15 research outputs found

    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

    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

    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

    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

    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

    Chemical inhibition of HSF1 synergizes with VX809 to improve F508del-CFTR function in patient-derived primary epithelium.

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    <p>(A) Short-circuit current analysis of human primary hBE cells (F508del/F508del, patient code CF006) treated with DMSO, 3 µM VX809, and 25 nM triptolide or a combination of VX809 and triptolide, for 96 h (daily dosing). The data is presented as fold change relative to the basal current seen with DMSO treatment, and shown as mean ± SD, <i>n</i>≥3 (replicated multiple times); * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (B) Representative short-circuit current (I<sub>sc</sub>) traces for DMSO, VX809, triptolide, or triptolide + VX809 treatment of primary hBE cells from (A). (C) Quantitative analysis of organoid swelling (shown in D) that is indicative of CFTR function over the period of 60 min. Organoids were obtained from two distinct F508del/F508del CF patients (CF4, CF22), and treated with DMSO, 3 µM VX809, 25 nM triptolide, or a combination of VX809 and triptolide. Experiments were repeated once and results are shown as a mean ± SD, <i>n</i>≥2; * and # indicate <i>p</i><0.05 relative to DMSO or VX809, respectively. (D) Representative images of organoids derived from patients (CF4 and CF22) at T = 0 or after stimulus with Forskolin/Genistein at T = 60 min treated with the indicated compounds. Scale bar represents 110 µm. The underlying data used to make (A–C) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p

    HSF1 silencing increases F508del folding, trafficking, and function and improves the phenotype of other misfolding diseases.

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    <p>(A) Immunoblot of indicated proteins following siHSF-1. (B) Short-circuit current (<i>I</i><sub>sc</sub>) analysis of CFTR in polarized CFBE cells expressing WT- or F508del-CFTR treated with the indicated siRNA or temperature corrected at 30°C. Channel activity was determined in response to forskolin and genistein (positive deflection) or with CF specific inhibitor 172 (Inh172) (negative deflection). The ** indicates p<0.05 and * indicates p<0.1 relative to control siRNA or DMSO for VX809 (<i>n</i>≥4). (C) Immunoblot of the indicated proteins in WT- and Z-AAT expressing IB3 cells (<i>n</i> = 3). (D) Immunoblot of the indicated proteins in Z-AAT expressing IB3 cells in response to siHSF1 treatment. Shown for the AAT are the immature (I), mature (M), and secreted (S) forms (<i>n</i> = 3). (E) Immunoblot of the indicated proteins from primary fibroblasts derived from WT- or mutant I1061T-NPC1 patients (<i>n</i> = 3). (F) Immunoblot of the digestion pattern before and after endo-H digestion of WT- or mutant I1061T-NPC1 stably expressed in Hela cells and treated with the indicated siRNA (<i>n</i> = 3). Immunoblot shows NPC1 endo-H resistant band (R) and sensitive band (S). Quantification represents total I1061T NPC1 (R+S) in control or siHSF1-treated cells, shown as percentage of control. Percentage of endo-H sensitive band (S) or resistant band (R) is shown in gray and black color, respectively (<i>n</i>≥3, * indicates <i>p</i><0.05). (G) Immunoblots of indicated proteins and quantification of HSF1 and HSF1-P on brain homogenates obtained from WT or AD mice of approximately 4 months (4 m; <i>n</i> = 3 for WT, <i>n</i> = 3 for AD), 9 months (9 m; <i>n</i> = 2 for WT, <i>n</i> = 2 for AD), and 16 months of age (16 m; <i>n</i> = 3 for WT, <i>n</i> = 3 for AD). Results were normalized to tubulin loading control and shown as fold change relative to WT-3m set to 1 (* indicates <i>p</i><0.05 relative to age-matched WT mice). (H) Immunoblot for APP (110 kDa), Aβ<sub>42</sub> toxic species (monomer, 4 kDa, and multimers from 6–12 kDa), and actin control on particulate fractions of brain homogenates obtained from WT (on the right) or AD mice. Controls shown include 0.5 µg recombinant Aβ<sub>42</sub>; control and Alzheimer disease samples from human brain homogenates. The underlying data used to make (B), (F) and (G) in this figure can be found in the supplementary file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001998#pbio.1001998.s008" target="_blank">Data S1</a>.</p

    Q-state management of MSR to correct human disease.

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    <p>Illustrated is the activation state of the HSR in response to acute stress (red) or to the MSR (blue) seen in disease. Acute HSR activation, seen during acute stress insults, protects from and/or corrects misfolding and rapidly returns to basal levels, allowing normal biology to resume. In misfolding disease, chronic activation of the HSR alters the normal, physiologic Q-state (Q<sup>n</sup>) because of the continued expression of misfolded protein. Once chronically elevated (Q*), the folding environment becomes maladaptive as it fails to return to the Q<sup>n</sup> (light yellow area). Down-regulation of the MSR by siHSF1, sip23, or triptolide promotes a reduction of the Q*, which now falls within the proteostasis buffering capacity (green line), promoting a more normal cellular folding environment. This effect can be further improved (purple line) when combined with protein fold correctors (pharmacologic chaperones; PCs) which impart improved thermodynamic stability to the fold, or proteostasis regulators (PRs) that improve protein Q-state biology, improving function of disease-related misfolded protein and its proteome's associated environment, promoting abrogation of the chronic stress and improving health.</p
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