Cancer Res Treat.  2006 Apr;38(2):92-98.

Overexpression of alpha-protease Inhibitor and Galectin-1 in Radiation-induced Early Phase of Pulmonary Fibrosis

• Im HS
• Kim HD
• Song JY
• Han Y
• Lee DY
• Kim CW
• Yun YS

Abstract

PURPOSE: Radiation-induced pulmonary fibrosis (RIF) is a significant complication of radiotherapy for lung cancer. Despite the large number of studies, the molecular mechanisms of RIF are poorly understood. Therefore, the complex protein expression pattern in RIF was characterized by identifying the proteins with an altered expression level after thorax irradiation using two-dimensional electrophoresis (2-DE) and mass spectrometry.
MATERIALS AND METHODS
A mouse model of RIF was used to examine the alteration of the lung proteome because of availability of murine data related to human cases and the abundance of murine fibrotic lung samples. A mouse model of RIF was induced in radiosensitive C57BL/6 mice. Twenty-one weeks after 25 Gy irradiation, hematoxylin-eosin staining and hydroxyproline assay confirmed the early-phase pulmonary fibrosis.
RESULTS
Lung samples from the irradiated and age-matched control mice were used to generate 16 high quality 2-DE gels containing approximately 1,000 spots. Of the 31 significantly up- or down-regulated protein spots, 17 were identified by MALDI-TOF/MS.
CONCLUSIONS
Two important upregulated proteins were found, the alpha-protease inhibitor and galectin-1, which might be used as potential markers for the early phase of RIF.

Keyword

Radiation-induced pulmonary fibrosis; proteomics; alpha-protease inhibitor; Galectin-1

MeSH Terms

Animals
Electrophoresis
Galectin 1*
Gels
Humans
Hydroxyproline
Lung
Lung Neoplasms
Mass Spectrometry
Mice
Proteome
Proteomics
Pulmonary Fibrosis*
Radiotherapy
Thorax
Galectin 1
Gels
Hydroxyproline
Proteome

Figure

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  Fig. 1 Experimental scheme.

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  Fig. 2 Confirmation of pulmonary fibrosis using H/E staining and the hydroxyproline assay. The mice were local thorax irradiated at a dose of 25 Gy (0.5 Gy/min). Twenty-one weeks later, severe collagen deposition and the initial phase of fibrosis developed. (A) Alveolar congestion, alveolar cell infiltration, and collagen lay-down can be seen in the 25 Gy irradiation slide (original magnification×40). (B) The increase in the level of collagen can be confirmed by the data (means±SD) representing two comparable experiments with four mice per group.

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  Fig. 3 2-DE of mouse lung. (A) representative proteome image of RIF. (B) normal lung tissue. (C) radiation-induced pulmonary fibrosis lung tissue. The proteins from the whole lung were extracted and separated on pH 3 to 10 linear immobilized pH-gradient strips, followed by a 10~16% sodium dodecyl sulfate/polyacrylamide gel. The gels were stained by silver staining. A total of 70 spots are tagged by numbers. (A) indicates the total up-regulated or down-regulated protein. The upper arrows indicate the protein spots corresponding to the α1-protease inhibitor, and the lower arrows indicate the protein spots corresponding to galectin-1.

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  Fig. 4 2-DE of murine α1-protease inhibitor. (A) Quantitation of the serine protease inhibitor in the normal lung tissues and RIF tissues. The protein spots that appeared in the 2DE gels were quantified using PDQuest computer software. The average intensities (n=8) of the spots corresponding to the α1-protease inhibitor in 2DE gels are shown. *p<0.005 compared to control. (B) 2DE gel images of α1-protease inhibitor. The arrow indicates the protein spot corresponding to the α1-protease inhibitor. (C) MALDI-TOF MS peptide mass spectrum of the tryptic digestion of the α1-protease inhibitor.

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  Fig. 5 2-DE of murine galectin-1. (A) Quantitation of galectin-1 in the normal lung tissues and RIF tissues. The protein spots that appeared in the 2DE gels were quantified using PDQuest computer software. The average intensities (n=8) of the spots corresponding to galectin-1 in 2DE gels are shown. *p<0.005 compared to control. (B) 2DE gel images of galectin-1. The arrow indicates the protein spot corresponding to galectin-1. (C) MALDI-TOF MS peptide mass spectrum of the tryptic digestion of the gallectin-1.

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