簡易檢索 / 詳目顯示

研究生: 謝磊
Hsieh, Lei
論文名稱: 利用iTRAQ化學標定方法進行乳癌細胞系MCF-7多重抗藥性之差異蛋白質體學分析
Differential proteomic analysis of multidrug resistance in breast cancer cell line MCF-7 by iTRAQ technology
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 99
中文關鍵詞: 差異蛋白質乳癌
英文關鍵詞: differential protein
DOI URL: http://doi.org/10.6345/NTNU202001590
論文種類: 學術論文
相關次數: 點閱:101下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 謝誌 i 中文摘要 ii Abstract iii 目錄 iv 表目錄 vii 圖目錄 viii 第一章 緒論 1 第一節 乳癌 1 第二節 乳腺癌細胞系MCF-7 1 第三節 乳癌荷爾蒙療法介紹 2 第四節 乳癌治療藥品 3 一、 Tamoxifen (泰莫西芬) 3 二、 Everolimus 4 三、 Palbociclib 6 第五節 質譜儀技術 7  高能碰撞解離(High energy collision dissociation, HCD) 8  軌道阱(Orbitrap)質量分析器 9 第六節 蛋白質身份鑑定 11 第七節 差異蛋白質體學 13 第八節 液相層析分離技術 22 一、 鹼性逆相層析法(Basic reverse phase chromatography, bRP) 23 二、 強陽離子交換層析法(Strong cationic exchange chromatography) 24 三、 液相等電點聚焦分離法(Solution isoelectric focusing) 25 第九節 研究動機 27 第二章 實驗材料與方法 29 第一節 樣品介紹 29 第二節 樣品製備 29 一、 四株抗藥性細胞株蛋白收集 30 二、 收集 medium 蛋白 31 第三節 樣品純化濃縮 31 第四節 蛋白質濃度測定 (Bradford protein assay) 32 第五節 蛋白質水解(In-solution digestion)和化學標定iTRAQ®試劑 33 第六節 第一維分離策略 35 一、 鹼性逆相層析法(Basic reverse phase chromatography, bRP) 35 二、 強陽離子交換層析法(Strong cationic exchange chromatography) 36 三、 液相等電點聚焦分離法(Solution isoelectric focusing) 38 第七節 自製碳18離心管柱 (C18 spin column) 去鹽 40 第八節 奈米級超高效能液相層析電噴灑串聯式質譜儀 (nanoLC ESI tandom mass spectrometry) 42 一、 超高效能液相層析 (Ultimate system 3000 nanoLC system, Thermo Fisher Scientific ) 42 二、 Orbitrap Fusion Lumos 軌道阱質譜儀 43 第九節 資料分析 (Data analysis) 46 第三章 結果與討論 48 第一節 蛋白質樣品濃度測定 48 第二節 iTRAQ 試劑標記 51 第三節 蛋白質身份鑑定及相對定量 52 第四節 鹼性逆向層析法分離樣品之質譜鑑定結果 53 第五節 強陽離子交換層析法分離樣品之質譜鑑定結果 57 第六節 等電聚焦分級分離儀分離樣品之質譜鑑定結果 61 第七節 比較三種不同分離方法之蛋白質及胜肽鑑定結果 64 第八節 不同樣品蛋白質相對定量 72 第九節 蛋白質相關生物作用途徑 77 第十節 相關差異蛋白質探討 84 第四章 結論與未來展望 89 第五章 文獻參考 90

    1. Hulka, B. S.; Moorman, P. G., Reprint of Breast cancer: hormones and other risk factors. Maturitas 2008, 61 (1-2), 203-213.
    2. Carter, P. J., Introduction to current and future protein therapeutics: a protein engineering perspective. Experimental cell research 2011, 317 (9), 1261-1269.
    3. Burdall, S. E.; Hanby, A. M.; Lansdown, M. R.; Speirs, V., Breast cancer cell lines: friend or foe? Breast cancer research 2003, 5 (2), 1-7.
    4. Horwitz, K.; Costlow, M.; McGuire, W., MCF-7: a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors. Steroids 1975, 26 (6), 785-795.
    5. Shirazi, F. H.; Zarghi, A.; Ashtarinezhad, A.; Kobarfard, F.; Nakhjavani, M.; Anjidani, N.; Zendehdel, R.; Arfaiee, S.; Shoeibi, S.; Mohebi, S., Remarks in successful cellular investigations for fighting breast cancer using novel synthetic compounds. INTECH Open Access Publisher Croatia: 2011.
    6. Sweeney, E. E.; McDaniel, R. E.; Maximov, P. Y.; Fan, P.; Jordan, V. C., Models and mechanisms of acquired antihormone resistance in breast cancer: significant clinical progress despite limitations. Hormone molecular biology and clinical investigation 2012, 9 (2), 143-163.
    7. Sørlie, T.; Perou, C. M.; Tibshirani, R.; Aas, T.; Geisler, S.; Johnsen, H.; Hastie, T.; Eisen, M. B.; Van De Rijn, M.; Jeffrey, S. S., Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences 2001, 98 (19), 10869-10874.
    8. Bange, J.; Zwick, E.; Ullrich, A., Molecular targets for breast cancer therapy and prevention. Nature medicine 2001, 7 (5), 548-552.
    9. Fisher, B.; Costantino, J. P.; Wickerham, D. L.; Cecchini, R. S.; Cronin, W. M.; Robidoux, A.; Bevers, T. B.; Kavanah, M. T.; Atkins, J. N.; Margolese, R. G., Tamoxifen for the prevention of breast cancer: current status of the National Surgical Adjuvant Breast and Bowel Project P-1 study. Journal of the National Cancer Institute 2005, 97 (22), 1652-1662.
    10. Lee, J. J.; Loh, K.; Yap, Y.-S., PI3K/Akt/mTOR inhibitors in breast cancer. Cancer biology & medicine 2015, 12 (4), 342.
    11. Fry, D. W.; Harvey, P. J.; Keller, P. R.; Elliott, W. L.; Meade, M.; Trachet, E.; Albassam, M.; Zheng, X.; Leopold, W. R.; Pryer, N. K., Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Molecular cancer therapeutics 2004, 3 (11), 1427-1438.
    12. Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D. J.; VanderWel, S. N.; Zhou, H.; Keller, P. R.; McNamara, D. J.; Sherry, D.; Zhu, T., Discovery of a potent and selective inhibitor of cyclin-dependent kinase 4/6. Journal of medicinal chemistry 2005, 48 (7), 2388-2406.
    13. Whittaker, S. R.; Mallinger, A.; Workman, P.; Clarke, P. A., Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacology & therapeutics 2017, 173, 83-105.
    14. Finn, R. S.; Aleshin, A.; Slamon, D. J., Targeting the cyclin-dependent kinases (CDK) 4/6 in estrogen receptor-positive breast cancers. Breast Cancer Research 2016, 18 (1), 17.
    15. Steger, G. G.; Gnant, M.; Bartsch, R., Palbociclib for the treatment of postmenopausal breast cancer–an update. Expert opinion on pharmacotherapy 2016, 17 (2), 255-263.
    16. Beaver, J. A.; Amiri-Kordestani, L.; Charlab, R.; Chen, W.; Palmby, T.; Tilley, A.; Zirkelbach, J. F.; Yu, J.; Liu, Q.; Zhao, L., FDA approval: palbociclib for the treatment of postmenopausal patients with estrogen receptor–positive, HER2-negative metastatic breast cancer. Clinical Cancer Research 2015, 21 (21), 4760-4766.
    17. Boér, K., Impact of palbociclib combinations on treatment of advanced estrogen receptor-positive/human epidermal growth factor 2-negative breast cancer. OncoTargets and therapy 2016, 9, 6119.
    18. Bouchal, P.; Roumeliotis, T.; Hrstka, R.; Nenutil, R.; Vojtesek, B.; Garbis, S. D., Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis. Journal of proteome research 2009, 8 (1), 362-373.
    19. Chen, Y.; Choong, L.-Y.; Lin, Q.; Philp, R.; Wong, C.-H.; Ang, B.-K.; Tan, Y.-L.; Hew, C.-L.; Shah, N.; Druker, B. J., Differential expression of novel tyrosine kinase substrates during breast cancer development. Molecular & Cellular Proteomics 2007, 6 (12), 2072-2087.
    20. Wolters, D. A.; Washburn, M. P.; Yates, J. R., An automated multidimensional protein identification technology for shotgun proteomics. Analytical chemistry 2001, 73 (23), 5683-5690.
    21. Gruber, K. A.; Stein, S.; Brink, L.; Radhakrishnan, A.; Udenfriend, S., Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues. Proceedings of the National Academy of Sciences 1976, 73 (4), 1314-1318.
    22. Gilar, M.; Olivova, P.; Daly, A. E.; Gebler, J. C., Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions. Journal of separation science 2005, 28 (14), 1694-1703.
    23. Yang, F.; Shen, Y.; Camp, D. G.; Smith, R. D., High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert review of proteomics 2012, 9 (2), 129-134.
    24. Stein, D. R.; Hu, X.; McCorrister, S. J.; Westmacott, G. R.; Plummer, F. A.; Ball, T. B.; Carpenter, M. S., High pH reversed‐phase chromatography as a superior fractionation scheme compared to off‐gel isoelectric focusing for complex proteome analysis. Proteomics 2013, 13 (20), 2956-2966.
    25. Spicer, V.; Ezzati, P.; Neustaeter, H.; Beavis, R. C.; Wilkins, J. A.; Krokhin, O. V., 3D HPLC-MS with reversed-phase separation functionality in all three dimensions for large-scale bottom-up proteomics and peptide retention data collection. Analytical chemistry 2016, 88 (5), 2847-2855.
    26. Washburn, M. P.; Wolters, D.; Yates, J. R., Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature biotechnology 2001, 19 (3), 242-247.
    27. Hao, P.; Qian, J.; Ren, Y.; Sze, S. K., Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus strong cation exchange (SCX) for fractionation of iTRAQ-labeled peptides. Journal of proteome research 2011, 10 (12), 5568-5574.
    28. Currie, E.; Schulze, A.; Zechner, R.; Walther, T. C.; Farese Jr, R. V., Cellular fatty acid metabolism and cancer. Cell metabolism 2013, 18 (2), 153-161.
    29. Tsaniras, S. C.; Kanellakis, N.; Symeonidou, I.; Nikolopoulou, P.; Lygerou, Z.; Taraviras, S. In Licensing of DNA replication, cancer, pluripotency and differentiation: an interlinked world?, Seminars in cell & developmental biology, Elsevier: 2014; pp 174-180.
    30. Otto, T.; Sicinski, P., Cell cycle proteins as promising targets in cancer therapy. Nature Reviews Cancer 2017, 17 (2), 93.
    31. Kuhajda, F. P., Fatty acid synthase and cancer: new application of an old pathway. Cancer research 2006, 66 (12), 5977-5980.
    32. Menendez, J. A.; Lupu, R., Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nature Reviews Cancer 2007, 7 (10), 763-777.
    33. Kuhajda, F. P.; Jenner, K.; Wood, F. D.; Hennigar, R. A.; Jacobs, L. B.; Dick, J. D.; Pasternack, G. R., Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proceedings of the National Academy of Sciences 1994, 91 (14), 6379-6383.
    34. Liu, Y., Fatty acid oxidation is a dominant bioenergetic pathway in prostate cancer. Prostate cancer and prostatic diseases 2006, 9 (3), 230-234.
    35. Wang, L.; Harris, T. E.; Roth, R. A.; Lawrence, J. C., PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding. Journal of Biological Chemistry 2007, 282 (27), 20036-20044.
    36. Glorieux, C.; Zamocky, M.; Sandoval, J. M.; Verrax, J.; Calderon, P. B., Regulation of catalase expression in healthy and cancerous cells. Free Radical Biology and Medicine 2015, 87, 84-97.
    37. Glorieux, C.; Calderon, P. B., Catalase down-regulation in cancer cells exposed to arsenic trioxide is involved in their increased sensitivity to a pro-oxidant treatment. Cancer cell international 2018, 18 (1), 1-9.
    38. Hiscott, J.; Pitha, P.; Genin, P.; Nguyen, H.; Heylbroeck, C.; Mamane, Y.; Algarte, M.; Lin, R., Triggering the interferon response: the role of IRF-3 transcription factor. Journal of interferon & cytokine research 1999, 19 (1), 1-13.
    39. Collins, S. E.; Noyce, R. S.; Mossman, K. L., Innate cellular response to virus particle entry requires IRF3 but not virus replication. Journal of virology 2004, 78 (4), 1706-1717.
    40. Hawkin, R.; Arends, M.; Ritchie, A.; Langdon, S.; Miller, W., Tamoxifen increases apoptosis but does not influence markers of proliferation in an MCF-7 xenograft model of breast cancer. The Breast 2000, 9 (2), 96-106.
    41. Lee, J.-H.; Takahashi, T.; Yasuhara, N.; Inazawa, J.; Kamada, S.; Tsujimoto, Y., Bis, a Bcl-2-binding protein that synergizes with Bcl-2 in preventing cell death. Oncogene 1999, 18 (46), 6183-6190.
    42. Rosati, A.; Graziano, V.; De Laurenzi, V.; Pascale, M.; Turco, M., BAG3: a multifaceted protein that regulates major cell pathways. Cell death & disease 2011, 2 (4), e141-e141.
    43. Pagano, M.; Pepperkok, R.; Verde, F.; Ansorge, W.; Draetta, G., Cyclin A is required at two points in the human cell cycle. The EMBO journal 1992, 11 (3), 961-971.
    44. Yam, C.; Fung, T.; Poon, R., Cyclin A in cell cycle control and cancer. Cellular and Molecular Life Sciences CMLS 2002, 59 (8), 1317-1326.
    45. Kostantin, E.; Hardy, S.; Valinsky, W. C.; Kompatscher, A.; de Baaij, J. H.; Zolotarov, Y.; Landry, M.; Uetani, N.; Martínez-Cruz, L. A.; Hoenderop, J. G., Inhibition of PRL-2· CNNM3 protein complex formation decreases breast cancer proliferation and tumor growth. Journal of Biological Chemistry 2016, 291 (20), 10716-10725.
    46. Morris, J. C.; Chiche, J.; Grellier, C.; Lopez, M.; Bornaghi, L. F.; Maresca, A.; Supuran, C. T.; Pouysségur, J.; Poulsen, S.-A., Targeting hypoxic tumor cell viability with carbohydrate-based carbonic anhydrase IX and XII inhibitors. Journal of medicinal chemistry 2011, 54 (19), 6905-6918.
    47. Chiche, J.; Ilc, K.; Laferriere, J.; Trottier, E.; Dayan, F.; Mazure, N. M.; Brahimi-Horn, M. C.; Pouysségur, J., Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH. Cancer research 2009, 69 (1), 358-368.
    48. Kuang, W. W.; Thompson, D. A.; Hoch, R. V.; Weigel, R. J., Differential screening and suppression subtractive hybridization identified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line. Nucleic acids research 1998, 26 (4), 1116-1123.
    49. Adam, P. J.; Boyd, R.; Tyson, K. L.; Fletcher, G. C.; Stamps, A.; Hudson, L.; Poyser, H. R.; Redpath, N.; Griffiths, M.; Steers, G., Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer. Journal of Biological Chemistry 2003, 278 (8), 6482-6489.
    50. Catalina-Rodriguez, O.; Kolukula, V. K.; York Tomita, A. P.; Palmieri, F.; Wellstein, A.; Byers, S.; Giaccia, A. J.; Glasgow, E.; Albanese, C.; Avantaggiati, M. L., The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis. Oncotarget 2012, 3 (10), 1220.
    51. Falasca, M.; Hughes, W. E.; Dominguez, V.; Sala, G.; Fostira, F.; Fang, M. Q.; Cazzolli, R.; Shepherd, P. R.; James, D. E.; Maffucci, T., The role of phosphoinositide 3-kinase C2α in insulin signaling. Journal of Biological Chemistry 2007, 282 (38), 28226-28236.
    52. Conte, N.; Charafe-Jauffret, E.; Delaval, B.; Adélaïde, J.; Ginestier, C.; Geneix, J.; Isnardon, D.; Jacquemier, J.; Birnbaum, D., Carcinogenesis and translational controls: TACC1 is down-regulated in human cancers and associates with mRNA regulators. Oncogene 2002, 21 (36), 5619-5630.
    53. Cully, M.; Shiu, J.; Piekorz, R. P.; Muller, W. J.; Done, S. J.; Mak, T. W., Transforming acidic coiled coil 1 promotes transformation and mammary tumorigenesis. Cancer research 2005, 65 (22), 10363-10370.
    54. Cavell, B. E.; Syed Alwi, S. S.; Donlevy, A. M.; Proud, C. G.; Packham, G., Natural product-derived antitumor compound phenethyl isothiocyanate inhibits mTORC1 activity via TSC2. Journal of natural products 2012, 75 (6), 1051-1057.
    55. Bhaskar, P. T.; Hay, N., The two TORCs and AKT. Developmental cell 2007, 12 (4), 487-502.
    56. Kawamoto, H.; Koizumi, H.; Uchikoshi, T., Expression of the G2-M checkpoint regulators cyclin B1 and cdc2 in nonmalignant and malignant human breast lesions: immunocytochemical and quantitative image analyses. The American journal of pathology 1997, 150 (1), 15.
    57. Yuan, J.; Yan, R.; Krämer, A.; Eckerdt, F.; Roller, M.; Kaufmann, M.; Strebhardt, K., Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene 2004, 23 (34), 5843-5852.
    58. Xie, X. H.; An, H. J.; Kang, S.; Hong, S.; Choi, Y. P.; Kim, Y. T.; Choi, Y. D.; Cho, N. H., Loss of Cyclin B1 followed by downregulation of Cyclin A/Cdk2, apoptosis and antiproliferation in Hela cell line. International journal of cancer 2005, 116 (4), 520-525.
    59. Rae, J. M.; Johnson, M. D.; Scheys, J. O.; Cordero, K. E.; Larios, J. M.; Lippman, M. E., GREB1 is a critical regulator of hormone dependent breast cancer growth. Breast cancer research and treatment 2005, 92 (2), 141-149.
    60. Sarafian, V.; Jadot, M.; Foidart, J. M.; Letesson, J. J.; Van den Brûle, F.; Castronovo, V.; Wattiaux, R.; Wattiaux‐De Coninck, S., Expression of Lamp‐1 and Lamp‐2 and their interactions with galectin‐3 in human tumor cells. International journal of cancer 1998, 75 (1), 105-111.
    61. Andrejewski, N.; Punnonen, E.-L.; Guhde, G.; Tanaka, Y.; Lüllmann-Rauch, R.; Hartmann, D.; Von Figura, K.; Saftig, P., Normal lysosomal morphology and function in LAMP-1-deficient mice. Journal of Biological Chemistry 1999, 274 (18), 12692-12701.
    62. Jiang, H.; Wu, J.; He, C.; Yang, W.; Li, H., Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation. Cell research 2009, 19 (4), 458-468.
    63. Jiang, H.; Luo, S.; Li, H., Cdk5 activator-binding protein C53 regulates apoptosis induced by genotoxic stress via modulating the G2/M DNA damage checkpoint. Journal of Biological Chemistry 2005, 280 (21), 20651-20659.
    64. Wang, J.; He, X.; Luo, Y.; Yarbrough, W. G., A novel ARF-binding protein (LZAP) alters ARF regulation of HDM2. Biochemical Journal 2006, 393 (2), 489-501.
    65. Bruening, W.; Giasson, B. I.; Klein‐Szanto, A. J.; Lee, V. M. Y.; Trojanowski, J. Q.; Godwin, A. K., Synucleins are expressed in the majority of breast and ovarian carcinomas and in preneoplastic lesions of the ovary. Cancer: Interdisciplinary International Journal of the American Cancer Society 2000, 88 (9), 2154-2163.
    66. Jia, T.; Liu, Y. E.; Liu, J.; Shi, Y. E., Stimulation of breast cancer invasion and metastasis by synuclein γ. Cancer research 1999, 59 (3), 742-747.
    67. Kong, Y.; Kejun, N.; Yin, Y., Identification and characterization of CAC1 as a novel CDK2-associated cullin. Cell Cycle 2009, 8 (21), 3552-3561.
    68. Choi, H.; Lee, S. H.; Um, S.-J.; Kim, E.-J., CACUL1 functions as a negative regulator of androgen receptor in prostate cancer cells. Cancer Letters 2016, 376 (2), 360-366.
    69. Westbrook, L.; Manuvakhova, M.; Kern, F. G.; Estes, N. R.; Ramanathan, H. N.; Thottassery, J. V., Cks1 regulates cdk1 expression: a novel role during mitotic entry in breast cancer cells. Cancer Research 2007, 67 (23), 11393-11401.
    70. Yang, R.; Müller, C.; Huynh, V.; Fung, Y. K.; Yee, A. S.; Koeffler, H. P., Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins. Molecular and cellular biology 1999, 19 (3), 2400-2407.

    無法下載圖示 電子全文延後公開
    2025/09/10
    QR CODE