Surgery Today

, Volume 36, Issue 12, pp 1085–1093

Protein Pattern Difference in the Colon Cancer Cell Lines Examined by Two-Dimensional Differential In-Gel Electrophoresis and Mass Spectrometry

  • Masafumi Katayama
  • Hiroshi Nakano
  • Atsuko Ishiuchi
  • Wenwen Wu
  • Ryuichi Oshima
  • Joe Sakurai
  • Hiroyuki Nishikawa
  • Susumu Yamaguchi
  • Takehito Otsubo



The pivotal metastatic processes of colorectal cancer (CRC) have yet to be fully investigated by a comprehensive all-inclusive protein analysis. We used two-dimensional differential in-gel electrophoresis (2D-DIGE) and liquid chromatography–tandem mass spectrometry (LC/MS/MS) to investigate the protein pattern changes during the metastasis of CRC. Two CRC cell lines were investigated: SW480 derived from the primary lesion and SW620 derived from lymph node metastasis in the same patient.


The two cell lines were compared using 2D-DIGE with a maleimide CyDye fluorescent protein labeling technique, which has an enhanced sensitivity for many proteins at a low concentration. A comprehensive proteomics analysis was performed by the dual-labeling method using Cy3 and Cy5 and by LC/MS/MS. In addition, an in vivo experiment of metastasis using nude mice was performed by the injection of the two cell lines into the spleen.


Among approximately 1 500 proteins, we detected 9 protein spots with definitively significant changes between the two cell lines. Three out of the nine proteins were validated by a Western blot analysis. Alpha-enolase and triosephosphate isomerase were significantly upregulated in SW620 in comparison to SW480. Annexin A2 (annexin II) was significantly downregulated in SW620 compared to SW480. Neither liver metastasis nor peritoneal dissemination was established in the metastatic experiment using SW480 but some liver and peritoneal metastases occurred in the experiment using SW620. An in vivo metastatic experiment using SW620 showed the expressions of alpha-enolase and triosephosphate isomerase to increase in the liver metastases in comparison to those in the splenic implanted lesion. The expressions of triosephosphate isomerase increased in the peritoneal lesions in comparison to those in the splenic implanted lesion.


2D-DIGE and LC/MS/MS techniques identified nine proteins that increased significantly more in SW620 than in SW480. The finding of our in vivo metastatic experiment suggests that alpha-enolase and triosephosphate isomerase, at least in part, may be associated with the metastatic process of these two cell lines.

Key words

Proteomics Colon cancer cell lines Alpha-enolase Triosephosphate isomerase 


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  1. 1.
    Greenlee, RT, Murray, T, Bolden, S, Wingo, PS 2000Cancer Statistics 2000CA Cancer J Clin50733PubMedGoogle Scholar
  2. 2.
    Ballantyne, GH, Quin, J 1993Surgical treatment of liver metastases in patients with colorectal cancerCancer71425266PubMedCrossRefGoogle Scholar
  3. 3.
    Weiss, L 2000Metastasis of cancer: a conceptual history from antiquity to the 1990sCancer Metastasis Rev19193383CrossRefGoogle Scholar
  4. 4.
    Fidler, IJ 2001Seed and soil revisited: contribution of the organ microenvironment to cancer metastasisSurg Oncol Clin North Am1025769Google Scholar
  5. 5.
    Ridley, A 2000Molecular switches in metastasisNature4064667PubMedCrossRefGoogle Scholar
  6. 6.
    Moustafa, AS, Nicolson, GL 1997Breast cancer metastasis-associated genes: prognostic significance and therapeutic implicationsOncol Res950525PubMedGoogle Scholar
  7. 7.
    Debies, MT, Welch, DR 2001Genetic basis of human breast cancer metastasisJ Mammary Gland Biol Neoplasia644151PubMedCrossRefGoogle Scholar
  8. 8.
    Nicolson, GL, Nawa, A, Moustafa, A, Taniguchi, S, Nishimori, K, Moustafa, A 2003Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulationClin Exp Metastasis201926PubMedCrossRefGoogle Scholar
  9. 9.
    Pandey, A, Mann, M 2000Proteomics to study genes and genomesNature40583746PubMedCrossRefGoogle Scholar
  10. 10.
    Alaiya, AA, Franzen, B, Auer, G, Linder, S 2000Cancer proteomics: from identification of novel markers to creation of artifical learning models for tumor classificationElectrophoresis2112107PubMedCrossRefGoogle Scholar
  11. 11.
    Dutt, MJ, Lee, KH 2000Proteomic analysisCurr Opin Biotechnol111769PubMedCrossRefGoogle Scholar
  12. 12.
    Lahm, HW, Langen, H 2000Mass spectrometry: A tool for the identification of proteins separated by gelsElectrophoresis2112107CrossRefGoogle Scholar
  13. 13.
    Wu, W, Tang, X, Hu, W, Lotan, R, Hong, WK, Mao, L 2002Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometryClin Exp Metastasis1931926PubMedCrossRefGoogle Scholar
  14. 14.
    Hanash, SM 2000Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current statusElectrophoresis2112029PubMedCrossRefGoogle Scholar
  15. 15.
    Gorg, A, Obermaier, C, Boguth, G, Harder, A, Scheibe, B, Wildgruber, R,  et al. 2000The current state of two-dimensional electrophoresis with immobilized pH gradientsElectrophoresis21103753PubMedCrossRefGoogle Scholar
  16. 16.
    Zhou, G, Li, H, DeCamp, D, Chen, S, Shu, H, Gong, Y,  et al. 20022D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markersMol Cell Proteomics111724PubMedCrossRefGoogle Scholar
  17. 17.
    Unlu, M, Morgan, ME, Minden, JS 1997Difference gel electrophoresis: a single gel method for detecting changes in protein extractsElectrophoresis1820717PubMedCrossRefGoogle Scholar
  18. 18.
    Tonge, R, Shaw, J, Middleton, B, Rowlinson, R, Rayner, S, Young, J,  et al. 2001Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technologyProteomics137796PubMedCrossRefGoogle Scholar
  19. 19.
    Leibovitz, A, Stinson, JC, McCombs, WB,3rd, McCoy, CE, Mazur, KC, Mabry, ND 1976Classification of human colorectal adenocarcinoma cell linesCancer Res3645629PubMedGoogle Scholar
  20. 20.
    Nishikawa, H, Ooka, S, Sato, K, Arima, K, Okamoto, J, Klevit, RE,  et al. 2004Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligaseJ Biol Chem279391624PubMedCrossRefGoogle Scholar
  21. 21.
    Van den Bergh, G, Arckens, L 2004Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomicsCurr Opin Biotechnol153843PubMedCrossRefGoogle Scholar
  22. 22.
    Semenza, GL, Jiang, BH, Leung, SW, Passantino, R, Concordet, JP, Maire, P,  et al. 1996Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1J Biol Chem2713252937PubMedCrossRefGoogle Scholar
  23. 23.
    Minet, E, Michel, G, Remacle, J, Michiels, C 2000Role of HIF-1 as a transcription factor involved in embryonic development, cancer progression and apoptosisInt J Mol Med52539PubMedGoogle Scholar
  24. 24.
    Montgomerie, JZ, Gracy, RW, Holshuh, HJ, Keyser, AJ, Bennett, CJ, Schick, DG 1997The 28K protein in urinary bladder, squamous metaplasia and urine is triosephosphate isomeraseClin Biochem306138PubMedCrossRefGoogle Scholar
  25. 25.
    Chen, G, Gharib, TG, Huang, CC, Thomas, DG, Shedden, KA, Taylor, JM,  et al. 2002Proteomic analysis of lung adenocarcinoma: identification of a highly expressed set of proteins in tumorsClin Cancer Res82298305PubMedGoogle Scholar
  26. 26.
    Mai, J, Waisman, DM, Sloane, BF 2000Cell surface complex of cathepsin B/annexin II tetramer in malignant progressionBiochim Biophys Acta147721530PubMedGoogle Scholar
  27. 27.
    Hajjar, KA, Acharya, SS 2000Annexin II and regulation of cell surface fibrinolysisAnn NY Acad Sci90226571PubMedCrossRefGoogle Scholar
  28. 28.
    Siever, DA, Erickson, HP 1997Extracellular annexin IIInt J Biochem Cell Biol29121923PubMedCrossRefGoogle Scholar
  29. 29.
    Mai, J, Finley, RL,Jr, Waisman, DM, Sloane, BF 2000Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cellsJ Biol Chem2751280612PubMedCrossRefGoogle Scholar
  30. 30.
    Yu, B, Li, SY, An, P, Zhang, YN, Liang, ZJ, Yuan, SJ,  et al. 2004Comparative study of proteome between primary cancer and hepatic metastatic tumor in colorectal cancerWorld J Gastroenterol1026526PubMedGoogle Scholar
  31. 31.
    Clauser, KR, Hall, SC, Smith, DM, Webb, JW, Andrews, LE, Tran, HM,  et al. 1995Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGEProc Natl Acad Sci USA2350726CrossRefGoogle Scholar
  32. 32.
    Lin, YH, Park, ZY, Lin, D, Brahmbhatt, AA, Rio, MC, Yates, JR,III,  et al. 2004Regulation of cell migration and survival by focal adhesion targeting of Lasp-1J Cell Biol16542132PubMedCrossRefGoogle Scholar
  33. 33.
    Herlevsen, M, Schmidt, DS, Miyazaki, K, Zöller, M 2003The association of the tetraspanin D6.1A with the α6β4 integrin supports cell motility and liver metastasis formationJ Cell Sci116437390PubMedCrossRefGoogle Scholar
  34. 34.
    Ying-Tao, Z, Yi-Ping, G, Lu-Sheng, S, Yi-Li, W 2005Proteomic analysis of differentially expressed proteins between metastatic and non-metastatic human colorectal carcinoma cell linesEur J Gastroenterol Hepatol1772532PubMedCrossRefGoogle Scholar
  35. 35.
    Brunagel, G, Schoen, RE, Bauer, AJ, Vietmeier, BN, Getzenberg, RH 2002Nuclear matrix protein alterations associated with colon cancer metastasis to the liverClin Cancer Res8303945PubMedGoogle Scholar
  36. 36.
    Friedman, DB, Hill, S, Keller, JW, Merchant, NB, Levy, SE, Coffey, RJ,  et al. 2004Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometryProteomics4793811PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Tokyo 2006

Authors and Affiliations

  • Masafumi Katayama
    • 1
  • Hiroshi Nakano
    • 1
  • Atsuko Ishiuchi
    • 1
  • Wenwen Wu
    • 2
  • Ryuichi Oshima
    • 1
  • Joe Sakurai
    • 1
  • Hiroyuki Nishikawa
    • 2
  • Susumu Yamaguchi
    • 1
  • Takehito Otsubo
    • 1
  1. 1.Division of Gastroenterological SurgerySt. Marianna University HospitalKawasakiJapan
  2. 2.Division of Breast and Endocrine SurgerySt. Marianna University HospitalKawasakiJapan

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