SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA. © 2013 Macmillan Publishers Limited All rights reserved.

Accumulation of the PX domain mutant Frank-ter Haar syndrome protein Tks4 in aggresomes

BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization.

RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells.

CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.

Elevated NCOR1 disrupts a network of dietary-sensing nuclear receptors in bladder cancer cells

Increasingly invasive bladder cancer cells lines displayed insensitivity toward a panel of dietary-derived ligands for members of the nuclear receptor superfamily. Insensitivity was defined through altered gene regulatory actions and cell proliferation and reflected both reduced receptor expression and elevated nuclear receptor corepressor 1 (NCOR1) expression. Stable overexpression of NCOR1 in sensitive cells (RT4) resulted in a panel of clones that recapitulated the resistant phenotype in terms of gene regulatory actions and proliferative responses toward ligand. Similarly, silencing RNA approaches to NCOR1 in resistant cells (EJ28) enhanced ligand gene regulatory and proliferation responses, including those mediated by peroxisome proliferator-activated receptor (PPAR) gamma and vitamin D receptor (VDR) receptors. Elevated NCOR1 levels generate an epigenetic lesion to target in resistant cells using the histone deacetylase inhibitor vorinostat, in combination with nuclear receptor ligands. Such treatments revealed strong-additive interactions toward the PPARgamma, VDR and Farnesoid X-activated receptors. Genome-wide microarray and microfluidic quantitative real-time, reverse transcription-polymerase chain reaction approaches, following the targeting of NCOR1 activity and expression, revealed the selective capacity of this corepressor to govern common transcriptional events of underlying networks. Combined these findings suggest that NCOR1 is a selective regulator of nuclear receptors, notably PPARgamma and VDR, and contributes to their loss of sensitivity. Combinations of epigenetic therapies that target NCOR1 may prove effective, even when receptor expression is reduced.

Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

Caractérisation de la fonction des complexes histone déacétylases Rpd3S et Set3C

La chromatine est essentielle au maintien de l’intégrité du génome, mais, ironiquement, constitue l’obstacle principal à la transcription des gènes. Plusieurs mécanismes ont été développés par la cellule pour pallier ce problème, dont l’acétylation des histones composant les nucléosomes. Cette acétylation, catalysée par des histones acétyl transférases (HATs), permet de réduire la force de l’interaction entre les nucléosomes et l’ADN, ce qui permet à la machinerie transcriptionnelle de faire son travail. Toutefois, on ne peut laisser la chromatine dans cet état permissif sans conséquence néfaste. Les histone déacétylases (HDACs) catalysent le clivage du groupement acétyle pour permettre à la chromatine de retrouver une conformation compacte. Cette thèse se penche sur la caractérisation de la fonction et du mécanisme de recrutement des complexes HDACs Rpd3S et Set3C. Le complexe Rpd3S est recruté aux régions transcrites par une interaction avec le domaine C-terminal hyperphosphorylé de Rpb1, une sous-unité de l’ARN polymérase II. Toutefois, le facteur d’élongation DSIF joue un rôle dans la régulation de cette association en limitant le recrutement de Rpd3S aux régions transcrites. L’activité HDAC de Rpd3S, quant à elle, dépend de la méthylation du résidu H3K36 par l’histone méthyltransférase Set2. La fonction du complexe Set3C n’est pas clairement définie. Ce complexe est recruté à la plupart de ses cibles par l’interaction entre le domaine PHD de Set3 et le résidu H3K4 di- ou triméthylé. Un mécanisme indépendant de cette méthylation, possiblement le même que pour Rpd3S, régit toutefois l’association de Set3C aux régions codantes des gènes les plus transcrits. La majorité de ces résultats ont été obtenus par la technique d’immunoprécipitation de la chromatine couplée aux biopuces (ChIP-chip). Le protocole technique et le design expérimental de ce type d’expérience fera aussi l’objet d’une discussion approfondie.

The anti-epileptic drug Valproic Acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC(50) 67±10 µg/ml) and LH-induced (93% suppression; IC(50) 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Exercise and skeletal muscle glucose transporter 4 expression: molecular mechanisms

1.      Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT-4) expression in human skeletal muscle.

2.      Glucose transporter isoform 4 gene expression is increased immediately following a single bout of exercise, and the GLUT-4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. Glucose transporter isoform enhancer factor and MEF2 DNA binding activities are increased following exercise, and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined.

3.      These studies find possible roles for histone deacetylase 5 (HDAC5), adenosine monophosphate–activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and p38 mitogen-activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation.

4.      Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterized by mitochondrial dysfunction and insulin resistance in skeletal muscle.

Effect of sex differences on human MEF2 regulation during endurance exercise

Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% VO2peak in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-{alpha} ({alpha}AMPK) Thr172 (P < 0.001), histone deacetylase 5 (HDAC5) Ser498 (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC1{alpha}) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1{alpha} on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.

Suberoylanilide hydroxamic acid radiosensitizes tumor hypoxic cells in vitro through the oxidation of nitroxyl to nitric oxide

The pharmacological effects of hydroxamic acids are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Previously, it was concluded that oxidation of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) by the metmyoglobin/H2O2 reaction system releases NO, which was based on spin trapping of NO and accumulation of nitrite. Reinvestigation of this system demonstrates the accumulation of N2O, which is a marker of HNO formation, at similar rates under normoxia and anoxia. In addition, the yields of nitrite that accumulated in the absence and the presence of O2 did not differ, implying that the source of nitrite is other than autoxidation of NO. In this system metmyoglobin is instantaneously and continuously converted into compound II, leading to one-electron oxidation of SAHA to its respective transient nitroxide radical. Studies using pulse radiolysis show that one-electron oxidation of SAHA (pKa=9.56 ± 0.04) yields the respective nitroxide radical (pKa=9.1 ± 0.2), which under all experimental conditions decomposes bimolecularly to yield HNO. The proposed mechanism suggests that compound I oxidizes SAHA to the respective nitroxide radical, which decomposes bimolecularly in competition with its oxidation by compound II to form HNO. Compound II also oxidizes HNO to NO and NO to nitrite. Given that NO, but not HNO, is an efficient hypoxic cell radiosensitizer, we hypothesized that under an oxidizing environment SAHA might act as a NO donor and radiosensitize hypoxic cells. Preincubation of A549 and HT29 cells with 2.5 μM SAHA for 24h resulted in a sensitizer enhancement ratio at 0.01 survival levels (SER0.01) of 1.33 and 1.59, respectively. Preincubation of A549 cells with oxidized SAHA had hardly any effect and, with 2mM valproic acid, which lacks the hydroxamate group, resulted in SER0.01=1.17. Preincubation of HT29 cells with SAHA and Tempol, which readily oxidizes HNO to NO, enhanced the radiosensitizing effect of SAHA. Pretreatment with SAHA blocked A549 cells at the G1 stage of the cell cycle and upregulated γ-H2AX after irradiation. Overall, we conclude that SAHA enhances tumor radioresponse by multiple mechanisms that might also involve its ability to serve as a NO donor under oxidizing environments.

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging

Replicative life span in Saccharomyces cerevisiae is increased by glucose (G1c) limitation [ calorie restriction (CR)] and by augmented NAD(+). Increased survival promoted by CR was attributed previously to the NAD(+)-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD(+) synthesis and salvage pathways (pnc1 Delta, npt1 Delta, and bna6 Delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1 Delta and bna6 Delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)