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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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rGEP binding and rGEP-mediated signaling transduction in HCC cells after suppression of GPC3 and EXT1. Cells were transfected with NC siRNA and siRNAs targeting GPC3 and EXT1 (HSS104149 and HSS103435, respectively, from Life Technologies). (A) The cells were detached at 48h posttransfection for rGEP binding assay. The detached cells were incubated with 3.2 µg rGEP, followed by detection of anti-His antibody and analysis in flow cytometry. The chart shows the relative percentage of binding by comparing the MFI of each transfectant to the untreated cells (P). The means and the standard deviations were obtained from three independent assays. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test. The binding properties of Hep3B and Hep3B-sh1 are the same. (B) Suppression of GPC3 and EXT1 in HCC cell reduced the rGEP-mediated signaling transduction. Two cell lines (HepG2 and GEP-suppressed Hep3B) were transfected with the siRNAs and were treated with or without 0.2 µg/ml rGEP for 5min. Cells were detached, fixed by formaldehyde, permeabilized by 70% methanol and stained with fluorescein-conjugated specific antibody against pAKT and pERK1/2. The graphs show the difference of MFI with and without rGEP induction, reflecting the increased amount of pAKT and pERK1/2 upon rGEP induction in different siRNA treated cells. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test.
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Figure 6: rGEP binding and rGEP-mediated signaling transduction in HCC cells after suppression of GPC3 and EXT1. Cells were transfected with NC siRNA and siRNAs targeting GPC3 and EXT1 (HSS104149 and HSS103435, respectively, from Life Technologies). (A) The cells were detached at 48h posttransfection for rGEP binding assay. The detached cells were incubated with 3.2 µg rGEP, followed by detection of anti-His antibody and analysis in flow cytometry. The chart shows the relative percentage of binding by comparing the MFI of each transfectant to the untreated cells (P). The means and the standard deviations were obtained from three independent assays. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test. The binding properties of Hep3B and Hep3B-sh1 are the same. (B) Suppression of GPC3 and EXT1 in HCC cell reduced the rGEP-mediated signaling transduction. Two cell lines (HepG2 and GEP-suppressed Hep3B) were transfected with the siRNAs and were treated with or without 0.2 µg/ml rGEP for 5min. Cells were detached, fixed by formaldehyde, permeabilized by 70% methanol and stained with fluorescein-conjugated specific antibody against pAKT and pERK1/2. The graphs show the difference of MFI with and without rGEP induction, reflecting the increased amount of pAKT and pERK1/2 upon rGEP induction in different siRNA treated cells. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test.

Mentions: The polymerase of HS, EXT1, and a liver cancer marker, GPC3, were suppressed in two liver cancer cell lines to determine the contribution of HS on rGEP binding and rGEP-mediated signaling transduction. The suppression and binding properties of parental Hep3B and Hep3B-sh1 are very similar and so only that of parental Hep3B were shown. After transfection of specific siRNA, mRNA level of GPC3 of the two cell lines was shown to decrease (Supplementary Figure S3A, available at Carcinogenesis Online). The reduction of protein level of GPC3 was demonstrated in immunoblotting (Supplementary Figure S3B, available at Carcinogenesis Online) and flow cytometry (Supplementary Figure S3C, available at Carcinogenesis Online). Suppression of EXT1 mRNA level and decrease of cell surface HS after EXT1 siRNA transfection are shown in Supplementary Figure S4, available at Carcinogenesis Online. These GPC3- and EXT1-depleted cells were demonstrated to have reduced capacity for rGEP binding (Figure 6A). Notably, the reductions of binding on the GPC3-suppressed cells of ~20–30% compared with the negative control (NC) indicated that other cell surface HSPGs might also interact with GEP. The residual binding of rGEP on EXT1-suppressed cells may reflect the presence of surface HS before knockdown took place while turnover rate of surface HSPGs varies. The decreased rGEP binding shown in NC-treated Hep3B maybe due to cell line-specific effect of lipofectamine, which decrease the amount of HS (Supplementary Figures S3 and S4, available at Carcinogenesis Online). Suppression of EXT1 in Hep3B-sh1 inhibited the rGEP-mediated activation of AKT and ERK1/2, whereas suppression of GPC3 inhibited the AKT activation (Figure 6B). These results suggested that GEP-HS interaction on HCC cell surface plays a determining role on rGEP-mediated signaling transduction in HCC, whereas GPC3 may mainly contribute to rGEP-mediated activation of AKT. In general, siRNA transfection did not affect the basal pAKT or pERK1/2 levels. However, in the transfection of siEXT1 to HepG2 cells, upregulation of pERK1/2 was observed compared with siRNA NC and parental (P) cells in the absence of rGEP (data not shown). This might indicate additional roles of EXT1 in HepG2 MAPK signaling cassette and interfere with rGEP-mediated signaling transduction. Therefore, rGEP-mediated ERK1/2 activation in siEXT1 HepG2 was not shown in Figure 6B.


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)

rGEP binding and rGEP-mediated signaling transduction in HCC cells after suppression of GPC3 and EXT1. Cells were transfected with NC siRNA and siRNAs targeting GPC3 and EXT1 (HSS104149 and HSS103435, respectively, from Life Technologies). (A) The cells were detached at 48h posttransfection for rGEP binding assay. The detached cells were incubated with 3.2 µg rGEP, followed by detection of anti-His antibody and analysis in flow cytometry. The chart shows the relative percentage of binding by comparing the MFI of each transfectant to the untreated cells (P). The means and the standard deviations were obtained from three independent assays. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test. The binding properties of Hep3B and Hep3B-sh1 are the same. (B) Suppression of GPC3 and EXT1 in HCC cell reduced the rGEP-mediated signaling transduction. Two cell lines (HepG2 and GEP-suppressed Hep3B) were transfected with the siRNAs and were treated with or without 0.2 µg/ml rGEP for 5min. Cells were detached, fixed by formaldehyde, permeabilized by 70% methanol and stained with fluorescein-conjugated specific antibody against pAKT and pERK1/2. The graphs show the difference of MFI with and without rGEP induction, reflecting the increased amount of pAKT and pERK1/2 upon rGEP induction in different siRNA treated cells. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test.
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Figure 6: rGEP binding and rGEP-mediated signaling transduction in HCC cells after suppression of GPC3 and EXT1. Cells were transfected with NC siRNA and siRNAs targeting GPC3 and EXT1 (HSS104149 and HSS103435, respectively, from Life Technologies). (A) The cells were detached at 48h posttransfection for rGEP binding assay. The detached cells were incubated with 3.2 µg rGEP, followed by detection of anti-His antibody and analysis in flow cytometry. The chart shows the relative percentage of binding by comparing the MFI of each transfectant to the untreated cells (P). The means and the standard deviations were obtained from three independent assays. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test. The binding properties of Hep3B and Hep3B-sh1 are the same. (B) Suppression of GPC3 and EXT1 in HCC cell reduced the rGEP-mediated signaling transduction. Two cell lines (HepG2 and GEP-suppressed Hep3B) were transfected with the siRNAs and were treated with or without 0.2 µg/ml rGEP for 5min. Cells were detached, fixed by formaldehyde, permeabilized by 70% methanol and stained with fluorescein-conjugated specific antibody against pAKT and pERK1/2. The graphs show the difference of MFI with and without rGEP induction, reflecting the increased amount of pAKT and pERK1/2 upon rGEP induction in different siRNA treated cells. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t-test.
Mentions: The polymerase of HS, EXT1, and a liver cancer marker, GPC3, were suppressed in two liver cancer cell lines to determine the contribution of HS on rGEP binding and rGEP-mediated signaling transduction. The suppression and binding properties of parental Hep3B and Hep3B-sh1 are very similar and so only that of parental Hep3B were shown. After transfection of specific siRNA, mRNA level of GPC3 of the two cell lines was shown to decrease (Supplementary Figure S3A, available at Carcinogenesis Online). The reduction of protein level of GPC3 was demonstrated in immunoblotting (Supplementary Figure S3B, available at Carcinogenesis Online) and flow cytometry (Supplementary Figure S3C, available at Carcinogenesis Online). Suppression of EXT1 mRNA level and decrease of cell surface HS after EXT1 siRNA transfection are shown in Supplementary Figure S4, available at Carcinogenesis Online. These GPC3- and EXT1-depleted cells were demonstrated to have reduced capacity for rGEP binding (Figure 6A). Notably, the reductions of binding on the GPC3-suppressed cells of ~20–30% compared with the negative control (NC) indicated that other cell surface HSPGs might also interact with GEP. The residual binding of rGEP on EXT1-suppressed cells may reflect the presence of surface HS before knockdown took place while turnover rate of surface HSPGs varies. The decreased rGEP binding shown in NC-treated Hep3B maybe due to cell line-specific effect of lipofectamine, which decrease the amount of HS (Supplementary Figures S3 and S4, available at Carcinogenesis Online). Suppression of EXT1 in Hep3B-sh1 inhibited the rGEP-mediated activation of AKT and ERK1/2, whereas suppression of GPC3 inhibited the AKT activation (Figure 6B). These results suggested that GEP-HS interaction on HCC cell surface plays a determining role on rGEP-mediated signaling transduction in HCC, whereas GPC3 may mainly contribute to rGEP-mediated activation of AKT. In general, siRNA transfection did not affect the basal pAKT or pERK1/2 levels. However, in the transfection of siEXT1 to HepG2 cells, upregulation of pERK1/2 was observed compared with siRNA NC and parental (P) cells in the absence of rGEP (data not shown). This might indicate additional roles of EXT1 in HepG2 MAPK signaling cassette and interfere with rGEP-mediated signaling transduction. Therefore, rGEP-mediated ERK1/2 activation in siEXT1 HepG2 was not shown in Figure 6B.

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