臺灣博碩士論文加值系統

二甲雙胍(metformin)是目前廣泛使用於治療第二型糖尿病的用藥。其降血糖機制包括抑制肝臟葡萄糖新生作用、增加肌肉對葡萄糖的攝取、促進對胰島素的敏銳度。近期研究認為 metformin 或許會降低癌症發生率，但其抑制癌症的機制還不清楚，特別是在抑制胃癌的部分。在本研究中我們將評估 metformin 抑制人類胃癌細胞(AGS cells)的能力並利用相對和絕對定量的等量異位標籤(isobaric Tagging for Relative and Absolute Quantitation)定量蛋白質體學進一步探討抑制癌症的機制。我們的研究發現metformin抑制胃癌細胞增生的能力會隨著劑量增加而增加，metformin 也會誘發胃癌細胞G0/G1週期停滯，抑制胃癌細胞的細胞移動及影響胃癌細胞的細胞骨架之分布。我們利用定量蛋白質體學技術鑑定並定量177個蛋白質，並依照他們在控制組與metformin處理組的相對表現量挑選出9個表現量有差異的蛋白質，包含4個表現量上升的蛋白質(Prohibitin-2, Heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B), Clathrin heavy chain 1, Macrophage migration inhibitory factor (MIF))及5個表現量下降的蛋白質(T-complex protein 1 subunit epsilon, 26S proteasome non-ATPase regulatory subunit 2 (PSMD2), Stress-induced-phosphoprotein 1 (STIP1), Polypyrimidine tract-binding protein 1, Adenylyl cyclase-associated protein 1 (CAP1)). 其中蛋白質 STIP1 、 CAP1 及 PSMD2 在先前的研究中被指出與癌細胞的細胞增生和移動具有相關性。因此我們利用西方墨點法驗證 STIP1 、 CAP1 及 PSMD2 蛋白質的表現量並發現蛋白質表現趨勢與蛋白質體的分析結果是一致的。這些結果意味著 metformin 可能藉由調控胃癌細胞中蛋白質 STIP1 、 CAP1 及 PSMD2 的表現去抑制細胞增生和細胞移動。雖然還需更多的動物實驗或臨床實驗來驗證 metformin 的抗癌效果，本研究或許能提供胃癌治療的發展一個新的方向。

Metformin is one of the most widely used anti-diabetic drugs in type II diabetes treatment. The mechanism of lowering blood glucose is believed to be involved in suppressing hepatic gluconeogenesis, increasing muscular glucose uptake and enhancing insulin sensitivity. Recent studies suggest that metformin may reduce cancer risk, but the mechanism of anticancer action remains unclear, especially in gastric cancer. In our studies, we aim to evaluate the anticancer effects of metformin on human gastric cancer AGS cells, and use isobaric Tagging for Relative and Absolute Quantitation (iTRAQ)-based quantitative proteomics to further study the anticancer mechanism. Our results showed that metformin inhibited AGS cell proliferation in a dose dependent manner, induced AGS cell cycle arrest at G0/G1 phase, suppressed cell migration ability, and affected the distribution of cytoskeleton. Using quantitative proteomics approach, we identified 177 differentially expressed proteins upon metformin treatment; among these, nine proteins are significantly altered in expression level, including four up-regulated proteins (Prohibitin-2, Heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B), Clathrin heavy chain 1, Macrophage migration inhibitory factor (MIF)) and five down-regulated proteins (T-complex protein 1 subunit epsilon, 26S proteasome non-ATPase regulatory subunit 2 (PSMD2), Stress-induced-phosphoprotein 1 (STIP1), Polypyrimidine tract-binding protein 1, Adenylyl cyclase-associated protein 1 (CAP1)). In previous study, STIP1, CAP1 and PSMD2 were reported associated with cancer cell proliferation and motility. The expression level of STIP1, CAP1 and PSMD2 were further validated by immunoblotting and consistent with the proteomic results. Our findings imply that metformin might inhibit cell proliferation and migration in AGS cells by suppressing STIP1, CAP1 and PSMD2. Although more animal and clinical studies are needed to confirm the effects of metformin, our studies may provide a new way for developing therapies for gastric cancer.

Contents
致謝 I
中文摘要 II
Abstract III
Contents V
Chapter 1 Introduction 1
1.1 Gastric cancer 1
1.2 Metformin 1
1.2.1 History 1
1.2.2 Functions and molecular mechanisms of metformin in anti-diabetics 2
1.2.3 Metformin and Cancer 3
1.3 Proteomics 5
1.3.1 Overview 5
1.3.2 iTRAQ-based quantitative proteomics 6
1.4 Aim 7
Chapter 2 Materials and Methods 8
2.1 Chemical 8
2.2 Cell culture 8
2.2.1 Culture medium preparation 8
2.2.2 Gastric cancer cell line AGS 9
2.3 Cell proliferation assay 9
2.3.1 MTT assay 9
2.3.2 xCELLigence Real-Time Cell Analyzer Dual-Plate (RTCA DP) system 10
2.3.3 Colony formation 10
2.4 Flow cytometry analysis 11
2.4.1 Cell cycle analysis by flow cytometry 11
2.4.2 Apoptosis analysis by flow cytometry 11
2.5 Cell migration assay 12
2.5.1 Transwell migration assay 12
2.5.2 xCELLigence RTCA DP system 13
2.6 Immunofluorescence staining 13
2.7 iTRAQ 14
2.7.1 Protein extraction 14
2.7.2 Digestion of proteins 15
2.7.3 iTRAQ labeling 16
2.7.4 LC-MS/MS analysis 16
2.7.5 Protein identification 17
2.7.6 Selection of differentially expressed proteins 18
2.8 Western blotting 20
2.9 RNA extraction and Reverse transcription 21
2.10 Real-time PCR 21
Chapter 3 Results 23
3.1 Metformin inhibits proliferation of human gastric cancer AGS cells 23
3.2 Metformin blocks cell cycle in G0/G1 phase 24
3.3 Metformin doesn’t induce AGS cells apoptosis 25
3.4 Metformin inhibits cell migration of AGS cells 25
3.5 Metformin induces reorganization of cytoskeletons in AGS cells 26
3.6 Identification of differentially expressed proteins by iTRAQ analysis 27
Chapter 4 Discussion 29
Chapter 5 Future work 34
Figures 35
Tables 54
Appendix 66
Poster 71
References 73

1.A. Jemal et al., Global cancer statistics. CA: a cancer journal for clinicians 61, 69 (Mar-Apr, 2011).
2.R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, 2013. CA: a cancer journal for clinicians 63, 11 (Jan, 2013).
3.D. Cunningham et al., Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer. The New England journal of medicine 355, 11 (Jul 6, 2006).
4.J. S. Macdonald et al., Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. The New England journal of medicine 345, 725 (Sep 6, 2001).
5.L. A. Witters, The blooming of the French lilac. Journal of Clinical Investigation 108, 1105 (2001).
6.D. G. Hardie, Metformin-acting through cyclic AMP as well as AMP? Cell metabolism 17, 313 (Mar 5, 2013).
7.S. Correia et al., Mechanisms of action of metformin in type 2 diabetes and associated complications: an overview. Mini reviews in medicinal chemistry 8, 1343 (Nov, 2008).
8.M. Stumvoll, N. Nurjhan, G. Perriello, G. Dailey, J. E. Gerich, Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. The New England journal of medicine 333, 550 (Aug 31, 1995).
9.D. Galuska, J. Zierath, A. Thorne, T. Sonnenfeld, H. Wallberg-Henriksson, Metformin increases insulin-stimulated glucose transport in insulin-resistant human skeletal muscle. Diabete & metabolisme 17, 159 (May, 1991).
10.W. W. Winder, D. G. Hardie, AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. The American journal of physiology 277, E1 (Jul, 1999).
11.S. Ali, V. Fonseca, Overview of metformin: special focus on metformin extended release. Expert opinion on pharmacotherapy 13, 1797 (Aug, 2012).
12.J. M. Moreno-Navarrete et al., OCT1 Expression in adipocytes could contribute to increased metformin action in obese subjects. Diabetes 60, 168 (Jan, 2011).
13.L. Chen et al., Role of organic cation transporter 3 (SLC22A3) and its missense variants in the pharmacologic action of metformin. Pharmacogenetics and genomics 20, 687 (Nov, 2010).
14.H. Motohashi et al., Gene expression levels and immunolocalization of organic ion transporters in the human kidney. Journal of the American Society of Nephrology : JASN 13, 866 (Apr, 2002).
15.M. Y. El-Mir, Dimethylbiguanide Inhibits Cell Respiration via an Indirect Effect Targeted on the Respiratory Chain Complex I. Journal of Biological Chemistry 275, 223 (2000).
16.J. M. Evans, L. A. Donnelly, A. M. Emslie-Smith, D. R. Alessi, A. D. Morris, Metformin and reduced risk of cancer in diabetic patients. BMJ 330, 1304 (Jun 4, 2005).
17.G. Libby et al., New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes. Diabetes care 32, 1620 (Sep, 2009).
18.D. Li, S. C. Yeung, M. M. Hassan, M. Konopleva, J. L. Abbruzzese, Antidiabetic therapies affect risk of pancreatic cancer. Gastroenterology 137, 482 (Aug, 2009).
19.Menendez, The anti-diabetic drug metformin suppresses the metastasis-associated protein CD24 in MDA-MB-468 triple-negative breast cancer cells. Oncology Reports 25, (2010).
20.K. Kato et al., The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo. Molecular cancer therapeutics 11, 549 (Mar, 2012).
21.I. Ben Sahra et al., The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene 27, 3576 (Jun 5, 2008).
22.Y. Storozhuk et al., Metformin inhibits growth and enhances radiation response of non-small cell lung cancer (NSCLC) through ATM and AMPK. British journal of cancer 108, 2021 (May 28, 2013).
23.X. Z. Zhou, Y. M. Xue, B. Zhu, J. P. Sha, [Effects of metformin on proliferation of human colon carcinoma cell line SW-480]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University 30, 1935 (Aug, 2010).
24.K. Kisfalvi, A. Moro, J. Sinnett-Smith, G. Eibl, E. Rozengurt, Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas 42, 781 (Jul, 2013).
25.A. Tomimoto et al., Metformin suppresses intestinal polyp growth in ApcMin/+ mice. Cancer science 99, 2136 (Nov, 2008).
26.B. Quinn et al., Inhibition of lung tumorigenesis by metformin is associated with decreased plasma IGF-I and diminished receptor tyrosine kinase signaling. Cancer Prev Res (Phila), (Jun 14, 2013).
27.V. N. Anisimov et al., Effect of metformin on life span and on the development of spontaneous mammary tumors in HER-2/neu transgenic mice. Experimental gerontology 40, 685 (Aug-Sep, 2005).
28.M. Zakikhani, R. Dowling, I. G. Fantus, N. Sonenberg, M. Pollak, Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer research 66, 10269 (Nov 1, 2006).
29.I. Ben Sahra et al., Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer research 71, 4366 (Jul 1, 2011).
30.M. N. Pollak, Investigating metformin for cancer prevention and treatment: the end of the beginning. Cancer discovery 2, 778 (Sep, 2012).
31.V. C. Wasinger et al., Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16, 1090 (Jul, 1995).
32.J. Cox, M. Mann, Quantitative, high-resolution proteomics for data-driven systems biology. Annual review of biochemistry 80, 273 (2011).
33.P. Alves et al., Advancement in protein inference from shotgun proteomics using peptide detectability. Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing, 409 (2007).
34.B. T. Chait, Chemistry. Mass spectrometry: bottom-up or top-down? Science 314, 65 (Oct 6, 2006).
35.P. L. Ross et al., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Molecular & cellular proteomics : MCP 3, 1154 (Dec, 2004).
36.H. R. F. a. G. E. Morris, Quantitative Proteomics Using iTRAQ labeling and mass spectrometry. (2012).
37.G. Koopman et al., Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415 (Sep 1, 1994).
38.I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of immunological methods 184, 39 (Jul 17, 1995).
39.W. T. Lin et al., Multi-Q: a fully automated tool for multiplexed protein quantitation. Journal of proteome research 5, 2328 (Sep, 2006).
40.M. Malumbres, M. Barbacid, Mammalian cyclin-dependent kinases. Trends in biochemical sciences 30, 630 (Nov, 2005).
41.M. Meyerson, E. Harlow, Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Molecular and cellular biology 14, 2077 (Mar, 1994).
42.C. J. Sherr, Mammalian G1 cyclins. Cell 73, 1059 (Jun 18, 1993).
43.F. A. Oberhammer, K. Hochegger, G. Froschl, R. Tiefenbacher, M. Pavelka, Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. The Journal of cell biology 126, 827 (Aug, 1994).
44.H. Yamaguchi, J. Wyckoff, J. Condeelis, Cell migration in tumors. Current opinion in cell biology 17, 559 (Oct, 2005).
45.D. Yamazaki, S. Kurisu, T. Takenawa, Regulation of cancer cell motility through actin reorganization. Cancer science 96, 379 (Jul, 2005).
46.T. D. Pollard, J. A. Cooper, Actin, a central player in cell shape and movement. Science 326, 1208 (Nov 27, 2009).
47.B. Wehrle-Haller, B. A. Imhof, Actin, microtubules and focal adhesion dynamics during cell migration. The international journal of biochemistry & cell biology 35, 39 (Jan, 2003).
48.Y. Matsuyama et al., Proteasomal non-catalytic subunit PSMD2 as a potential therapeutic target in association with various clinicopathologic features in lung adenocarcinomas. Molecular carcinogenesis 50, 301 (Apr, 2011).
49.C. L. Tsai et al., Secreted stress-induced phosphoprotein 1 activates the ALK2-SMAD signaling pathways and promotes cell proliferation of ovarian cancer cells. Cell reports 2, 283 (Aug 30, 2012).
50.K. Yamazaki et al., Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility. Laboratory investigation; a journal of technical methods and pathology 89, 425 (Apr, 2009).
51.P. Bertuccio et al., Recent patterns in gastric cancer: a global overview. International journal of cancer. Journal international du cancer 125, 666 (Aug 1, 2009).
52.Y. Shao, Y. Qu, S. Dang, B. Yao, M. Ji, MiR-145 inhibits oral squamous cell carcinoma (OSCC) cell growth by targeting c-Myc and Cdk6. Cancer cell international 13, 51 (2013).
53.S. Shirali et al., Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 34, 1085 (Apr, 2013).
54.Z. Xing et al., Fangchinoline Induces G1 Arrest in Breast Cancer Cells Through Cell-Cycle Regulation. Phytotherapy research : PTR, (Feb 11, 2013).
55.A. Nurnberg, T. Kitzing, R. Grosse, Nucleating actin for invasion. Nature reviews. Cancer 11, 177 (Mar, 2011).
56.J. J. Bravo-Cordero, L. Hodgson, J. Condeelis, Directed cell invasion and migration during metastasis. Current opinion in cell biology 24, 277 (Apr, 2012).
57.A. V. Hubberstey, E. P. Mottillo, Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 16, 487 (Apr, 2002).
58.K. Moriyama, I. Yahara, Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. Journal of cell science 115, 1591 (Apr 15, 2002).
59.E. Bertling et al., Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Molecular biology of the cell 15, 2324 (May, 2004).
60.O. O. Odunuga, V. M. Longshaw, G. L. Blatch, Hop: more than an Hsp70/Hsp90 adaptor protein. BioEssays : news and reviews in molecular, cellular and developmental biology 26, 1058 (Oct, 2004).
61.G. L. Blatch, Stress-inducible, Murine Protein mSTI1. CHARACTERIZATION OF BINDING DOMAINS FOR HEAT SHOCK PROTEINS AND IN VITRO PHOSPHORYLATION BY DIFFERENT KINASES. Journal of Biological Chemistry 272, 1876 (1997).
62.S. Tomida et al., Identification of a metastasis signature and the DLX4 homeobox protein as a regulator of metastasis by combined transcriptome approach. Oncogene 26, 4600 (Jul 5, 2007).
63.H. A. Hirsch, D. Iliopoulos, P. N. Tsichlis, K. Struhl, Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer research 69, 7507 (Oct 1, 2009).
64.A. Vazquez-Martin et al., Metformin regulates breast cancer stem cell Ontogeny by transcriptional regulation of the Epithelial-Mesenchymal Transition (EMT) status. Cell Cycle 9, 3807 (2010).
65.M. F. McCarty, Metformin may antagonize Lin28 and/or Lin28B activity, thereby boosting let-7 levels and antagonizing cancer progression. Medical hypotheses 78, 262 (Feb, 2012).
66.R. Rattan, R. Ali Fehmi, A. Munkarah, Metformin: an emerging new therapeutic option for targeting cancer stem cells and metastasis. Journal of oncology 2012, 928127 (2012).
67.C. Oliveras-Ferraros et al., Micro(mi)RNA expression profile of breast cancer epithelial cells treated with the anti-diabetic drug metformin: Induction of the tumor suppressor miRNA let-7a and suppression of the TGFβ-induced oncomiR miRNA-181a. Cell Cycle 10, 1144 (2011).
68.D. Iliopoulos, H. A. Hirsch, K. Struhl, An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell 139, 693 (Nov 13, 2009).
69.M. E. Peter, Let-7 and miR-200 microRNAs: guardians against pluripotency and cancer progression. Cell Cycle 8, 843 (Mar 15, 2009).
70.B. Boyerinas, S. M. Park, A. Hau, A. E. Murmann, M. E. Peter, The role of let-7 in cell differentiation and cancer. Endocrine-related cancer 17, F19 (Mar, 2010).
71.T. C. Chang et al., Lin-28B transactivation is necessary for Myc-mediated let-7 repression and proliferation. Proceedings of the National Academy of Sciences of the United States of America 106, 3384 (Mar 3, 2009).
72.S. Dangi-Garimella et al., Raf kinase inhibitory protein suppresses a metastasis signalling cascade involving LIN28 and let-7. The EMBO journal 28, 347 (Feb 18, 2009).
73.A. Salminen, J. M. Hyttinen, K. Kaarniranta, AMP-activated protein kinase inhibits NF-kappaB signaling and inflammation: impact on healthspan and lifespan. J Mol Med (Berl) 89, 667 (Jul, 2011).
74.P. M. Danzon, At what price? Nature 449, 176 (Sep 13, 2007).
75.C. R. Chong, D. J. Sullivan, Jr., New uses for old drugs. Nature 448, 645 (Aug 9, 2007).
76.J. Boonstra, J. A. Post, Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337, 1 (Aug 4, 2004).
77.Wu, Y.-H., Hu, C.-W., Chien, C.-W., Chen, Y.-J., Huang, H.-C. and Juan, H.-F., Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin. PLoS ONE (2013)(in press)