27 research outputs found

    Combined effect of eIF3H silencing and SAHA treament.

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    <p>(<b>A–B</b>) Immunoblot analysis of HIF-1α, p53, eIF3H and GAPDH protein expression in cell lysates following siRNA-mediated silencing of eIF3H (sieIF3H) in presence or absence of SAHA+DFO (<b>A</b>) or SAHA+MG132 (<b>B</b>) in HuH7 cells. Quantitative analysis of the level of HIF-1α in response to silencing of the indicated eIF3H in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to DFO treatment+Scr control (<b>A</b>) MG132 treatment+Scr control (<b>B</b>) (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control+DFO (<b>A</b>) or Scr control+MG132 (<b>B</b>) (black bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using SAHA+MG132 (<b>B</b>) or SAHA+DFO (grey bar) as the reference. In all panels GAPDH is used as loading control.</p

    SAHA represses HIF-1α induction in response to hypoxic mimics.

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    <p>(<b>A</b>) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA, DMSO, DMSO+150 µM cobalt chloride (CoCl<sub>2</sub>) or SAHA+150 µM cobalt chloride (CoCl<sub>2</sub>) for 24 h. (<b>B</b>) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA, DMSO, DMSO+500 µM dimethyloxallyl glycine (DMOG) or SAHA+500 µM dimethyloxallyl glycine (DMOG) for 24 h. (<b>C</b>) Immunoblot analysis of HIF-1α, p53 and GAPDH protein expression from cell lysates following treatment of HuH7 cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 100 µM desferrioxamine (DFO) for 18 h. (<b>D</b>) Immunoblot analysis of HIF-1α, p53, HDAC7 and GAPDH protein expression from cell lysates following treatment of HuH7 cells after SAHA treatment at the indicate concentration is shown in combination with 50 µM MG132 for 4 h. (<b>E</b>) and Hep3B cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 50 µM MG132 for 4 h. (<b>F</b>) Immunoblot analysis of HIF-1α, p53, HDAC7 and GAPDH protein expression as well as the splicing of LC3 following treatment of HuH7 cells with 5 µM SAHA or DMSO for 24 h in the presence or absence of 50 µM MG132 for 4 h. (<b>G</b>) Immunoblot analyses of HIF-1α, p53 as well as the splicing of LC3 following treatment of HuH7 cells with 50 µM MG132+5 µM SAHA or DMSO in presence or absence of 10 mM ammonium chloride (NH<sub>4</sub>Cl) for 8 h. In all panels GAPDH is used as loading control and HDAC7 is used as control to SAHA treatment.</p

    A model for SAHA mediated effects on HIF-1α translation.

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    <p>In non-treated conditions (left panel-A), HIF-1α translation is controlled by HDAC9 and then HIF-1α mRNA is translated in protein. Following SAHA treatment (middle panel-B), HDAC9 is inhibited and we suggest that SAHA could promote mRNA and protein expression of an unknown protein (referred as X) that controls HIF-1α translation. As a result of this, the unknown protein could repress specifically HIF-1α translation. Upon the combined treatment SAHA and eIF3G silencing (right panel-C), we suggest that eIF3G may play a regulatory role in translation of the mRNA coding the unknown protein. By consequence, the silencing of eIF3G results in the inhibition of the unknown protein translation. As the expression of this unknown protein is down regulated, HIF-1α translation is not repressed anymore and could be translated <i>de</i><i>novo</i>.</p

    The Histone Deacetylase Inhibitor, Vorinostat, Represses Hypoxia Inducible Factor 1 Alpha Expression through Translational Inhibition

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    <div><p>Hypoxia inducible factor 1α (HIF-1α) is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi) block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA) and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor - eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.</p></div

    SAHA did not affect the level of HIF-1α mRNA.

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    <p>(<b>A and D</b>) qRT-PCR analysis of p53 mRNA level in HuH7 cell line following the indicated concentration of SAHA (<b>A</b>) or HDAC9 and HDAC10 silencing (<b>D</b>). Data is shown as the fold change of the ratio of p53 to GAPDH mRNA relative to that seen for DMSO (0 µM) (<b>A</b>) or scrambled (Scr) siRNA control (<b>D</b>) (mean ± SD, n = 3). In all panels, asterisk indicates p<0.05, as determined by two-tailed t-test using scrambled siRNA (Scr) (<b>D</b>) or DMSO (0 mM) (<b>A</b>) as the reference. (<b>B–C</b>) qRT-PCR analysis of HIF-1α mRNA level in HuH7 cell line following the indicated concentration of SAHA (<b>B</b>) or HDAC9 and HDAC10 silencing (<b>C</b>). Data is shown as the fold change of the ratio of HIF-1α to GAPDH mRNA relative to that seen for DMSO (0 µM) (<b>B</b>) or scrambled (Scr) siRNA control (<b>C</b>) (mean ± SD, n = 3). In all panels, asterisk indicates p<0.05, as determined by two-tailed t-test using scrambled siRNA (Scr) (<b>C</b>) or DMSO (0 µM) (<b>B</b>) as the reference.</p

    eIF3G silencing reversed SAHA effect on HIF-1α repression in response to hypoxic mimic.

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    <p>Immunoblot analysis of HIF-1α and GAPDH protein expression in cell lysates following siRNA-mediated silencing of eIF3 A-M, eIF4 E, G1–3 and eIF5 in HuH7 cells in presence or absence of SAHA+MG132. Quantitative analysis of the level of HIF-1α in response to silencing of the indicated eIF in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to MG132 treatment alone (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control (grey bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using MG132 (black bar) as the reference. In all panels GAPDH is used as loading control.</p

    Silencing of HDAC9 represses HIF-1α induction.

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    <p>(<b>A</b>) Immunoblot analysis of HIF-1α, HDAC7 and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDACs 1–11 in HuH7 cells. Quantitative analysis (lower) of the level of HIF-1α in response to silencing of the indicated HDAC in HuH7 cells. Data shown denote the fold change in HIF-1α protein expression relative to scramble (Scr) control (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control (black bar) as the reference and # indicates p<0.05 as determined by two-tailed t-test using HDAC9 siRNA as the reference. (<b>B</b>) Immunoblot analysis of p53 and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDACs 1–11 in HuH7 cells. Quantitative analysis (lower) of the level of p53 in response to silencing of the indicated HDAC in HuH7 cells. Data shown denote the fold change in p53 protein expression relative to scramble (Scr) control (black bar) (mean ± SD, n = 3). Asterisks indicates p<0.05 as determined by two-tailed t-test using Scr control as the reference. (<b>C</b>) Immunoblot analysis of HIF-1α and GAPDH protein expression in cell lysates following siRNA-mediated silencing of HDAC9 (siHDAC9) in the presence of 5 µM SAHA+50 µM MG132 in HuH7 cells. In all panels GAPDH is used as loading control and HDAC7 is used as control to SAHA treatment.</p

    Inhibition of FGFR signaling suppresses tumor lymphangiogenesis and VEGF-C expression.

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    <p>(A) Left Panel, 66c14 tumor cells are observed into the lumens of VEGFR-3-positive lymphatic vessels (green) in 66c14 control tumors (white arrows in both left image and right zoomed-inset). Right panel, cytokeratin-stained 66c14 tumor cells (green) are detectable in axillary lymph nodes of 66c14 control cells-bearing mice (white arrows in both left image and right zoomed-inset), confirming the invasion mechanism via the lymphatic system of the 66c14 cells. (B) Left panel, representative images of VEGFR-3 (green) and DAPI (blue) staining of parental, empty plasmid (Control) and FGFR-2DN-expressing 66c14 tumors sections. White arrows indicate lumenized lymphatic vessels or isolated lymphatic endothelial cells in controls (Control and Parental) and FGFR-2DN tumors, respectively. Right panel, quantification of VEGFR-3-positive lymphatic vessel density (VD) demonstrates a density decrease in FGFR2-DN (R-2DN) expressing 66c14 tumor as compared to parental (Par.) or control (Ctrl) tumors. (C) Upper panel, FGFR-2DN-expressing 66c14 (66c14 FGFR-2DN) tumors exhibit a decrease in podoplanin-positive lymphatic vessel (green) density compared to control groups (66c14 Control and Parental). White arrows confirm the presence of lumenized lymphatic vessels or isolated lymphatic endothelial cells in controls and FGFR-2DN tumors, respectively. Bottom panel, quantification of podoplanin-positive lymphatic vessel density (VD) confirms a density decrease in FGFR2-DN (R-2DN) expressing 66c14 tumor as compared to parental (Par.) or control (Ctrl) tumors. (D) VEGF-C and PDGF-B (black and white bars, respectively) mRNA quantification of 66c14 tumor by qRT-PCR shows uniquely a VEGF-C expression decrease in 66c14 FGFR-2DN-expressing (R-2DN) versus control tumors (Ctrl; 66c14 control and Par; parental). (Scale Bars, 200 µm in A–C, *p<0.05 versus respective control groups).</p

    <i>In vitro</i> lymphangiogenesis is inhibited by FGFR-2DN-expressing 66c14 tumor cells.

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    <p>(<b>A</b>) Co-culture of human lymphatic endothelial cells and mouse mammary 66c14 carcinoma tumor cells induces lymphatic vessel-like tubes in the presence of 66c14 control cells (A1) and FGFR-2DN clone C4-expressing cells in combination with VEGFs (A4) while no vascular structure is observed in FGFR-2DN clone C4 (A2) or clone C22-expressing cells (A3), control cells pretreated with VEGFRs/Fc chimera (A5) or with FGFR inhibitor PD-173074 (A6). (B) Lymphatic tube formation was quantified and expressed as fold change as compared to control condition (A1). (C) <i>In vitro</i> lymphatic tubes formation was induced in the presence of supernatant from control or parental, but not from FGFR-2DN C4 and C22-expressing 66c14 carcinoma cells. (Scale bars, 200 µm in A and C, # and *p<0.05 versus respective control group).</p

    Blockade of Fibroblast Growth Factor Signaling suppresses VEGF-C expression in 66c14 cancer cells.

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    <p>(A) Left panel, decrease in VEGF-C mRNA expression is detected in FGFR-2DN-expressing 66c14 tumor cells (clones C4, C22) as compared to empty plasmid-transfected group (Control). Right panel, inhibition of VEGF-C protein secretion in FGFR-2DN-expressing 66c14 cell supernatant (C4 and C22) was confirmed by western blotting, and normalized to control group using coomassie blue (C.B.) staining of the membrane as loading control. (B) The FGFR inhibitor PD-173074 inhibits VEGF-C mRNA expression in a dose dependent manner in 66c14 tumor cells. (C) Specific siRNA-mediated inhibition of FGFR-2, but not of FGFR-1, expression reduces VEGF-C mRNA level in 66c14 tumor cells. (*p<0.05 versus respective control group).</p
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