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

<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.

<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.

<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

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

<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 (CoCl₂) or SAHA+150 µM cobalt chloride (CoCl₂) 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 (NH₄Cl) 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.

<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

<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

Silencing of HDAC9 represses HIF-1α induction.

<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

Amyloid dye-binding properties of REF and SRPP.

<p><b>A.</b> Samples (top image) and absorbance spectra in presence of Congo red (CR). <b>B.</b> Birefringence of REF red aggregates under visible light (top) and cross-polarized light (bottom). <b>C.</b> ThT binding spectra. <b>D.</b> Bis-ANS binding spectra.</p

Proposed model of REF and SRPP interaction with the monolayer membrane of the rubber particle.

<p>SRPP is bound to the surface of the Small Rubber Particle (SRP) while REF is inserted into the membrane of the Large Rubber Particle (LRP). Other proteins are also transmembrane proteins such as cis-prenyl transferases (CPT) or rubber transferases able to condensate and elongate natural rubber from isopentenyl and dimethylallyl diphosphates.</p

Phylogenetic analysis of REF and SRPP protein family.

<p>A BlastP was realized on REF P15252 at <a href="http://www.phylogeny.fr" target="_blank">http://www.phylogeny.fr</a>. The accession numbers of each 24 aligned sequences with ClustalW (<a href="http://www.genome.jp/tools/clustalw/program" target="_blank">http://www.genome.jp/tools/clustalw/program</a>) were as follows: REF <i>Hevea brasiliensis</i> P15252, REF <i>Hevea brasiliensis</i> AEH05970 (2), REF <i>Hevea brasiliensis</i> AAR11448 (3), SRPP <i>Hevea brasiliensis</i> O82803, SRPP <i>Hevea brasiliensis</i> AAO66432 (2), RLP1 <i>Hevea brasiliensis</i> AAP46159, RLP2 <i>Hevea brasiliensis</i> AAP46160, REF <i>Ricinus communis</i> XP_002512427, REF <i>Arabidopsis thaliana</i> NP_187201, SRP <i>Vitis riparia</i> Q9SW70, REF <i>Morus alba</i> ACV90044, REF <i>Amblyomma maculatum</i> AEO33677, SRP <i>Ipomoea batatas</i> ABP35522, SRP <i>Oryza sativa</i> AAO72547, SRP <i>Zea mays</i> ACG39345, REF <i>Selaginella moellendorffii</i> XP_002969776, SRPP <i>Parthenium argentatum</i> AAQ11374, SRP <i>Capsicum annuum</i> ADI60300, SRPP <i>Ricinus communis</i> XP_002514917, REF <i>Populus trichocarpa</i> XP_002319520, REF <i>Arabidopsis lyrata</i> XP_002882419, SRP <i>Glycine max</i> XP_003543052, REF/SRPP-like protein <i>Medicago truncatula</i> XP_003593563. Evolutionary analyses were conducted in MEGA5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048065#pone.0048065-Tamura1" target="_blank">[65]</a> using the Neighbor-Joining method, the bootstrap test (1000 replicates) and the p-distance method. All positions containing gaps and missing data were eliminated. On the right, characterized proteins are presented. SRPP: Small Rubber Particle Protein; REF: Rubber Elongation Factor; SRP: Stress-related protein; RLP: REF-like stress related protein. Both REF and SRPP proteins used in this study are framed. <i>Hevea brasiliensis</i> protein family is framed by a gray dashed box.</p