Abstract
To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L. This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Furthermore, one-dimensional Western blot analysis showed consistently increased CK19 expression in progressively more metastatic cells. Immunohistochemical study on 102 human HCC specimens revealed that more patients in the CK19-positive group had overt intrahepatic metastases (satellite nodules, p < 0.05; vascular tumor emboli, p < 0.001; tumor node metastatis staging, p < 0.001). CK19 fragment CYFRA 21-1 levels measured in sera from nude mice model of human HCC metastasis with radioimmunoassay increased in parallel with tumor progression and rose remarkably when pulmonary metastases occurred. The results demonstrated that overexpression of CK19 in HCC cells is related to metastatic behavior. Serum CK19 level might reflect the pathological progression in some HCC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases.
Highlights
To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry
Proteomic Analysis of MHCC97-H and MHCC97-L Cells and Cytokeratin 19 (CK19) Differential Expression—From cell culture to 2-DE, the experiments were repeated three times and silver-stained gels were analyzed by PDQuest software
Verification of CK19 Differential Expression by Western Blot and Immunofluorescene Assay—To confirm the above proteomic result, we employed two-dimensional Western blot, which has the advantage of enhanced sensitivity and the ability to detect protein isoforms assigned from post-translational modifications
Summary
OVEREXPRESSION OF CYTOKERATIN 19 CORRELATES WITH HEPATOCELLULAR CARCINOMA METASTASIS*. Among the currently available techniques, proteomics permits the analysis of thousands of modified or unmodified proteins simultaneously and becomes increasingly popular for identifying biomarkers for early detection, classification, and prognosis of tumors, as well as pinpointing targets for improved treatment outcomes [6] We had used this approach to identify differentially expressed proteins between human HCC and normal liver cell line and investigated malignant growthassociated proteins in human hepatoma cells transfected with antisense-epidermal growth factor receptor [7, 8]. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L by proteomic approach This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Serum CK19 fragment CYFRA 21-1 levels in nude mice model demonstrated that CK19 might be a useful marker for predicting the possibility of tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases
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