Abstract
BackgroundFibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved.MethodsThe MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively.ResultsBoth of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity.ConclusionsPolypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.
Highlights
Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains
The study of FN expression on cancer cells has determined that decreased FN expression is closely associated with cancer growth and metastasis [8,9,10], which illustrates that an increase of FN expression in cancer cells may facilitate the reduction of cancer cell metastasis, implying that FN may have the potential for a significant clinical application
Purification and Heparin Affinity of rhFNHN29 and rhFNHC36 Polypeptides The N-terminal heparin-binding domain polypeptide of FN consists of five I homologous structures (Figure 1A), containing 237 amino acids (Ser46-Gly282)
Summary
Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. Obstacles exist for the clinical application of FN, including deficiency of blood plasma, danger of blood-infection disease, and difficulty in engineering the synthesis of the whole-molecule FN with a molecular weight as large as 420 kDa. FN contains several active sites that serve as scaffoldings for cell anchorage [11], known as the heparinbinding domains, collagen-binding domain, fibrin-binding domain and cell-binding domain, respectively. FN contains several active sites that serve as scaffoldings for cell anchorage [11], known as the heparinbinding domains, collagen-binding domain, fibrin-binding domain and cell-binding domain, respectively These domains are involved in a diverse array of cell functions including adhesion, migration, differentiation, apoptosis, morphous change, haemostasis and reparation of damage, etc. The CH50 polypeptide that contains Cell I and Hep II dual domain fragment of FN has been shown to play a role involving inhibition of tumor growth, invasion and angiogenesis [13]. The FNIII14 peptide effectively inhibited the adhesion and metastasis of lymphoma cells [14]
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