乳癌是目前世界上常見的癌症之ㄧ，也是婦女因癌症死亡的主要種類。當前有需要發展出能作為偵測早期乳癌的有潛力生物標記分子。本實驗利用蛋白質體技術2D-DIGE輔以MALDI-TOF檢測，探討人類正常乳腺細胞MCF-10A與人類乳癌細胞MCF-7、MDA-MB-231在受到無血清環境刺激下蛋白質體表現的差異，並藉此尋找可能作為生物標記的分子。首先我們將人類正常乳腺細胞MCF-10A與人類乳癌細胞MCF-7、MDA-MB-231培養在無血清的環境中，經過30小時後收集培養液，從中純化出蛋白質。後續利用2D-DIGE的方法，我們調查了三種細胞在條件血清的處理下，各自蛋白質表現。透過軟體Decyder與comassie blue染色法的協助，得以挑選出表現量差距達1.5倍以上的蛋白質，結果約175個具有表現差異的蛋白質點被選出。採用質譜儀MALDI-TOF鑑定這些蛋白質，最終確立了90的蛋白質點。另外，還利用了西方點墨法再度確認這些結果，最後證實該蛋白質體技術的可信度。而在這些90個蛋白質中我們有興趣於那些癌細胞生長、演進、細胞存活等相關蛋白質，並挑選cathepsin D 和flavin reductase 做生物功能性的測試。使用這兩種蛋白質抑制物處理細胞，以MTT assay觀察細胞存活率。結果發現在低濃度抑制物時三株細胞存活率有些許差異，但在高濃度時皆造成細胞死亡。顯示這兩種抑制物或其作用的蛋白質不適合直接做為治療乳癌的目標。

Breast cancer is one of the most common cancers in the world, and it is the leading cancer-related death in women. There is a need in discovery of prospective biomarkers in early detection for breast cancer. In this experiment, we applied proteomic technology 2D-DIGE coupled with MALDI-TOF mass spectrometry to investigate the proteome profile of MCF-10A human normal mammary cell and MCF-7、MDA-MB-231 human breast cancer cells under serum-free medium treatment, and hopefully screen out any potent biomarkers. Firstly, we incubated MCF-10A human mammary cell and MCF-7、MDA-MB-231 breast cancer cells in serum-free medium for 30 hours, and then collected the medium which is the source where proteins are purified from. Secondly, we utilized 2D-DIGE technique to investigate the protein expression profiles of each three cell lines under conditioned medium treatment. With the aid of the software Decyder and comassie blue stain, we are able to screen and pick out 175 protein spots that were differentially expressed above 1.5 fold. Thirdly, 90 proteins from these differentially expressed proteins are identified by MALDI-TOF mass spectrometry. Moreover, Western blot validation results proved this strategy as a reliable tool for searching biomarker candidates. Last but not the least, we are interested in those proteins that are related to tumor physiology such as tumor cell growth、tumor progression、cell survival and choose cathepsin D and flavin reductase for further biofunctional assay. We treated MCF-10A、MCF-7 and MDA-MB-231 with the protein inhibitors of cathepsin D and flavin reductase ,respectively. Observing by MTT assay, we found that there is a slightly different survival rate between the cells at low concentration of protein inhibitors, but death to all cells at a certain higher concentration, suggesting that both protein inhibitors and cathepsin D and flavin reductase may not be suitable as a direct therapeutic target for breast cancer.

參考文獻

Aitken,A. (1996). 14-3-3 and its possible role in co-ordinating multiple signalling pathways. Trends Cell Biol. 6, 341-347.
Balakirev,M.Y., Tcherniuk,S.O., Jaquinod,M., and Chroboczek,J. (2003). Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517-522.
Benes,P., Vetvicka,V., and Fusek,M. (2008). Cathepsin D--many functions of one aspartic protease. Crit Rev. Oncol. Hematol. 68, 12-28.
Cabezon,T., Celis,J.E., Skibshoj,I., Klingelhofer,J., Grigorian,M., Gromov,P., Rank,F., Myklebust,J.H., Maelandsmo,G.M., Lukanidin,E., and Ambartsumian,N. (2007). Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer. Int. J. Cancer 121, 1433-1444.
Celis,J.E., Gromov,P., Cabezon,T., Moreira,J.M., Ambartsumian,N., Sandelin,K., Rank,F., and Gromova,I. (2004). Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery. Mol. Cell Proteomics. 3, 327-344.
Doerks,T., Strauss,M., Brendel,M., and Bork,P. (2000). GRAM, a novel domain in glucosyltransferases, myotubularins and other putative membrane-associated proteins. Trends Biochem. Sci. 25, 483-485.
Donato,R. (2003). Intracellular and extracellular roles of S100 proteins. Microsc. Res. Tech. 60, 540-551.
Dupont,A., Tokarski,C., Dekeyzer,O., Guihot,A.L., Amouyel,P., Rolando,C., and Pinet,F. (2004). Two-dimensional maps and databases of the human macrophage proteome and secretome. Proteomics. 4, 1761-1778.
Fiedler,T.A., Karpova,T.S., Fleig,U., Young,M.E., Cooper,J.A., and Hegemann,J.H. (2002). The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton. Mol. Genet. Genomics 268, 190-205.
Inoue,Y., Kawamoto,S., Shoji,M., Hashizume,S., Teruya,K., Katakura,Y., and Shirahata,S. (2000). Properties of ras-amplified recombinant BHK-21 cells in protein-free culture. Cytotechnology 33, 21-26.
Jenkinson,S.R., Barraclough,R., West,C.R., and Rudland,P.S. (2004). S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis. Br. J. Cancer 90, 253-262.
Jin,Z.G., Melaragno,M.G., Liao,D.F., Yan,C., Haendeler,J., Suh,Y.A., Lambeth,J.D., and Berk,B.C. (2000). Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ. Res. 87, 789-796.
Kylaniemi,M.K., Haveri,A., Vuola,J.M., Puolakkainen,M., and Lahesmaa,R. (2009). Gene expression signatures characterizing the development of lymphocyte response during experimental Chlamydia pneumoniae infection. Microb. Pathog. 46, 235-242.
Liotta,L.A. and Kohn,E.C. (2001). The microenvironment of the tumour-host interface. Nature 411, 375-379.
Muramatsu,T. and Miyauchi,T. (2003). Basigin (CD147): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion. Histol. Histopathol. 18, 981-987.
Pupa,S.M., Menard,S., Forti,S., and Tagliabue,E. (2002). New insights into the role of extracellular matrix during tumor onset and progression. J. Cell Physiol 192, 259-267.
Sarasin,A. (2003). An overview of the mechanisms of mutagenesis and carcinogenesis. Mutat. Res. 544, 99-106.
Spinazzi,R., Albertin,G., Nico,B., Guidolin,D., Di Liddo,R., Rossi,G.P., Ribatti,D., and Nussdorfer,G.G. (2006). Urotensin-II and its receptor (UT-R) are expressed in rat brain endothelial cells, and urotensin-II via UT-R stimulates angiogenesis in vivo and in vitro. Int. J. Mol. Med. 18, 1107-1112.
Spitzner,M., Martins,J.R., Soria,R.B., Ousingsawat,J., Scheidt,K., Schreiber,R., and Kunzelmann,K. (2008b). Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells. J. Biol. Chem. 283, 7421-7428.
Spitzner,M., Martins,J.R., Soria,R.B., Ousingsawat,J., Scheidt,K., Schreiber,R., and Kunzelmann,K. (2008a). Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells. J. Biol. Chem. 283, 7421-7428.
Takahashi,K., Totsune,K., Murakami,O., Arihara,Z., Noshiro,T., Hayashi,Y., and Shibahara,S. (2003). Expression of urotensin II and its receptor in adrenal tumors and stimulation of proliferation of cultured tumor cells by urotensin II. Peptides 24, 301-306.
Thiery,J.P. and Sleeman,J.P. (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131-142.
Vercoutter-Edouart,A.S., Lemoine,J., Le,B., X, Louis,H., Boilly,B., Nurcombe,V., Revillion,F., Peyrat,J.P., and Hondermarck,H. (2001). Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells. Cancer Res. 61, 76-80.
Wiseman,B.S. and Werb,Z. (2002). Stromal effects on mammary gland development and breast cancer. Science 296, 1046-1049.
Xiao,S., Li,D., Zhu,H.Q., Song,M.G., Pan,X.R., Jia,P.M., Peng,L.L., Dou,A.X., Chen,G.Q., Chen,S.J., Chen,Z., and Tong,J.H. (2006). RIG-G as a key mediator of the antiproliferative activity of interferon-related pathways through enhancing p21 and p27 proteins. Proc. Natl. Acad. Sci. U. S. A 103, 16448-16453.
Xue,H., Lu,B., and Lai,M. (2008). The cancer secretome: a reservoir of biomarkers. J. Transl. Med. 6, 52.
Zhu,D., Cardenas,M.E., and Heitman,J. (1996). Calcineurin mutants render T lymphocytes resistant to cyclosporin A. Mol. Pharmacol. 50, 506-511.
Zwickl,H., Traxler,E., Staettner,S., Parzefall,W., Grasl-Kraupp,B., Karner,J., Schulte-Hermann,R., and Gerner,C. (2005). A novel technique to specifically analyze the secretome of cells and tissues. Electrophoresis 26, 2779-2785.