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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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Reduction of integrin expression and MMP activity by rhFNHN29 and rhFNHC36. (A-1), Representative immunoblots for integrin αv, β3 and β1 proteins. (A-2), The group data represents the mean ± SD (n = 3). The densitometry data were normalized to β-actin. * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA. (B-1), Analysis of MMP activity. Supernatants from MHCC97H cells cultured with Mn2+ in the absence or presence of different concentrations of rhFNHN29 and rhFNHC36 were performed by Gelatin Zymography. (B-2), The group data of enzymolysis strip volume represent mean ± SD (n = 3). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA.
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Figure 6: Reduction of integrin expression and MMP activity by rhFNHN29 and rhFNHC36. (A-1), Representative immunoblots for integrin αv, β3 and β1 proteins. (A-2), The group data represents the mean ± SD (n = 3). The densitometry data were normalized to β-actin. * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA. (B-1), Analysis of MMP activity. Supernatants from MHCC97H cells cultured with Mn2+ in the absence or presence of different concentrations of rhFNHN29 and rhFNHC36 were performed by Gelatin Zymography. (B-2), The group data of enzymolysis strip volume represent mean ± SD (n = 3). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA.

Mentions: To verify whether the changes in integrin and MMP expression contributed to the reduction of adhesion and invasion of MHCC97H cells by intervention of rhFNHN29 and rhFNHC36, we evaluated the expression of related integrins and activity of MMP-2 and MMP-9 in MHCC97H cells. As shown in Figure 6A, rhFNHN29 and rhFNHC36 affected the expression of integrin αv, β3 and β1 in a dose-dependent manner. Furthermore, integrin αv and β3 expression were almost lost as the cells were treated with rhFNHC36 at a high concentration (200 μg/ml). This indicated that the effectiveness of rhFNHC36 was stronger than that of rhFNHN29. In addition, the impact of rhFNHN29 and rhFNHC36 on activity of proMMP-2 was not significant, but the impact of rhFNHC36 on activity of actMMP-9 and proMMP-9 was significant with concentration-dependence (Figure 6B). Taken together, these results account for the decrease of adhesion and invasion of MHCC97H cells.


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)

Reduction of integrin expression and MMP activity by rhFNHN29 and rhFNHC36. (A-1), Representative immunoblots for integrin αv, β3 and β1 proteins. (A-2), The group data represents the mean ± SD (n = 3). The densitometry data were normalized to β-actin. * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA. (B-1), Analysis of MMP activity. Supernatants from MHCC97H cells cultured with Mn2+ in the absence or presence of different concentrations of rhFNHN29 and rhFNHC36 were performed by Gelatin Zymography. (B-2), The group data of enzymolysis strip volume represent mean ± SD (n = 3). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Reduction of integrin expression and MMP activity by rhFNHN29 and rhFNHC36. (A-1), Representative immunoblots for integrin αv, β3 and β1 proteins. (A-2), The group data represents the mean ± SD (n = 3). The densitometry data were normalized to β-actin. * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA. (B-1), Analysis of MMP activity. Supernatants from MHCC97H cells cultured with Mn2+ in the absence or presence of different concentrations of rhFNHN29 and rhFNHC36 were performed by Gelatin Zymography. (B-2), The group data of enzymolysis strip volume represent mean ± SD (n = 3). * denotes a statistically significant difference compared to the control (P < 0.05) and # denotes a statistically significant difference compared to rhFNHN29 at the same concentration (P < 0.05) by One-Way ANOVA.
Mentions: To verify whether the changes in integrin and MMP expression contributed to the reduction of adhesion and invasion of MHCC97H cells by intervention of rhFNHN29 and rhFNHC36, we evaluated the expression of related integrins and activity of MMP-2 and MMP-9 in MHCC97H cells. As shown in Figure 6A, rhFNHN29 and rhFNHC36 affected the expression of integrin αv, β3 and β1 in a dose-dependent manner. Furthermore, integrin αv and β3 expression were almost lost as the cells were treated with rhFNHC36 at a high concentration (200 μg/ml). This indicated that the effectiveness of rhFNHC36 was stronger than that of rhFNHN29. In addition, the impact of rhFNHN29 and rhFNHC36 on activity of proMMP-2 was not significant, but the impact of rhFNHC36 on activity of actMMP-9 and proMMP-9 was significant with concentration-dependence (Figure 6B). Taken together, these results account for the decrease of adhesion and invasion of MHCC97H cells.

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