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N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells.

Tang NH, Chen YL, Wang XQ, Li XJ, Wu Y, Zou QL, Chen YZ - BMC Cancer (2010)

Bottom Line: The 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fujian Institute of Hepatobiliary Surgery, Union Hospital, Fujian Medical University, Fuzhou, China.

ABSTRACT

Background: 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. 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.

Methods: The 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.

Results: Both 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.

Conclusions: Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

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Related in: MedlinePlus

Heparin affinity analysis of rhFNHN29 and rhFNHC36 polypeptides. (A), The chromatography showed the peak fractions (Arrow) of eluted rhFNHN29 and rhFNHC36 polypeptides on a column (1 ml) of HiTrap Heparin HP at a flow rate of 1.5 ml/min. (B), LC/MS analysis indicated the elution position of molecular weight for purified rhFNHN29 (27.897 kDa) and rhFNHC36 polypeptides (31.051 kDa).
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Figure 2: Heparin affinity analysis of rhFNHN29 and rhFNHC36 polypeptides. (A), The chromatography showed the peak fractions (Arrow) of eluted rhFNHN29 and rhFNHC36 polypeptides on a column (1 ml) of HiTrap Heparin HP at a flow rate of 1.5 ml/min. (B), LC/MS analysis indicated the elution position of molecular weight for purified rhFNHN29 (27.897 kDa) and rhFNHC36 polypeptides (31.051 kDa).

Mentions: Upon obtaining yeast expression vectors pPIC9K-FNHN29 and pPIC9K-FNHC36, we successfully established yeast strains GS115-FNHN29 and KM71-FNHC36 that exhibited high expression. The purity of rhFNHN29 and rhFNHC36 polypeptides, which were purified from fermentation supernatant through centrifugalization, ultrafiltration, ion exchange chromatography, and sieve chromatography (Figure 1B), was over 95%. Western blot confirmed the detection of rhFNHN29 and rhFNHC36 with FN polyclonal antibody (Figure 1C). Heparin affinity chromatography indicated that rhFNHN29 and rhFNHC36 display the activity of binding heparin (Figure 2A). Mass spectrometry analysis provided evidence that the molecular weights were 27.9 kDa and 31.0 kDa, respectively (Figure 2B).


N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells.

Tang NH, Chen YL, Wang XQ, Li XJ, Wu Y, Zou QL, Chen YZ - BMC Cancer (2010)

Heparin affinity analysis of rhFNHN29 and rhFNHC36 polypeptides. (A), The chromatography showed the peak fractions (Arrow) of eluted rhFNHN29 and rhFNHC36 polypeptides on a column (1 ml) of HiTrap Heparin HP at a flow rate of 1.5 ml/min. (B), LC/MS analysis indicated the elution position of molecular weight for purified rhFNHN29 (27.897 kDa) and rhFNHC36 polypeptides (31.051 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965728&req=5

Figure 2: Heparin affinity analysis of rhFNHN29 and rhFNHC36 polypeptides. (A), The chromatography showed the peak fractions (Arrow) of eluted rhFNHN29 and rhFNHC36 polypeptides on a column (1 ml) of HiTrap Heparin HP at a flow rate of 1.5 ml/min. (B), LC/MS analysis indicated the elution position of molecular weight for purified rhFNHN29 (27.897 kDa) and rhFNHC36 polypeptides (31.051 kDa).
Mentions: Upon obtaining yeast expression vectors pPIC9K-FNHN29 and pPIC9K-FNHC36, we successfully established yeast strains GS115-FNHN29 and KM71-FNHC36 that exhibited high expression. The purity of rhFNHN29 and rhFNHC36 polypeptides, which were purified from fermentation supernatant through centrifugalization, ultrafiltration, ion exchange chromatography, and sieve chromatography (Figure 1B), was over 95%. Western blot confirmed the detection of rhFNHN29 and rhFNHC36 with FN polyclonal antibody (Figure 1C). Heparin affinity chromatography indicated that rhFNHN29 and rhFNHC36 display the activity of binding heparin (Figure 2A). Mass spectrometry analysis provided evidence that the molecular weights were 27.9 kDa and 31.0 kDa, respectively (Figure 2B).

Bottom Line: The 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fujian Institute of Hepatobiliary Surgery, Union Hospital, Fujian Medical University, Fuzhou, China.

ABSTRACT

Background: 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. 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.

Methods: The 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.

Results: Both 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.

Conclusions: Polypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Show MeSH
Related in: MedlinePlus