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Vol.47 No.7 contents | Japanese/English |
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- Original Article -
Validation Analysis Using Immunohistochemistry of Non-small Cell Lung Cancer-associated Proteins Detected by Two-dimensional Polyacrylamide Gel Electrophoresis
Masakazu Kojika1,2, Takashi Hirano1, Jun Matsubayashi2, Ran Guo1, Yunbo Gong1, Takeshi Kawamura3, Hiroaki Kataba1, Tatsuo Ohira1, Kiyoshi Mukai2, Harubumi Kato11First Department of Surgery, 2Department of Diagnostic Pathology, 3Clinical Proteome Center, Tokyo Medical University, Japan
Objective. We set out to detect primary lung cancer (PLC)-associated proteins using 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis and to identify the protein molecules for the exploration of biomarkers reflecting the biological characteristics of lung cancer. Study Design. We prepared samples for 2-DE from representative histological types of primary lung cancer; squamous cell carcinoma, adenocarcinoma and small cell carcinoma. We selected 8 cases of well or moderately differentiated squamous cell carcinoma confirmed postoperativly to possess typical histological features of primary lung squamous cell carcinoma. The mixture that contained equal amounts of these 8 samples was taken to be a standard sample of lung squamous cell carcinoma. Similary, we selected 8 cases of well or moderately differentiated adenocarcinoma to prepare a standard sample of primary lung adenocarcinoma. We also mixed specimens of 4 cases of small cell carcinoma, and prepared a standard sample of small cell carcinoma. We then labeled each standard sample with a fluorescent dye (either Cy2, Cy3 or Cy5) according to the manufacturer's protocol. Furthermore, using mass spectrometry we identified protein molecules that were detected in 2-DE analysis, followed by validation analysis by immunohistochemistry. Results. We detected 19 kinds of squamous cell carcinoma-associated spots (eSq), 16 kinds of adenocarcinoma-associated spots (eAd) and 17 kinds of neuroendocrine carcinoma-associated spots (eNE). From these spots, 6 kinds of eSq-protein molecules and 8 kinds of eAd-protein molecules were identified. Eight protein molecules out of these 14 identified molecules were cytokeratin (CK) molecules. CK5, CK6A, CK6C, CK6D and CK17 were identified as eSq-protein molecules, and furthermore CK8, CK18 and CK19 were identified as eAd-protein molecules. The results of a validation analysis using immunohistochemistry indicated a high possibility that CK5, CK5/6 and CK17 are biomarkers for squamous cell carcinoma, and that CK18 is a biomarker for adenocarcinoma. We succeeded in showing a significant relationship between the histopathological differentiation of PLC and the expression of CKs. Conclusion. There is a strong possibility that the biological characteristics of lung carcinoma may be clarified by classification based upon proteomic analysis, and the results of proteomic analysis may be applied to the selection of therapeutic strategies of PLC.
key words: Proteomics, Cytokeratin, 2-dimensional polyacrylamide gel electrophoresis, Squamous cell carcinoma-associated protein, Adenocarcinoma-associated protein
Received: August 27, 2007
Accepted: October 16, 2007
JJLC 47 (7): 861-869, 2007
