Expression And Effects Of Connective Tissue Growth Factor On Human Primary Hepatocellular Carcinoma

Objective: The aims of this study were to determine the expression characteristics of connective tissue growth factor (CTGF) in human hepatocellular carcinoma (HCC) in histology and to elucidate the roles of CTGF on HCC development in vitro.Background: CTGF is a cysteine-rich matricellular protein that has been implicated in regulating diverse processes in vivo, including angiogenesis, embryogenesis, chondrogenesis, fibrogenesis, and tumorigenesis. A recent study in xenograft animal model has described that CTGF acts as a downstream mediator for TGF-β1-induced tumor growth in HCC. However, the role of CTGF in tumorigenesis of human HCC remains elusive.Methods: Liver samples from 36 patients (who underwent hepatic resection for the first HCC between 2006 and 2009) and 6 normal individuals were examined for TGF-β1 and CTGF mRNA by in situ hybridization. Computer imaging analysis was performed to measure integrated optimal density (IOD) of CCN2 mRNA-positive cells in carcinoma foci and surrounding stroma respectively. CTGF was assessed for its stimulation of HepG2 cell adhesion, migration, invasion by using commercial kits; and cell cycle analysis by flow cytometry.Results: In situ hybridization analysis showed that a significance increase of TGF-β1 mRNA was mainly detected in connective tissue and vesculatures around carcinoma foci, with a concomitant expression of TGF-β1 and CTGF mRNA in those areas and that 9.4-fold enhanced expression of CTGF mRNA in the surrounding stroma (IOD: 60.2±28.7) and only 1.9-fold expression of CTGF mRNA in carcinoma foci (IOD: 12.3±6.0) in comparison with normal controls (IOD: 6.4±2.3) (P<0.01). The expression of CCN2 mRNA detected in the surrounding stroma was 4.8-fold higher than that in carcinoma foci. Adhesion of HepG2 cells to CTGF was seen in a dose-dependent manner with an optimal concentration of 2.0μg/ml. Incubation of HepG2 cells with CTGF at concentration of 100 ng /ml induced a significant migratory (4.0-fold) and invasion (5.7-fold) effect. Furthermore, CTGF acted as a downstream mediater for TGF-β1-induced HepG2 cell invasion because the TGF-β1-induced cell invasion was abrogated in the presence of neutralizing CTGF antibody. Cell cycle analysis showed that addition of 100 ng ml CTGF to HepG2 cell culture medium resulted in more cells transitioning into S phase.Conclusion: CTGF is overexpressed in HCC, especially in surrounding stroma, and it also significantly induces human HCC cell adhesion, migration, invasion and cell cycle progress. These data support a role for CTGF in the development of HCC and highlight CTGF as potential novel therapeutic targets.