of selected gene expression via luciferase assay. Finally, investigate tumor growth of different HCC in vivo via cancer cell xenografted nude mice model. |
