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.

1.
Akin S, Can G, Aydiner A, Ozdilli K, Durna Z (2010) Quality of life, symptom experience and distress of lung cancer patients undergoing chemotherapy. Eur J Oncol Nurs 14:400-409.

Amarasena IU, Walters JA, Wood-Baker R, Fong K (2008) Platinum versus non-platinum chemotherapy regimens for small cell lung cancer. Cochrane Database Syst Rev CD006849.

Bian J, Sun Y (1997) Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. Mol Cell Biol 17:6330-6338.

Biesalski HK, Bueno de Mesquita B, Chesson A, Chytil F, Grimble R, Hermus RJ, Kohrle J, Lotan R, Norpoth K, Pastorino U, Thurnham D (1998) European Consensus Statement on Lung Cancer: risk factors and prevention. Lung Cancer Panel. CA Cancer J Clin 48:167-176.

Bruckl WM, Lampert S, Zirlik S, Hahn EG, Wiest GH (2008) [Drug therapy for small-cell lung cancer (SCLC)--new molecular strategies for therapy]. Pneumologie 62:23-30.

Chambers AF, Groom AC, MacDonald IC (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2:563-572.

Chen S, Flower A, Ritchie A, Liu J, Molassiotis A, Yu H, Lewith G (2010) Oral Chinese herbal medicine (CHM) as an adjuvant treatment during chemotherapy for non-small cell lung cancer: A systematic review. Lung Cancer 68:137-145.
Chetty C, Lakka SS, Bhoopathi P, Rao JS (2010) MMP-2 alters VEGF expression via alphaVbeta3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells. Int J Cancer 127:1081-1095.

Choi JH, Han EH, Hwang YP, Choi JM, Choi CY, Chung YC, Seo JK, Jeong HG (2010) Suppression of PMA-induced tumor cell invasion and metastasis by aqueous extract isolated from Prunella vulgaris via the inhibition of NF-kappaB-dependent MMP-9 expression. Food Chem Toxicol 48:564-571.

Chung AS, Ferrara N (2011) Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol 27:563-584.

Clauss M (2000) Molecular biology of the VEGF and the VEGF receptor family. Semin Thromb Hemost 26:561-569.

Corcoran ML, Hewitt RE, Kleiner DE, Jr., Stetler-Stevenson WG (1996) MMP-2: expression, activation and inhibition. Enzyme Protein 49:7-19.

Dong J, Su SY, Wang MY, Zhan Z (2010) Shenqi fuzheng, an injection concocted from Chinese medicinal herbs, combined with platinum-based chemotherapy for advanced non-small cell lung cancer: a systematic review. J Exp Clin Cancer Res 29:137.

Elena I. Deryugina, James P. Quigley (2006) Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 25:9-34.

Eto H, Toriyama K, Itakura H (1994) A clinicpathological study of hepatocellular carcinoma in Nagasaki, south-western Japan: the association of hepatitis B and C viruses. Southeast Asian J Trop Med Public Health 25:88-92.

Feng L, Au-Yeung W, Xu YH, Wang SS, Zhu Q, Xiang P (2011) Oleanolic acid from Prunella Vulgaris L. induces SPC-A-1 cell line apoptosis via regulation of Bax, Bad and Bcl-2 expression. Asian Pac J Cancer Prev 12:403-408.

Fidler IJ (2003) The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat Rev Cancer 3:453-458.

Gao H, Lamusta J, Zhang WF, Salmonsen R, Liu Y, O'Connell E, Evans JE, Burstein S, Chen JJ (2011) Tumor Cell Selective Cytotoxicity and Apoptosis Induction by an Herbal Preparation from Brucea javanica. North Amer J Med Sci 4:62-66.

Girard N, Geriniere L, Blandin S, Perrot E, Souquet PJ (2004) [French experience of gefitinib in non-small cell bronchial carcinoma]. Rev Mal Respir 21:934-942.

Goldberg GI, Strongin A, Collier IE, Genrich LT, Marmer BL (1992) Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem 267:4583-4591.

Hailfinger S, Jaworski M, Marx-Stoelting P, Wanke I, Schwarz M (2007) Regulation of P53 stability in p53 mutated human and mouse hepatoma cells. Int J Cancer 120:1459-1464.

Howard EW, Bullen EC, Banda MJ (1991) Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2. J Biol Chem 266:13064-13069.

Hua J, Muschel RJ (1996) Inhibition of matrix metalloproteinase 9 expression by a ribozyme blocks metastasis in a rat sarcoma model system. Cancer Res 56:5279-5284.

Jespersen C, Doller A, Akool el S, Bachmann M, Muller R, Gutwein P, Muhl H, Pfeilschifter J, Eberhardt W (2009) Molecular mechanisms of nitric oxide-dependent inhibition of TPA-induced matrix metalloproteinase-9 (MMP-9) in MCF-7 cells. J Cell Physiol 219:276-287.

John A, Tuszynski G (2001) The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathol Oncol Res 7:14-23.

Kang JH, Han IH, Sung MK, Yoo H, Kim YG, Kim JS, Kawada T, Yu R (2008) Soybean saponin inhibits tumor cell metastasis by modulating expressions of MMP-2, MMP-9 and TIMP- 2. Cancer Lett 261:84-92.

Kim SH, Huang CY, Tsai CY, Lu SY, Chiu CC, Fang K (2012) The Aqueous Extract of Prunella vulgaris Suppresses Cell Invasion and Migration in Human Liver Cancer Cells by Attenuating Matrix Metalloproteinases. Am J Chin Med 40:643-656.

Klein T, Bischoff R (2011) Physiology and pathophysiology of matrix metalloproteases. Amino Acids 41:271-290.

Larcher F, Murillas R, Bolontrade M, Conti CJ, Jorcano JL (1998) VEGF/VPF overexpression in skin of transgenic mice induces angiogenesis, vascular hyperpermeability and accelerated tumor development. Oncogene 17:303-311.

Liang CZ, Zhang JK, Shi Z, Liu B, Shen CQ, Tao HM (2012) Matrine induces caspase-dependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/FasL and downregulation of Bcl-2. Cancer Chemotherapy and Pharmacology 69:317-331

Liu J, Zhan M, Hannay JA, Das P, Bolshakov SV, Kotilingam D, Yu D, Lazar AF, Pollock RE, Lev D (2006) Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis. Mol Cancer Res 4:803-810.

Loffek S, Schilling O, Franzke CW (2010) Biological role of matrix metalloproteinases: a critical balance. The Euro Resp J 38:191-208

Lu AP, Jia HW, Xiao C, Lu QP (2004) Theory of traditional Chinese medicine and therapeutic method of diseases. World J Gastroenterol 10:1854-1856.

Maragoudakis ME, Tsopanoglou NE, Andriopoulou P (2002) Mechanism of thrombin-induced angiogenesis. Biochem Soc Trans 30:173-177.

McCulloch M, See C, Shu XJ, Broffman M, Kramer A, Fan WY, Gao J, Lieb W, Shieh K, Colford JM, Jr. (2006) Astragalus-based Chinese herbs and platinum-based chemotherapy for advanced non-small-cell lung cancer: meta-analysis of randomized trials. J Clin Oncol 24:419-430.

McWhinney SR, Goldberg RM, McLeod HL (2009) Platinum neurotoxicity pharmacogenetics. Mol Cancer Ther 8:10-16.

Mou H, Zheng Y, Zhao P, Bao H, Fang W, Xu N (2011) Celastrol induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria- and Fas/FasL-mediated pathways. Toxicol In Vitro 25:1027-1032.

Nykamp V (2010) Surgical treatment of non-small cell lung cancer. S D Med Spec No:57-59.

Rosti G, Carminati O, Monti M, Tamberi S, Marangolo M (2006) Chemotherapy advances in small cell lung cancer. Ann Oncol 17 Suppl 5:v99-102.

Rouhi P, Lee SL, Cao Z, Hedlund EM, Jensen LD, Cao Y (2010) Pathological angiogenesis facilitates tumor cell dissemination and metastasis. Cell Cycle 9:913-917.

Seki K, Chisaka M, Eriguchi M, Yanagie H, Hisa T, Osada I, Sairenji T, Otsuka K, Halberg F (2005) An attempt to integrate Western and Chinese medicine: rationale for applying Chinese medicine as chronotherapy against cancer. Biomed Pharmacother 59 Suppl 1:S132-140.

Shu X, McCulloch M, Xiao H, Broffman M, Gao J (2005) Chinese herbal medicine and chemotherapy in the treatment of hepatocellular carcinoma: a meta-analysis of randomized controlled trials. Integr Cancer Ther 4:219-229.

Stuttfeld E, Ballmer-Hofer K (2009) Structure and function of VEGF receptors. Int Union Biochem Mol Biol Life 61:915-922.

Suzuki K, Matsubara H (2011) Recent advances in p53 research and cancer treatment. J Biomed Biotechnol 2011:978312.

Toyosaka A, Okamoto E, Mitsunobu M, Oriyama T, Nakao N, Miura K (1996) Intrahepatic metastases in hepatocellular carcinoma: evidence for spread via the portal vein as an efferent vessel. Am J Gastroenterol 91:1610-1615.

Travis WD, Travis LB, Devesa SS (1995) Lung cancer. Cancer 75:191-202.

VanderBorght A, Valckx A, Van Dun J, Grand-Perret T, De Schepper S, Vialard J, Janicot M, Arts J (2006) Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells. Oncogene 25:6672-6677.

Xu ZY, Jin CJ, Zhou CC, Wang ZQ, Zhou WD, Deng HB, Zhang M, Su W, Cai XY (2011) Treatment of advanced non-small-cell lung cancer with Chinese herbal medicine by stages combined with chemotherapy. J Cancer Res Clin Oncol 137:1117-1122.

Zhang S, Cao Z, Tian H, Shen G, Ma Y, Xie H, Liu Y, Zhao C, Deng S, Yang Y, Zheng R, Li W, Zhang N, Liu S, Wang W, Dai L, Shi S, Cheng L, Pan Y, Feng S, Zhao X, Deng H, Yang S, Wei Y (2011) SKLB1002, a novel potent inhibitor of VEGF receptor 2 signaling, inhibits angiogenesis and tumor growth in vivo. Clin Cancer Res 17:4439-4450.

Zhang Y, Zhang H, Yu P, Liu Q, Liu K, Duan H, Luan G, Yagasaki K, Zhang G (2009) Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration. Cytotechnology 59:191-200.

Zitka O, Kukacka J, Krizkova S, Huska D, Adam V, Masarik M, Prusa R, Kizek R (2010) Matrix metalloproteinases. Curr Med Chem 17:3751-3768.

2.
Chan WM, Tsoi H, Wu CC, Wong CH, Cheng TC, Li HY, Lau KF, Shaw PC, Perrimon N, Chan HY (2011) Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1. Hum Mol Genet 20:1738-1750.

Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, Yang L, Kotin RM, Paulson HL, Davidson BL (2005) RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A 102:5820-5825.

Menzies FM, Huebener J, Renna M, Bonin M, Riess O, Rubinsztein DC (2010) Autophagy induction reduces mutant ataxin-3 levels and toxicity in a mouse model of spinocerebellar ataxia type 3. Brain 133:93-104.

Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069-1075.

Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein DC (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36:585-595.

Sarkar S, Ravikumar B, Floto RA, Rubinsztein DC (2009) Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ 16:46-56.

Shao J, Diamond MI (2007) Polyglutamine diseases: emerging concepts in pathogenesis and therapy. Hum Mol Genet 16 Spec No. 2:R115-123.

Wang CE, Zhou H, McGuire JR, Cerullo V, Lee B, Li SH, Li XJ (2008) Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin. J Cell Biol 181:803-816.