10 research outputs found

    Stable vector-mediated, inducible knockdown of Egr1.

    No full text
    <p>To achieve an inducible knockdown of Egr1, we firstly transfected SK-HEP-1 cells with pCDNA6/TR which express tet repressor. Stable cells were selected using Blasticidin before co-transfecting SK-HEP-1 T-REx cells with tetracycline–regulated pSUPER.retro.neo+GFP tet RNAi construct, which was directed against a sequence containing Egr1. Stable cells were selected using G418. After selection, clones were analyzed for downregulation of Egr1 after 24 h of tetracycline treatment. Ten-fold down-regulation of Egr1 was detected by immunoblotting in pSUPER.retro.neo+GFP tet/Egr1 clones (<b>6A</b>) but not in mock transfected clones (<b>6B</b>). The graph compares the signal intensities obtained from Egr1 bands.</p

    Heparin inhibits HGF-induced Egr1 expression and the Egr1 promoter activity in a dose-dependent manner.

    No full text
    <p>HGF-induced activation of Egr1 mRNA and protein level (<b>2A, 2C</b>) and the effect of heparin on the HGF induced Egr1 expression in SK-HEP-1 cells were examined by RT-PCR and western blotting (<b>2B, 2D</b>). Egr1 transcriptional activity was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with pGL2-Luc-B-Egr1 plasmid construct and the pRL-TK <i>Renilia</i> luciferase. Relative luciferase activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042717#s4" target="_blank">Materials and Methods</a>. The firefly luciferase activity was normalized to <i>Renilia</i> luciferase activity (<b>2E, 2F</b>). Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.</p

    Overexpression of Egr1 induces MMP-9 activation, cell invasion and cell migration of HCC cells.

    No full text
    <p>The effect of Egr1 on the cell invasion of HCC cell lines were examined using SNU-449 transiently transfected with pCMV-6-AC-GFP Egr1 overexpression vector (<b>8A</b>). Equal amounts of wild type and Egr1 overexpressing SNU-449 cells were used for migration (<b>8B</b>) and invasion (<b>8C</b>) assay in the presence and absence of HGF. Results are expressed as fold differences of matrigel invaded cells. Zymographic gel showing the active MMP-9 bands in 24 h conditioned medium of cultured Egr1-overexpressing SNU-449 cells (<b>8D</b>). The graph compares the signal intensities obtained from zymography gels.</p

    Heparin inhibits HGF-induced p-Met, p-MAPK, MMP-2 and MMP-9 activation in SK-HEP-1.

    No full text
    <p>In order to examine the time dependent effects of HGF, overnight starved SK-HEP-1 cells were grown in presence or absence of HGF for the time periods indicated. Total cell lysates were then analyzed by immunoblotting. Blots were probed with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 and anti-calnexin antibodies. From top to bottom: HGF stimulated c-Met phosphorylation; the amounts of c-Met protein present whole cell lysates; HGF induced ERK1/2 phosphorylation and ERK1/2 and c-Met total protein levels. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The bottom panel verifies equal protein loading among the lanes (<b>3A</b>). SK-HEP1 cells left untreated or treated with HGF for 2 hours in the presence and absence of heparin. Total protein lysates were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 antibodies, and anti-calnexin antibodies. The first panel shows the level of tyrosine phosphorylated c-Met; the amount of c-Met protein present in the each lane is shown in the second panel. The third and fourth panels show the amount of phosphorylated ERK1/2 level and the level of ERK1/2 protein in the whole cell lysates, respectively. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The lower panels show the amount of calnexin as a loading control (<b>3B</b>). Zymographic gel showing active MMP-9 and MMP-2 bands in 24 h conditioned medium from cultured SK-HEP-1 cells left untreated or stimulated with HGF and heparin (<b>3C, 3D</b>).</p

    Heparin Inhibits Hepatocyte Growth Factor Induced Motility and Invasion of Hepatocellular Carcinoma Cells through Early Growth Response Protein 1

    Get PDF
    <div><p>The Hepatocyte Growth Factor (HGF)/c-Met signaling pathway regulates hepatocyte proliferation, and pathway aberrations are implicated in the invasive and metastatic behaviors of hepatocellular carcinoma (HCC). In addition to c-Met, heparin acts as a co-receptor to modulate pathway activity. Recently, anti-metastatic and anti-cancer effects of heparin have been reported. However, the role of heparin in the regulation of HGF signaling remains controversial and the effects of heparin on HGF-induced biological responses during hepatocarcinogenesis is not yet defined. In this study we determined the effects of heparin on HGF-induced activities of HCC cells and the underlying molecular mechanisms. Here, we report for the first time that heparin inhibits HGF-induced adhesion, motility and invasion of HCC cells. In addition, heparin reduced HGF-induced activation of c-Met and MAPK in a dose-dependent manner, as well as decreased transcriptional activation and expression of Early growth response factor 1 (Egr1). HGF-induced MMP-2 and MMP-9 activation, and MT1-MMP expression, also were inhibited by heparin. Stable knockdown of Egr1 caused a significant decrease in HGF-induced invasion, as well as the activation and expression of MMPs. Parallel to these findings, the overexpression of Egr1 increased the invasiveness of HCC cells. Our results suggest that Egr1 activates HGF-induced cell invasion through the regulation of MMPs in HCC cells and heparin inhibits HGF-induced cellular invasion via the downregulation of Egr1. Therefore, heparin treatment might be a therapeutic approach to inhibit invasion and metastasis of HCC, especially for patients with active HGF/c-Met signaling.</p></div

    The c-Met inhibitor SU11274 blocks HGF-induced Egr1 expression and Egr1 promoter activity.

    No full text
    <p>HGF-induced activation of Egr1 and the effect of c-Met inhibition on HGF-induced Egr1 expression in SK-HEP-1 cells were examined by immunoblotting and luciferase reporter assays (<b>5A, 5B</b>). Transcriptional activity of Egr1 was measured by luciferase reporter assay after transient transfection of SK-HEP-1 with a pGL2-Luc-B-Egr1 plasmid construct and pRL-TK <i>Renilia</i> luciferase. Relative luciferase activity was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042717#s4" target="_blank">Materials and Methods</a>. The firefly luciferase activity was normalized to <i>Renilia</i> luciferase activity. Results are representative of two independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.</p

    Heparin inhibits HGF/c-Met interaction.

    No full text
    <p>HGF binding to c-Met was measured in a Biacore T-100. HGF strongly binds to c-Met (black line), <b>and the binding decreased upon</b> heparin treatment in a dose-dependent manner (100 nM Heparin blue line, 1 µM heparin red line). Heparin alone did not show any significant binding to c-Met.</p

    The mechanism of heparin inhibition of HGF-induced cell motility and invasion.

    No full text
    <p>Activation of HGF/c-Met signaling in HCC cells leads to MAPK signaling (i), and up-regulation of Egr1 (ii), which in turn induces MMP-2 and MMP-9 activation and MT1-MMP expression (iii), and increased cell motility and invasion (iv). Heparin treatment results in the inhibition of HGF-induced cellular invasion via repression of HGF-induced c-Met activation, as well as inhibition of MAPK signaling, downregulation of Egr1 expression, and MMP activation (v).</p

    Knockdown of Egr1 decreases HGF-mediated invasion and activation of MMP2, MMP9, and expression of MT1-MMP.

    No full text
    <p>SK-HEP-1 TREx cells were obtained by stable transfection of pCDNA-6/TR. Then SK-HEP-1 TREx cells were transfected with recombinant pSUPER-retro.neo+GFP vector expressing Egr-1 targeted shRNA or empty pSUPER-retro.neo+GFP vector using Fugene as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042717#s4" target="_blank">Materials and Methods</a>. Knockdown of Egr1 in Egr1-shRNA-expressing SK-HEP-1 stable cell lines were induced by tetracycline (<b>7A</b>). Equal amount of tetracycline induced and un-induced Egr1-shRNA-expressing SK-HEP-1 cells were used for migration and invasion assays in the presence and absence of HGF (<b>7B, 7C</b>). Results are expressed as fold differences of matrigel invaded cells and are representative of two or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups. Zymographic gel showing active MMP-2 and-9 bands in 24 h conditioned medium of cultured Egr1-shRNA-expressing SK-HEP-1 cells left untreated or stimulated with tetracycline and/or HGF (<b>7D, 7E</b>). Protein levels of MT1-MMP were studied in tetracyclin and/or HGF-induced SK-HEP-1-Egr1 cells (<b>7F</b>). Densitometric analysis of gelatin zymography and immunoblots showed that HGF induces MT1-MMP expression and MMP-2 and -9 activation, whereas silencing of Egr1 blocks HGF-induced MMPs up-regulation.</p

    Heparin and HGF do not effects Egr1 expression, motility or invasion in Egr1 over-expressed SNU-449 cell lines.

    No full text
    <p>The effects of heparin and HGF on Egr1 expression in Egr1 over-expressing SNU-449 cells was analyzed by immunoblotting (<b>9A</b>). Following serum deprivation, Egr1 overexpressed SN-449 cells were left untreated (0) or treated with HGF in the presence or absence of heparin, then invasion and migration assays were performed using the Roche xCELLigence System (<b>9B, 9C</b>). Results are representative of three or more independent experiments performed in triplicate. Bars indicate standard error of the mean (SEM), asterisks (*) indicate statistically significant differences between the indicated groups.</p
    corecore