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Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Yip CW, Cheung PF, Leung IC, Wong NC, Cheng CK, Fan ST, Cheung ST - Carcinogenesis (2014)

Bottom Line: Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction.Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT.Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Centre for Cancer Research and.

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Binding properties of rGEP on liver cancer cell lines. (A) Confocal images of rGEP binding on HCC cell surface. Hep3B and HepG2 were incubated with rGEP, specific GEP antibody, anti-rabbit second antibody and Texas Red-conjugated WGA. The cells were then fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole. (B) Cell surface-binding capacity of Hep3B (left panel) and HepG2 (right panel) for rGEP. Detached cells were incubated with different amounts (0.4–3.2 µg) of rGEP at 4°C and the cell surface binding of rGEP was detected by FITC-anti-His antibody. The histograms show fluorescent intensity of cells and the shaded peaks are isotypic controls. The bar charts show the geometric mean fluorescent intensity (MFI) of different samples. (C) Neutralization of rGEP binding by GEP mAb (×GEP). GEP mAb or mouse IgG were premixed with 0.8 µg rGEP. This rGEP-antibody mixture was added to EDTA-detached Hep3B for binding. The histogram shows the fluorescent intensity of cells bound with pretreated rGEP. The bar chart shows the percentage of cells in the positive region in different samples. Untreated cells detected by isotypic control antibody (IC) and anti-His antibody (basal) are also shown. (D) Competitive binding assay of rGEP. Different growth factors were preincubated with detached Hep3B at 4°C, followed by the binding of 0.8 µg rGEP, detection of anti-His antibody and quantification in flow cytometry. The bar chart shows the percentage of cells in the positive region where the IC and the anti-His basal levels are also shown. The percentages shown are the means and standard deviations obtained from three independent assays. Asterisks represent significant differences from the ‘rGEP only’ at 95% level according to Student’s t-test.
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Figure 2: Binding properties of rGEP on liver cancer cell lines. (A) Confocal images of rGEP binding on HCC cell surface. Hep3B and HepG2 were incubated with rGEP, specific GEP antibody, anti-rabbit second antibody and Texas Red-conjugated WGA. The cells were then fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole. (B) Cell surface-binding capacity of Hep3B (left panel) and HepG2 (right panel) for rGEP. Detached cells were incubated with different amounts (0.4–3.2 µg) of rGEP at 4°C and the cell surface binding of rGEP was detected by FITC-anti-His antibody. The histograms show fluorescent intensity of cells and the shaded peaks are isotypic controls. The bar charts show the geometric mean fluorescent intensity (MFI) of different samples. (C) Neutralization of rGEP binding by GEP mAb (×GEP). GEP mAb or mouse IgG were premixed with 0.8 µg rGEP. This rGEP-antibody mixture was added to EDTA-detached Hep3B for binding. The histogram shows the fluorescent intensity of cells bound with pretreated rGEP. The bar chart shows the percentage of cells in the positive region in different samples. Untreated cells detected by isotypic control antibody (IC) and anti-His antibody (basal) are also shown. (D) Competitive binding assay of rGEP. Different growth factors were preincubated with detached Hep3B at 4°C, followed by the binding of 0.8 µg rGEP, detection of anti-His antibody and quantification in flow cytometry. The bar chart shows the percentage of cells in the positive region where the IC and the anti-His basal levels are also shown. The percentages shown are the means and standard deviations obtained from three independent assays. Asterisks represent significant differences from the ‘rGEP only’ at 95% level according to Student’s t-test.

Mentions: After demonstrating the exogenous rGEP could trigger intracellular signaling pathways in liver cancer cell lines, we reasoned that rGEP might interact with specific binding partner(s) on the cell surface. Therefore, purified rGEP was added to Hep3B and HepG2 and cell surface binding was detected by GEP antibody (Santa Cruz). The confocal images of the cells showed rGEP (green) localized on plasma membrane (red) (Figure 2A). Besides, detached HCC cells were incubated with purified rGEP at 4°C to quantify the rGEP binding by flow cytometry. In Figure 2B, rGEP was demonstrated to bind to both Hep3B and HepG2 from 0.4 to 3.2 µg without achieving plateau. Similar binding effects were demonstrated in Hep3B-sh1 and Huh-7 cells (data not shown). The binding of rGEP could be neutralized by preincubating the protein with GEP mAb, confirming the binding is specific (Figure 2C).


Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Yip CW, Cheung PF, Leung IC, Wong NC, Cheng CK, Fan ST, Cheung ST - Carcinogenesis (2014)

Binding properties of rGEP on liver cancer cell lines. (A) Confocal images of rGEP binding on HCC cell surface. Hep3B and HepG2 were incubated with rGEP, specific GEP antibody, anti-rabbit second antibody and Texas Red-conjugated WGA. The cells were then fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole. (B) Cell surface-binding capacity of Hep3B (left panel) and HepG2 (right panel) for rGEP. Detached cells were incubated with different amounts (0.4–3.2 µg) of rGEP at 4°C and the cell surface binding of rGEP was detected by FITC-anti-His antibody. The histograms show fluorescent intensity of cells and the shaded peaks are isotypic controls. The bar charts show the geometric mean fluorescent intensity (MFI) of different samples. (C) Neutralization of rGEP binding by GEP mAb (×GEP). GEP mAb or mouse IgG were premixed with 0.8 µg rGEP. This rGEP-antibody mixture was added to EDTA-detached Hep3B for binding. The histogram shows the fluorescent intensity of cells bound with pretreated rGEP. The bar chart shows the percentage of cells in the positive region in different samples. Untreated cells detected by isotypic control antibody (IC) and anti-His antibody (basal) are also shown. (D) Competitive binding assay of rGEP. Different growth factors were preincubated with detached Hep3B at 4°C, followed by the binding of 0.8 µg rGEP, detection of anti-His antibody and quantification in flow cytometry. The bar chart shows the percentage of cells in the positive region where the IC and the anti-His basal levels are also shown. The percentages shown are the means and standard deviations obtained from three independent assays. Asterisks represent significant differences from the ‘rGEP only’ at 95% level according to Student’s t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4216055&req=5

Figure 2: Binding properties of rGEP on liver cancer cell lines. (A) Confocal images of rGEP binding on HCC cell surface. Hep3B and HepG2 were incubated with rGEP, specific GEP antibody, anti-rabbit second antibody and Texas Red-conjugated WGA. The cells were then fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole. (B) Cell surface-binding capacity of Hep3B (left panel) and HepG2 (right panel) for rGEP. Detached cells were incubated with different amounts (0.4–3.2 µg) of rGEP at 4°C and the cell surface binding of rGEP was detected by FITC-anti-His antibody. The histograms show fluorescent intensity of cells and the shaded peaks are isotypic controls. The bar charts show the geometric mean fluorescent intensity (MFI) of different samples. (C) Neutralization of rGEP binding by GEP mAb (×GEP). GEP mAb or mouse IgG were premixed with 0.8 µg rGEP. This rGEP-antibody mixture was added to EDTA-detached Hep3B for binding. The histogram shows the fluorescent intensity of cells bound with pretreated rGEP. The bar chart shows the percentage of cells in the positive region in different samples. Untreated cells detected by isotypic control antibody (IC) and anti-His antibody (basal) are also shown. (D) Competitive binding assay of rGEP. Different growth factors were preincubated with detached Hep3B at 4°C, followed by the binding of 0.8 µg rGEP, detection of anti-His antibody and quantification in flow cytometry. The bar chart shows the percentage of cells in the positive region where the IC and the anti-His basal levels are also shown. The percentages shown are the means and standard deviations obtained from three independent assays. Asterisks represent significant differences from the ‘rGEP only’ at 95% level according to Student’s t-test.
Mentions: After demonstrating the exogenous rGEP could trigger intracellular signaling pathways in liver cancer cell lines, we reasoned that rGEP might interact with specific binding partner(s) on the cell surface. Therefore, purified rGEP was added to Hep3B and HepG2 and cell surface binding was detected by GEP antibody (Santa Cruz). The confocal images of the cells showed rGEP (green) localized on plasma membrane (red) (Figure 2A). Besides, detached HCC cells were incubated with purified rGEP at 4°C to quantify the rGEP binding by flow cytometry. In Figure 2B, rGEP was demonstrated to bind to both Hep3B and HepG2 from 0.4 to 3.2 µg without achieving plateau. Similar binding effects were demonstrated in Hep3B-sh1 and Huh-7 cells (data not shown). The binding of rGEP could be neutralized by preincubating the protein with GEP mAb, confirming the binding is specific (Figure 2C).

Bottom Line: Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction.Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT.Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Centre for Cancer Research and.

Show MeSH
Related in: MedlinePlus