1. 探討夏枯草抑制非小細胞肺癌細胞轉移機制 2.篩選以細胞自噬去除神經細胞堆積多麩胺酸的小分子新穎化合物

Abstract

1. 癌症是由於體細胞複製脫離正常的調控而不斷快速複製異常細胞的現象。異常增生的細胞形成腫瘤侵入週邊組織持續擴大並影響原發部位的器官功能及壓迫周遭器官；而癌細胞進入人體的血液循環以及淋巴系統轉移至其它部位增生，更是具有威脅性。 癌細胞的轉移，與分解細胞外基質(extracellular matrix, ECM)，以及促進血管新生的能力有很大的關聯。而在這些過程中，基質金屬蛋白酶(matrix metalloproteinase, MMP)扮演了一個十分重要的角色。 本研究利用中草藥處理人類肺腺癌細胞株(A549、H460及H1299)以及以化學合成藥物處理p53 mutation 之肝癌細胞株(Huh7)。實驗首先利用gelatin zymography assay篩選出具有抑制MMP-9與MMP-2活性能力之藥物，並以western blot觀察細胞懸浮液MMP-9與MMP-2的表現量。接著觀察藥物是否對細胞有毒性，再以wound healing assay觀察癌細胞移動能力的影響；最後以invasion assay觀察中藥對腫瘤細胞的侵入能力的影響。綜合以上的實驗結果看出，夏枯草能夠對肺癌細胞的MMP-9與MMP-2活性產生抑制效果，而且可以抑制肺癌細胞的入侵以及移動能力，在H460細胞株上的效果較為顯著，且藥效不會因p53的狀態而受到影響。另外本次實驗並沒有在所試驗的化學合成藥物中觀察到具抑制MMP活性的合成藥物。

2. 細胞自噬(autophagy)是細胞一個重要的維持細胞生存的方式。細胞透過這個過程，清除細胞中非必要的物質，以取得胺基酸等營養物質，也避免細胞異常的物質累積。自噬過程中，細胞會將目標物質包覆在雙層膜節構中形成自噬體(autophagosome)，再與溶酶體(lysosome)融合，降解目標物質，並且回收營養物質和能源。 Polyglutamine (polyQ) disease是一種在神經性退化症，其主要的病因是在基因中出現CAG或CUG的重複序列的異常增加。目前已經證明藉由調控自噬作用的藥物在polyQ disease，如亨丁頓氏症 (Huntington’s disease)小鼠模型可以減輕其毒性，因此增強自噬作用的藥物，可能會是對這種疾病的治療方式。 本論文轉殖了結合不同長度polyQ的綠色螢光蛋白基因之SK細胞株，將穩定的細胞株用各種合成化學物質複合物進行處理之後，使用螢光染色，觀察自噬體是否增加且觀察對細胞存活的影響。本論文初步篩選出了三種能夠誘導細胞自噬增加並減少polyQ堆積的小分子化合物。未來研究會再繼續確認細胞自噬對清除細胞polyQ堆積的機制。1. Cancer cells grow and duplicate unregulated that form malignant tumors. The capability of cell invasion and migration from the origin site (metastasis) to nearby parts is the most threating. Cancer metastasis starts with the degradation of extracellular matrix (ECM). Both invasion to nearby tissue of cancer cell and inducement to angiogenesis relies on the matrix metalloproteinase (MMP) activity for ECM degradation. The thesis focused on treating cell lines, A549 (wild type p53 human adenocarcinoma), H460 (human large cell lung carcinoma) and H1299 (p53 deleted human adenocarcinoma) with Chinese herb medicine (CHM), and then detect changes of MMP-9 and MMP-2 activities by evaluating gelatin zymography. Both gelatin zymography and western blot to find out whether CHM can inhibit the MMP-9 and MMP-2 expression. The work is also to figure out whether MMP-9 and MMP-2 inhibition affects the migration and invasion capacity of the cancer cells. We have found that the aqueous extract of Prunella vulgaris can affects MMP-9 and MMP-2 activities in human lung carcer cells, and inhibit the invasion and migration of cancer cells. The other goal of this thesis is to find out new CHM‘s that are capable of inhibiting tumor angiogenesis and metastasis by evaluating MMP-9 and MMP-2 activities in human hepatocarcinoma cells.

2. Cells digest unwanted substance by autophagy, a procedure that could reuse building blocks from unwanted substances and clarify poisonous substance. The recycled substrate is covered by lipid bilayer, forming autophagsome that combines lysosome for digestion. Polyglutamine (polyQ) disease is a set of genetic disorder caused by the increased numbers of CAG or CUG repeats in some neurodegenerative diseases. Drugs that induced up-regulation of autophagy have been proved decrease the toxicity of polyQ aggregation in mouse model of Huntington’s disease. Thus, the autophagy-inducing drugs promise to be an effective therapy of polyQ diseases. The thesis used SK cell lines transfected with green fluorescent protein conjugated with different length of polyQ. The purpose is to find out whether the autophagsome can be increased by treatment of different compounds and whether they affect the viabilities of the cells. Three compounds were found capable of inducing cell autophagy and decreasing polyQ aggregration in cell models. In the future, the research will focus on how the selected drugs affect autophagy and test the drugs in animal model.