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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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Related in: MedlinePlus

Purity and signaling transduction of rGEP. (A) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. (B) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t-test.
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Figure 1: Purity and signaling transduction of rGEP. (A) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. (B) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t-test.

Mentions: rGEP fused with C-terminal HIS-tag was expressed from a stable clone of pSecTag2/Hygro-rGEP transfected COS-1. Secreted rGEP was collected from the medium and purified to ho mogeneity. Molecular weights of rGEP were estimated to ~90 and 70 kDa, respectively, before and after the cleavage of the N-glycan (Figure 1A; Supplementary Figure S1, available at Carcinogenesis Online). A range of 0.1–0.4 µg/ml rGEP was found to trigger ph osphorylation of AKT and ERK1/2 in HepG2 and Hep3B-sh1 cells dose dependently (Figure 1B; Supplementary Figure S2, available at Carcinogenesis Online). These activations could be counteracted by pretreating the cells with PI3K inhibitor wortmannin and MEK inhibitor U0126 (Figure 1B). The activation of intracellular signaling pathways implicated that the rGEP is biologically functional and these signaling pathways maybe regulated through cell surface-binding partner of 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)

Purity and signaling transduction of rGEP. (A) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. (B) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP 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 1: Purity and signaling transduction of rGEP. (A) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. (B) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t-test.
Mentions: rGEP fused with C-terminal HIS-tag was expressed from a stable clone of pSecTag2/Hygro-rGEP transfected COS-1. Secreted rGEP was collected from the medium and purified to ho mogeneity. Molecular weights of rGEP were estimated to ~90 and 70 kDa, respectively, before and after the cleavage of the N-glycan (Figure 1A; Supplementary Figure S1, available at Carcinogenesis Online). A range of 0.1–0.4 µg/ml rGEP was found to trigger ph osphorylation of AKT and ERK1/2 in HepG2 and Hep3B-sh1 cells dose dependently (Figure 1B; Supplementary Figure S2, available at Carcinogenesis Online). These activations could be counteracted by pretreating the cells with PI3K inhibitor wortmannin and MEK inhibitor U0126 (Figure 1B). The activation of intracellular signaling pathways implicated that the rGEP is biologically functional and these signaling pathways maybe regulated through cell surface-binding partner of 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