臺灣博碩士論文加值系統

肝癌是目前最惡性的癌症之一且其癌症致死率在台灣甚至是全世界都高居第三位
。肝癌高致死率主要原因是由於治療後的高復發率以及其本身高轉移能力。然而，目前尚無有效抑制肝癌轉移的藥物。因此，找尋或開發具高效能且低副作用的藥物是目前在治療肝癌上一個重要的議題。在日本、中國以及台灣，由於草藥本身低副作用且低毒性，已被廣泛的使用在治療不同疾病，或作為保健食品。所以，在治療癌症方面草藥一直被認為是最具潛力的藥物來源。在此研究結果中，發現NTU04以及MSL-UH這兩種草藥萃取物在低濃度的狀態下就能有效地抑制肝癌細胞的侵襲力。在明膠基質金屬蛋白&;#37238;活性測試 (Gelatin zymography assay) 實驗中發現NTU04能隨著濃度的增加而減低基質金屬蛋白&;#37238;-2 (MMP2) 的活性。進一步發現，NTU04所造成基質金屬蛋白&;#37238;-2的活性降低是透過抑制基質金屬蛋白&;#37238;-2的基因表現及增加其金屬蛋白&;#37238;組織抑制因子-1 (TIMP1) 的基因表現。進一步證明，NTU04透過Erk訊息傳遞路徑而影響金屬蛋白&;#37238;組織抑制因子-1的表現。為了找尋有效成分，利用中效能液相層析法將MSL-UH草藥萃取液作分層，發現在F3、F4以及F5能有效地抑制肝癌細胞的侵襲轉移能力。同時也發現，將F3、F4以及F5依混合後餵食動物也可以導致腫瘤的生長受到抑制。總而言論，NTU04以及MSL-UH草藥萃取液是透過影響基質金屬蛋白&;#37238;-2的活性，同時也調控金屬蛋白&;#37238;組織抑制因子-1的表現量，進而能有效地抑制肝癌細胞的轉移侵襲能力。而本篇研究在未來在治療肝細胞癌方面提供一種新的藥物來源，並期望其能增進對轉移性肝癌的治療效率。

Hepatocellular carcinoma (HCC) is the third common causes of cancer mortality all over the world and in Taiwan. The high mortality of HCC is often caused by high recurrence and metastasis. However, there’s currently no effective therapeutic agent that can inhibit HCC metastasis. Therefore, to identify a new drug or compound with low cytotoxicity and high efficacy is imperative for cancer therapy. Herbal extracts have been used in traditional medicine for a long time and shown with low cytotoxicity and side effects. Thus, herbal extracts may serve as good sources to isolate effective components or compounds for cancer therapy. In the study, two herbal extracts (NTU04 and NTU43) were identified and able to inhibit HCC cell invasion with low cytotoxicity. Moreover, the results showed that NTU04 could significantly reduce matrix metalloprotease-2 (MMP-2) activities in the conditioned media of Huh7 cells at a dose-dependent manner. NTU04-reduced gelatinolytic activity of MMP-2 was at least due to down-regulation of MMP2 expression and up-regulation of tissue inhibitor of metalloprotease-1 (TIMP1) expression. Moreover, the effect of NTU04 on up-regulating TIMP-1 expression was via Erk1/2 signaling. I then used ethanol to extract a Taiwanese herb, a close species to NTU04 and named the extract as MSL-UH. MSL-UH also could inhibit HCC cell invasion with less cytotoxicity. Using liquid chromatography, MSL-UH were divided into nine fractions. The third, fourth and fifth fractions (F3, F4 and F5) can more effectively inhibit HCC cell invasion than the other six fractions. Moreover, the mixture of MSL-UH F3, F4 and F5 significantly suppressed the tumor growth of Huh7 cells in xenografted mouse model. In conclusion, NTU04 and MSL-UH can inhibit the invasion of HCC cells, partly through inhibiting MMP2 activity and up-regulating TIMP1. The data indicate that NTU04 and MSL-UH may contain effective compounds or components that can inhibit HCC cell invasion and be useful for chemoprevention or therapeutic purposes.

致謝.......................................................I
摘要.....................................................III
Abstract.................................................IV
Chapter 1. Introduction...................................1
1.1 Liver cancer..........................................2
1.2 Cancer progression and metastasis.....................2
1.3 Roles of matrix metalloproteinases (MMPs) and their cognate tissue inhibitors of metalloproteinases (TIMPs) in cancer metastasis.........................................3
1.4 Signaling pathways....................................4
1.4.1 FAK signaling pathway...............................4
1.4.2 PI3K/AKT/mTOR signaling pathway.....................5
1.4.3 MAPK/ERk signaling..................................6
1.5 Herbal extracts: NTU04 and MSL-UH.....................7
1.6 Research motivation...................................8
Chapter 2. Materials and Methods..........................9
2.1 Materials.........................................10
2.2 Methods...........................................13
Chapter 3. Results.......................................22
3.1 Effects of NTU04 on the cell viability and growth in
Huh7 cells. ......................................23
3.2 Effects of NTU04 on HCC cell invasion. ...........23
3.3 Effects of NTU04 on the gelatinolytic activities of
MMP2/9 and the expression levels of MMP2/9 and their
cognate inhibitor TIMPs in Huh7 cells. ...........24
3.4 Role of TIMP1 in NTU04-treated Huh7 cell invasion.25
3.5 Identification of molecular mechanisms in which NTU04
could up-regulate TIMP1 expression, leading to
inhibition of HCC cell invasion. .................25
3.6 Effects of NTU04 on the gelatinolytic activities of
recombinant active MMP2. .........................26
3.7 Identification of novel proteases and protease
inhibitors which were affected by NTU04 for
inhibiting Huh7 cell invasion. ...................26
3.8 Effects of MSL-UH on the cell viability and growth in
Huh7 cells. ......................................27
3.9 Examination of MSL-UH effect on HCC cell invasion.28
3.10 Identification of effective herbal compounds and
components which could suppress HCC cell invasion.
.................................................28
3.11 Effects of the third, fourth and fifth fractions of
MSL-UH on the cell viability and invasion of Huh7
cells. ..........................................29
3.12 Effects of F3-5 fractions of MSL-UH extract on tumor
volumes, tumor mass and body weight in xenografted
mouse model. ....................................30
Chapter 4. Discussion....................................32
Chapter 5. Figures.......................................36
Chapter 6. References....................................67

1 Worldwide Cancer Mortality. Cancer Research 2013.

2 中華民國衛生福利部國民健康署. 民國101年主要死因分析 2013.

3 Ashwin Ananthakrishnan MD, M.P.H. et al. Epidemiology of Primary and Secondary liver cancers. SEMINARS IN INTERVENTIONAL RADIOLOGY 2006; 23: 47-63.

4 Irfan Ahmed DNL. Malignant tumours of the liver. Surgery 2009; 27: 30-37.

5 El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557-2576.

6 Severi T, van Malenstein H, Verslype C, van Pelt JF. Tumor initiation and progression in hepatocellular carcinoma: risk factors, classification, and therapeutic targets. Acta Pharmacol Sin 2010; 31: 1409-1420.

7 Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: Worldwide incidence and trends. Gastroenterology 2004; 127: S5-S16.

8 Josep M. Llovet MD, Sergio Ricci, M.D., Vincenzo Mazzaferro, M.D. et al. Sorafenib in Advanced Hepatocellular carcinoma. Th e New England Journal o f Medicine 2008; 359: 378-390.

9 Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nature Reviews Cancer 2004; 4: 197-205.

10 Yokota J. Tumor progression and metastasis. Carcinogenesis 2000; 21: 497-503.

11 Wirtz D, Konstantopoulos K, Searson PC. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature Reviews Cancer 2011; 11: 512-522.

12 Rao JS. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 2003; 3: 489-501.

13 Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002; 2: 161-174.

14 Curry TE, Jr., Osteen KG. The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle. Endocrine Reviews 2003; 24: 428-465.

15 Birkedal-Hansen H, W. G. I. Moore MKB, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA. Matrix Metalloproteinases: A Review. Critical Reviews in Oral Biology and Medicine, 1993; 4: 197-250.

16 A.M. P, Beurden S-v, Hoff JWVd. Zymographic techniques for the analysis of MMPs and their inhibitors. BioTechniques 2005; 38: 73-83.

17 The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the development of esophageal cancer. Folia Histochem Cytobiol 2012; 50: 12-19.

18 Wart HEV, Birkedal-Hansent H. The cysteine switch: A principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc Natl Acad Sci USA 1990; 87: 5578-5582.

19 Keith Brew ea. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta 2000; 1477: 267-283.

20 Radomski A, Jurasz P, Sanders EJ, Overall CM, Bigg HF, Edwards DR et al. Identification, regulation and role of tissue inhibitor of metalloproteinases-4 (TIMP-4) in human platelets. British Journal of Pharmacology 2002; 137: 1330-1338.

21 Lee S-J, Yoo HJ, Bae YS, Kim H-J, Lee S-T. TIMP-1 inhibits apoptosis in breast carcinoma cells via a pathway involving pertussis toxin-sensitive G protein and c-Src. Biochemical and Biophysical Research Communications 2003; 312: 1196-1201.

22 Rho SB, Chung BM, Lee JH. TIMP-1 regulates cell proliferation by interacting with the ninth zinc finger domain of PLZF. Journal of Cellular Biochemistry 2007; 101: 57-67.

23 Sounni NE, Janssen M, Foidart JM, Noel A. Membrane type-1 matrix metalloproteinase and TIMP-2 in tumor angiogenesis. Matrix Biology 2003; 22: 55-61.

24 Li Y, Ma J, Guo Q, Duan F, Tang F, Zheng P et al. Overexpression of MMP-2 and MMP-9 in esophageal squamous cell carcinoma. Diseases of the Esophagus 2009; 22: 664-667.

25 Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005; 6: 56-68.

26 Navarro AI, Rico B. Focal adhesion kinase function in neuronal development. Current Opinion in Neurobiology 2014; 27C: 89-95.

27 McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer : a new therapeutic opportunity. Nat Rev Cancer 2005; 5: 505-515.

28 Zhao J, Guan JL. Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev 2009; 28: 35-49.

29 Porta C, Paglino C, Mosca A. Targeting PI3K/Akt/mTOR Signaling in Cancer. Frontiers in Oncololy 2014; 4: 1-11.

30 Phin S, Moore MW, Cotter PD. Genomic Rearrangements of PTEN in Prostate Cancer. Frontiers in Oncology 2013; 3: 1-9.

31 Toker A, Marmiroli S. Signaling specificity in the Akt pathway in biology and disease. Advances in Biological Regulation 2014; 55C: 28-38.

32 Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treatment Reviews 2004; 30: 193-204.

33 Houede N, Pourquier P. Targeting the genetic alterations of the PI3K-AKT-mTOR pathway: Its potential use in the treatment of bladder cancers. Pharmacol Ther 2014.

34 Chang F, Steelman LS, Lee JT, Shelton JG, Navolanic PM, Blalock WL et al. Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 2003; 17: 1263-1293.

35 Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene 2007; 26: 3279-3290.

36 Kim HJ, Bar-Sagi D. Modulation of signalling by Sprouty: a developing story. Nat Rev Mol Cell Biol 2004; 5: 441-450.

37 Wu TY, Chen CP, Jinn TR. Traditional Chinese medicines and Alzheimer''s disease. Taiwan J Obstet Gynecol 2011; 50: 131-135.

38 Shane-McWhorter L. Biological complementary therapies: a focus on botanical products in diabetes. Diabetes Spectrum 2001; 14: 199-208.

39 Martin KW. Herbal medicines for treatment of bacterial infections: a review of controlled clinical trials. Journal of Antimicrobial Chemotherapy 2003; 51: 241-246.

40 Mainardi T, Kapoor S, Bielory L. Complementary and alternative medicine: herbs, phytochemicals and vitamins and their immunologic effects. J Allergy Clin Immunol 2009; 123: 283-294.

41 Kim D, Choi J, Kim MJ, Kim SH, Cho SH, Kim S. Reconstitution of anti-allergic activities of PG102 derived from Actinidia arguta by combining synthetic chemical compounds. Exp Biol Med (Maywood) 2013; 238: 631-640.

42 Adams LS, Seeram NP, Hardy ML, Carpenter C, Heber D. Analysis of the interactions of botanical extract combinations against the viability of prostate cancer cell lines. Evid Based Complement Alternat Med 2006; 3: 117-124.

43 Ndagijimana A, Wang X, Pan G, Zhang F, Feng H, Olaleye O. A review on indole alkaloids isolated from Uncaria rhynchophylla and their pharmacological studies. Fitoterapia 2013; 86: 35-47.

44 Hsieh C-L, Chen M-F, Li T-C. Anticonvulsant effect of Uncaria rhynchophylla (Miq) Jack. in rats with kainic acid-induced epileptic seizure. American Journal of Chinese Medicine 1999; 27: 257-264.

45 Lee JS, Kim J, Kim BY, Lee HS, Ahn JS, Chang YS. Inhibition of Phospholipase Cc1 and Cancer Cell Proliferation by Triterpene Esters from Uncaria rhynchophylla. J Nat Prod 2000; 63: 753-756.

46 Hidekazu Nakabayashi KT, Keiko Miyano, et al. Growth of Human Hepatoma Cell Lines with Differentiated Functions in Chemically Defined Medium. Cancer Research 1982; 42: 3858-3863.

47 KNOWLES BB, C.C. HOWE, and D.P. ADEN. Human Hepatocellular Carcinoma Cell Lines Secrete the Major Plasma Proteins and Hepatitis B Surface Antigen. Science 1980; 209: 497-499.

48 Mersch-Sundermann V, Knasmuller S, Wu XJ, Darroudi F, Kassie F. Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 2004; 198: 329-340.

49 Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003; 3: 362-374.

50 Overall CM, Lopez-Otin C. Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2002; 2: 657-672.

51 Golubovskaya VM, Kwe FA, Cance WG. Focal adhesion kinase and cancer. Histol Histopathol 2009; 24: 503-510.

52 Xia Y, Yeddula N, Leblanc M, Ke E, Zhang Y, Oldfield E et al. Reduced cell proliferation by IKK2 depletion in a mouse lung-cancer model. Nature Cell Biology 2012; 14: 257-265.

53 Xiao LJ, Lin P, Lin F, Liu X, Qin W, Zou HF et al. ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion. International Journal of Oncology 2012; 40: 1714-1724.

54 Duan, Q., et al. LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures. Nucleic Acids Research, 2014; 12: 449-460.

55 Binder BR, Mihaly J, Prager GW. uPAR – uPA – PAI-1 interactions and signaling: A vascular biologist’s view. Thrombosis and Haemostasis 2007; 97: 336-342.

56 Duffy MJ. Urokinase Plasminogen Activator and Its Inhibitor,PAI-1, as Prognostic Markers in Breast Cancer: From Pilot to Level I Evidence Etudies. Clinical Chemistry 2002; 48: 1194-1197.

57 Lee C-C, Huang T-S. Plasminogen Activator Inhibitor-1: The Expression, Biological Functions, and Effects on Tumorigenesis and Tumor Cell Adhesion and Migration. Journal of Cancer Molecules 2005; 1: 25-36.

58 Flavin RJ, Pettersson A, Hendrickson WK, Fiorentino M, Finn SP, Kunz L et al. SPINK1 Protein Expression and Prostate Cancer Progression. Clinical Cancer Research 2014.

59 Robert C, Soria J-C, Spatz A, Le Cesne A, Malka D, Pautier P et al. Cutaneous side-effects of kinase inhibitors and blocking antibodies. The Lancet Oncology 2005; 6: 491-500.

60 Bourboulia, D. and W.G. Stetler-Stevenson. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion. Seminar Cancer Biology 2010; 20: 161-168.

61 Fingleton, B. MMPs as therapeutic targets-still a viable option? Seminar in Cell and Development Biology 2008; 19: 61-68.

62 Smyth, E. The trouble with inhibitors. Nature 2003: 1-4.

63 Van Lint, P. and C. Libert. Chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation. Journal of Leukocyte Biology 2007; 82: 1375-1381.

64. Gordon, G.M., et al. Cytokines and signaling pathways regulating matrix metalloproteinase-9 (MMP-9) expression in corneal epithelial cells. Journal of Cellular Physiology 2009; 221: 402-411.

65 Roberts, P.J. and C.J. Der. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007; 26: 3291-3310.

66 Li, Z., M.H. Theus, and L. Wei. Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells. Development Growth and Differentiation 2006; 48: 513-523.

67 Fernandez-Serra, M., et al. Role of the ERK-mediated signaling pathway in mesenchyme formation and differentiation in the sea urchin embryo. Development Biology 2004; 268: 384-402.