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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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Expression of purified rhFNHN29 and rhFNHC36 polypeptides. (A), Schematic structure of FN. FN is made up of a series of repeating homology units of three different types (FNI, FNII and FNIII). Yellow rectangles represent FNI modules, blue rhombus represent FNII modules and red ovals represent specific FNIII modules. RhFNHN29 containing N-terminal heparin-binding domain (HBD) is comprised of the FNI 1-5 modules, and rhFNHC36 containing C-terminal HBD is comprised of the FNIII12-14 modules. (B), Purified rhFNHN29 and rhFNHC36 polypeptides were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (C), The purified rhFNHN29 and rhFNHC36 polypeptides were analysed by Western blot. Immunoreactive fragments of 27.9 kDa and 31.0 kDa indicated their antigenicity.
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Figure 1: Expression of purified rhFNHN29 and rhFNHC36 polypeptides. (A), Schematic structure of FN. FN is made up of a series of repeating homology units of three different types (FNI, FNII and FNIII). Yellow rectangles represent FNI modules, blue rhombus represent FNII modules and red ovals represent specific FNIII modules. RhFNHN29 containing N-terminal heparin-binding domain (HBD) is comprised of the FNI 1-5 modules, and rhFNHC36 containing C-terminal HBD is comprised of the FNIII12-14 modules. (B), Purified rhFNHN29 and rhFNHC36 polypeptides were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (C), The purified rhFNHN29 and rhFNHC36 polypeptides were analysed by Western blot. Immunoreactive fragments of 27.9 kDa and 31.0 kDa indicated their antigenicity.

Mentions: The N-terminal heparin-binding domain polypeptide of FN consists of five I homologous structures (Figure 1A), containing 237 amino acids (Ser46-Gly282). The length of the DNA sequence encoding this polypeptide is 711 bp (403 bp-1113 bp). In this study, the PCR amplified fragment was 741 bp, and the double-digested fragment was 729 bp. The C-terminal heparin-binding domain polypeptide of FN consists of three III homologous structures (III-12, III-13 and III-14) (Figure 1A), containing 272 amino acids (Tyr1720-Tyr1991). The length of the DNA sequence encoding this polypeptide is 816 bp (5428 bp-6244 bp). In this study, the PCR amplified fragment was 835 bp, and the double-digested fragment was 828 bp. We named the two polypeptides rhFNHN29 and rhFNHC36, respectively.


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)

Expression of purified rhFNHN29 and rhFNHC36 polypeptides. (A), Schematic structure of FN. FN is made up of a series of repeating homology units of three different types (FNI, FNII and FNIII). Yellow rectangles represent FNI modules, blue rhombus represent FNII modules and red ovals represent specific FNIII modules. RhFNHN29 containing N-terminal heparin-binding domain (HBD) is comprised of the FNI 1-5 modules, and rhFNHC36 containing C-terminal HBD is comprised of the FNIII12-14 modules. (B), Purified rhFNHN29 and rhFNHC36 polypeptides were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (C), The purified rhFNHN29 and rhFNHC36 polypeptides were analysed by Western blot. Immunoreactive fragments of 27.9 kDa and 31.0 kDa indicated their antigenicity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Expression of purified rhFNHN29 and rhFNHC36 polypeptides. (A), Schematic structure of FN. FN is made up of a series of repeating homology units of three different types (FNI, FNII and FNIII). Yellow rectangles represent FNI modules, blue rhombus represent FNII modules and red ovals represent specific FNIII modules. RhFNHN29 containing N-terminal heparin-binding domain (HBD) is comprised of the FNI 1-5 modules, and rhFNHC36 containing C-terminal HBD is comprised of the FNIII12-14 modules. (B), Purified rhFNHN29 and rhFNHC36 polypeptides were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (C), The purified rhFNHN29 and rhFNHC36 polypeptides were analysed by Western blot. Immunoreactive fragments of 27.9 kDa and 31.0 kDa indicated their antigenicity.
Mentions: The N-terminal heparin-binding domain polypeptide of FN consists of five I homologous structures (Figure 1A), containing 237 amino acids (Ser46-Gly282). The length of the DNA sequence encoding this polypeptide is 711 bp (403 bp-1113 bp). In this study, the PCR amplified fragment was 741 bp, and the double-digested fragment was 729 bp. The C-terminal heparin-binding domain polypeptide of FN consists of three III homologous structures (III-12, III-13 and III-14) (Figure 1A), containing 272 amino acids (Tyr1720-Tyr1991). The length of the DNA sequence encoding this polypeptide is 816 bp (5428 bp-6244 bp). In this study, the PCR amplified fragment was 835 bp, and the double-digested fragment was 828 bp. We named the two polypeptides rhFNHN29 and rhFNHC36, respectively.

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