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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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GEP interacts with HS on cell surface of liver cancer cell line. (A–C) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. (D) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.
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Figure 3: GEP interacts with HS on cell surface of liver cancer cell line. (A–C) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. (D) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.

Mentions: Because PDGF-AA, PDGF-BB and FGF-2 have been shown to interact with cell surface HS, we speculated it as a potential binding partner of rGEP. Different amounts of heparinase III were used to cleave HS from cell surface of liver cancer cell lines to determine its role in the binding of rGEP. Depletion of cell surface HS by the treatment of heparinase III was confirmed in flow cytometry (Figure 3A) and this depletion dramatically reduced the rGEP binding capacity of the cell line (Figure 3B) as well as the expression level of GEP on the cell surface (Figure 3C), implicating a direct interaction between HS and GEP on the HCC cell surface. On the other hand, heparin, a highly sulfated structural analogues of HS (23), was used to displace the bound rGEP from cell surface HS. In Figure 3D, heparin displaces the bound rGEP from the cell surface in a dose-dependent manner. Therefore, these results implicated that the presence of HS is essential for cell surface binding of both rGEP and endogenous secretory GEP.


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)

GEP interacts with HS on cell surface of liver cancer cell line. (A–C) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. (D) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: GEP interacts with HS on cell surface of liver cancer cell line. (A–C) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. (D) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.
Mentions: Because PDGF-AA, PDGF-BB and FGF-2 have been shown to interact with cell surface HS, we speculated it as a potential binding partner of rGEP. Different amounts of heparinase III were used to cleave HS from cell surface of liver cancer cell lines to determine its role in the binding of rGEP. Depletion of cell surface HS by the treatment of heparinase III was confirmed in flow cytometry (Figure 3A) and this depletion dramatically reduced the rGEP binding capacity of the cell line (Figure 3B) as well as the expression level of GEP on the cell surface (Figure 3C), implicating a direct interaction between HS and GEP on the HCC cell surface. On the other hand, heparin, a highly sulfated structural analogues of HS (23), was used to displace the bound rGEP from cell surface HS. In Figure 3D, heparin displaces the bound rGEP from the cell surface in a dose-dependent manner. Therefore, these results implicated that the presence of HS is essential for cell surface binding of both rGEP and endogenous secretory GEP.

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