Histone Deacetylase Gene Expression Research Articles

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

N-acetyl l-aspartate and Triacetin modulate tumor suppressor MicroRNA and class I and II HDAC gene expression induce apoptosis in Glioblastoma cancer cells in vitro

Glioblastoma multiforme (GBM), grade IV glioma and is aggressive, malignant primary brain cancer. Altered expression and activity of epigenetic proteins such as histone deacetylases (HDACs) are involved in GBM metastasis. Also, acetates are important to brain metabolites that regulate cell proliferation and apoptosis. Here, we have examined the effect of the acetates on the cell-cycle. U87MG cancer cells treated with N-acetyl l-aspartate (NAA) and sodium acetate have exhibited G1 phase cell-cycle arrest whereas U87MG cells treated with Triacetin (TA), and potassium acetate has induced G2/M cell cycle arrest. We have observed inhibition of histone deacetylase (HDAC) mRNA levels in acetate treated U87MG cells. Interestingly, acetates-treated U87MG cells have shown a significant reduction in the mRNA level of class II HDACs than class I HDACs. Acetate treated cells have exhibited an enhanced expression of various microRNAs such as miR-15b, miR-92, miR-101, miR-155, miR-199, miR-200, miR-223, miR-16, and miR-17 that are involved in the inhibition of cancer cell proliferation, invasion, migration, and angiogenesis. Further, these acetate molecules regulate genes involved in mammalian target of rapamycin complex 2 (mTORC2) such as mammalian stress-activated protein kinase-interacting protein (mSIN1), protein observed with Rictor 2 (Protor 2), and protein kinase C α (PKCα). The present study reveals the possible involvement of the mTORC2 complex during acetate-mediated HDAC inhibition, as well as microRNA modulation. Furthermore, molecular modeling studies were employed to understand the binding mode of these acetate molecules to mTOR, Rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), and HDAC-8 proteins. Thus in this study, we have identified the pivotal role of acetates in the modulation of mTOR complex, epigenetic genes and provide structural as well as functional insights that will help in future drug discovery against GBM cancer therapy.

In silico analysis of maize HDACs with an emphasis on their response to biotic and abiotic stresses.

Histone deacetylases (HDACs) are key epigenetic factors in regulating chromatin structure and gene expression in multiple aspects of plant growth, development, and response to abiotic or biotic stresses. Many studies on systematic analysis and molecular function of HDACs in Arabidopsis and rice have been conducted. However, systematic analysis of HDAC gene family and gene expression in response to abiotic and biotic stresses has not yet been reported. In this study, a systematic analysis of the HDAC gene family in maize was performed and 18 ZmHDACs distributed on nine chromosomes were identified. Phylogenetic analysis of ZmHDACs showed that this gene family could be divided into RPD3/HDA1, SIR2, and HD2 groups. Tissue-specific expression results revealed that ZmHDACs exhibited diverse expression patterns in different tissues, indicating that these genes might have diversified functions in growth and development. Expression pattern of ZmHDACs in hormone treatment and inoculation experiment suggested that several ZmHDACs might be involved in jasmonic acid or salicylic acid signaling pathway and defense response. Interestingly, HDAC genes were downregulated under heat stress, and immunoblotting results demonstrated that histones H3K9ac and H4K5ac levels were increased under heat stress. These results provide insights into ZmHDACs, which could help to reveal their functions in controlling maize development and responses to abiotic or biotic stresses.

Molecular Mechanism of the Protective Effect of Zerumbone on Lipopolysaccharide-Induced Inflammation of THP-1 Cell-Derived Macrophages

Unregulated inflammatory responses lead to massive production of macrophages-activating inflammatory cytokines and chemokines, which may induce diabetes, cancer, and atherosclerosis. Macrophages differentiated from human monocyte (THP-1) have been extensively used in in vitro inflammation models in recent studies. Zerumbone is a major component of the essential oil of Zingiber zerumbet Smith, a type of wild ginger. In this study, we investigated the effects of zerumbone on the secretion of pro-inflammatory cytokines and its underlying mechanistic regulation in lipopolysaccharide (LPS)-activated inflammation of THP-1 cell-derived macrophages. Nuclear factor (NF)-κB and toll-like receptors (TLRs) are known to play important roles in inflammation and immunity. If pathogens enter the host, TLRs recognize the pathogens and signal for the activation of NF-κB to induce inflammatory gene products, such as cytokines. We demonstrated that zerumbone inhibits the secretion of pro-inflammatory cytokines and the induction of NF-κB p65 in LPS-activated inflammation of THP-1 cell-derived macrophages. In addition, zerumbone significantly inhibited mRNA and protein levels of TLR-2/4, and the expression of myeloid differentiation factor 88 (MyD88) adaptor proteins in the LPS-activated inflammation of THP-1 cell-derived macrophages. Moreover, we showed that zerumbone (1-10 μM) regulated histone deacetylase (HDAC) activity and the expression of HDAC genes. H3K9ac, H3K27ac, and H3K4me2 are inducible histone marks that activate gene expression. Treatment with LPS upregulated H3K9ac, H3K27ac, and H3K4me2 in THP-1 cell-derived macrophages; however, this upregulation was decreased by zerumbone treatment. Therefore, these results provide evidence that zerumbone may have therapeutic benefits for chronic inflammatory diseases.

Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque.

PurposeEpigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronie's disease (PD) or normal tunica albuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque.Materials and MethodsFor differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation.ResultsThe mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein.ConclusionsThese findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

Cationic lipid-based nanoparticles mediate functional delivery of acetate to tumor cells in vivo leading to significant anticancer effects.

Metabolic reengineering using nanoparticle delivery represents an innovative therapeutic approach to normalizing the deregulation of cellular metabolism underlying many diseases, including cancer. Here, we demonstrated a unique and novel application to the treatment of malignancy using a short-chain fatty acid (SCFA)-encapsulated lipid-based delivery system – liposome-encapsulated acetate nanoparticles for cancer applications (LITA-CAN). We assessed chronic in vivo administration of our nanoparticle in three separate murine models of colorectal cancer. We demonstrated a substantial reduction in tumor growth in the xenograft model of colorectal cancer cell lines HT-29, HCT-116 p53+/+ and HCT-116 p53−/−. Nanoparticle-induced reductions in histone deacetylase gene expression indicated a potential mechanism for these anti-proliferative effects. Together, these results indicated that LITA-CAN could be used as an effective direct or adjunct therapy to treat malignant transformation in vivo.

Epigenetic Regulation of Matrix Metalloproteinase-1 and -3 Expression in Mycobacterium tuberculosis Infection.

In pulmonary tuberculosis (TB), the inflammatory immune response against Mycobacterium tuberculosis (Mtb) is associated with tissue destruction and cavitation, which drives disease transmission, chronic lung disease, and mortality. Matrix metalloproteinase (MMP)-1 is a host enzyme critical for the development of cavitation. MMP expression has been shown to be epigenetically regulated in other inflammatory diseases, but the importance of such mechanisms in Mtb-associated induction of MMP-1 is unknown. We investigated the role of changes in histone acetylation in Mtb-induced MMP expression using inhibitors of histone deacetylases (HDACs) and histone acetyltransferases (HAT), HDAC siRNA, promoter-reporter constructs, and chromatin immunoprecipitation assays. Mtb infection decreased Class I HDAC gene expression by over 50% in primary human monocyte-derived macrophages but not in normal human bronchial epithelial cells (NHBEs). Non-selective inhibition of HDAC activity decreased MMP-1/-3 expression by Mtb-stimulated macrophages and NHBEs, while class I HDAC inhibition increased MMP-1 secretion by Mtb-stimulated NHBEs. MMP-3 expression, but not MMP-1, was downregulated by siRNA silencing of HDAC1. Inhibition of HAT activity also significantly decreased MMP-1/-3 secretion by Mtb-infected macrophages. The MMP-1 promoter region between −2,001 and −2,942 base pairs from the transcriptional start site was key in control of Mtb-driven MMP-1 gene expression. Histone H3 and H4 acetylation and RNA Pol II binding in the MMP-1 promoter region were increased in stimulated NHBEs. In summary, epigenetic modification of histone acetylation via HDAC and HAT activity has a key regulatory role in Mtb-dependent gene expression and secretion of MMP-1 and -3, enzymes which drive human immunopathology. Manipulation of epigenetic regulatory mechanisms may have potential as a host-directed therapy to improve outcomes in the era of rising TB drug resistance.

Epigenetic Regulation of Tumor Necrosis Factor (Tnf) in Skeletal Muscle by Chronic Caloric Restriction

Caloric restriction (CR), without malnutrition, is considered an effective dietary intervention to extend lifespan and quality of life. Such effects include those related to musculoskeletal health where chronic CR restores the phenotype of aged individuals to that of the young. We demonstrated that CR reverted the effect of a high fat diet and effectively reducing body mass. In skeletal muscle of the CR group, nutrient sensing and degradation pathways were activated. This could impact the canonical inflammatory marker TNF and related pathways, which are involved in the metabolism and physiology of the muscle. However, the epigenetic mechanisms elicited by CR on muscle inflammation remain elusive. In our study, 4‐week old male Sprague‐Dawley rats were fed a high fat diet (HF, 45% Kcal from fat) ad libitum for 4 months. Animals were then divided into HF ad libitum, maintenance group fed a control diet (ML, with 100% energy requirement at baseline adjusted with body weight), and the caloric restriction group (CR, 25% energy reduction). A control group (AL) was fed a control diet ad libitum. The dietary intervention continued for six months before animals were sacrificed and skeletal muscle samples were collected for gene expression and epigenetic analyses. In the CR group, gene expression was increased for the p50 subunit of the inflammatory marker Nfκb, but a marked decrease in gene expression occurred for Ikbkɛ, Tnf, Ifnγ, Il‐1b and Il‐6 compared to both the HF and ML groups. Gene expression of DNA methyltransferase Dnmt1 was reduced by CR to the level similar to AL, whilst Dnmt3a was increased in ML and CR groups which were both previously exposed to HF. Moreover, gene expression of histone deacetylases (Hdac1, Hdac3), and H3K4 methyltransferase (Kmt2b) were upregulated in ML and CR groups, eliciting a lasting HF effect to part of the histone modifying machinery. On the other hand, histone methylases Suv39h1 and Ezh2 were downregulated by CR. CpG density analysis for the Tnf rat gene revealed two CpG‐rich regions within the gene body in intron 1–2 (182 bp) and exon 4 (146 bp), as well as two highly dense CpG islands at the 3′‐downstream region (100 bp and 480 bp). Further analysis was performed to confirm the methylation and histone modification status within these regions. The findings strongly indicate that chronic CR regulates myogenesis and wasting through transcriptional targeting of Tnf by recruiting DNA methyltranferases and histone modifiers at different regions with the TNF locus.

A seasonal switch in histone deacetylase gene expression in the hypothalamus and their capacity to modulate nuclear signaling pathways

Seasonal animals undergo changes in physiology and behavior between summer and winter conditions. These changes are in part driven by a switch in a series of hypothalamic genes under transcriptional control by hormones and, of recent interest, inflammatory factors. Crucial to the control of transcription are histone deacetylases (HDACs), generally acting to repress transcription by local histone modification. Seasonal changes in hypothalamic HDAC transcripts were investigated in photoperiod-sensitive F344 rats by altering the day-length (photoperiod). HDAC4, 6 and 9 were found to change in expression. The potential influence of HDACs on two hypothalamic signaling pathways that regulate transcription, inflammatory and nuclear receptor signaling, was investigated. For inflammatory signaling the focus was on NF-κB because of the novel finding made that its expression is seasonally regulated in the rat hypothalamus. For nuclear receptor signaling it was discovered that expression of retinoic acid receptor beta was regulated seasonally. HDAC modulation of NF-κB-induced pathways was examined in a hypothalamic neuronal cell line and primary hypothalamic tanycytes. HDAC4/5/6 inhibition altered the control of gene expression (Fos, Prkca, Prkcd and Ptp1b) by inducers of NF-κB that activate inflammation. These inhibitors also modified the action of nuclear receptor ligands thyroid hormone and retinoic acid. Thus seasonal changes in HDAC4 and 6 have the potential to epigenetically modify multiple gene regulatory pathways in the hypothalamus that could act to limit inflammatory pathways in the hypothalamus during long-day summer-like conditions.

Nalmefene is effective at reducing alcohol seeking, treating alcohol-cocaine interactions and reducing alcohol-induced histone deacetylases gene expression in blood.

The opioid antagonist nalmefene (selincro®) was approved for alcohol-related disorders by the European Medicines Agency in 2013. However, there have been no studies regarding the effectiveness of nalmefene when alcohol is used in combination with cocaine. Using operant alcohol self-administration in Wistar rats and qRT-PCR, we evaluated (i) the dose-response curve for s.c. and p.o. nalmefene; (ii) the effects of nalmefene with increasing concentrations of alcohol; (iii) the efficacy of nalmefene on cocaine-potentiated alcohol responding; and (iv) the gene expression profiles of histone deacetylases (Hdac1-11) in peripheral blood in vivo and in the prefrontal cortex, heart, liver and kidney post mortem. S.c. (0.01, 0.05, 0.1mg·kg(-1) ) and p.o. (10, 20, 40mg·kg(-1) ) nalmefene dose-dependently reduced alcohol-reinforced responding by up to 50.3%. This effect of nalmefene was not dependent on alcohol concentration (10, 15, 20%). Cocaine potentiated alcohol responding by approximately 40% and nalmefene (0.05mg·kg(-1) ) reversed this effect of cocaine. Alcohol increased Hdac gene expression in blood and nalmefene prevented the increases in Hdacs 3, 8, 5, 7, 9, 6 and 10. In the other tissues, alcohol and nalmefene either did not alter the gene expression of Hdacs, as in the prefrontal cortex, or a tissue-Hdac-specific effect was observed. Nalmefene might be effective as a treatment for alcohol-dependent patients who also use cocaine. Also, the expression of Hdacs in peripheral blood might be useful as a biomarker of alcohol use and drug response.

Histone deactylase gene expression profiles are associated with outcomes in blunt trauma patients.

Treatment with histone deacetylase (HDAC) inhibitors, such as valproic acid, increases survival in animal models of trauma and sepsis. Valproic acid is a pan-inhibitor that blocks most of the known HDAC isoforms. Targeting individual HDAC isoforms may increase survival and reduce complications, but little is known of the natural history of HDAC gene expression following trauma. We hypothesized that distinct HDAC isoform gene expression patterns would be associated with differences in outcomes following trauma. Twenty-eight-day longitudinal HDAC leukocyte gene expression profiles in 172 blunt trauma patients were extracted from the Inflammation and the Host Response to Injury (Glue Grant) data set. Outcome was classified as complicated (death or no recovery by Day 28, n = 51) or uncomplicated (n = 121). Mixed modeling was used to compare the HDAC expression trajectories between the groups, corrected for Injury Severity Score (ISS), base deficit, and volume of blood products transfused during the initial 12 hours following admission. Weighted gene correlation network analysis identified modules of genes with significant coexpression, and HDAC genes were mapped to these modules. Biologic function of these modules was investigated using the Gene Ontology database. Elevated longitudinal HDAC expression trajectories for HDAC1, HDAC3, HDAC6, and HDAC11 were associated with complicated outcomes. In contrast, suppressed expression of Sirtuin 3 (SIRT3) was associated with adverse outcome (p < 0.01). Weighted gene correlation network analysis identified significant coexpression of HDAC and SIRT genes with genes involved in ribosomal function and down-regulation of protein translation in response to stress (HDAC1), T-cell signaling, and T-cell selection (HDAC3) as well as coagulation and hemostasis (SIRT3). No coexpression of HDAC11 was identified. Expression trajectories of HDAC1, HDAC3, HDAC6, HDAC11, and SIRT3 correlate with outcomes following trauma and may potentially serve as biomarkers. They may also be promising targets for pharmacologic intervention. The effects of HDAC and SIRT gene expression in trauma may be mediated through pathways involved in ribosomal and T-cell function as well as coagulation and hemostasis. Prognostic study, level III.

Methylation and gene expression of histone deacetylases 6 in systemic lupus erythematosus.

The purpose of this study is to investigate the role of methylation in the histone deacetylases 6 (HDAC6) promoter and HDAC6 messenger RNA (mRNA) expression in the pathogenesis of systemic lupus erythematosus (SLE). Direct bisulfite-polymerase chain reaction (PCR) sequencing was performed to detect the HDAC6 promoter methylation in 33 patients with SLE and 35 healthy controls. The HDAC6 mRNA expression was measured in 93 SLE patients and 84 healthy controls by using the method of quantitative real-time PCR. This study demonstrated that the methylation rates at HDAC6-680, -660 and -658 were significantly increased in the SLE patients compared with healthy controls (P = 0.041, 0.034 and 0.029, respectively). The SLE patients also had lower HDAC6 mRNA expression than the controls (P = 0.031). However, there was no significant difference in HDAC6 mRNA expression between patients with active and inactive SLE. The SLE patients had higher methylation in the HDAC6 promoter and lower HDAC6 mRNA expression than the controls. These changes may be related to the susceptibility of SLE. However, they are not associated with the disease activity of SLE.

Differential expression of HDAC and HAT genes in atrophying skeletal muscle.

Histone deacetylase (HDAC) proteins, which counter the activity of histone acetyltransferases (HATs), are necessary for normal muscle atrophy in response to several pathophysiological conditions. Despite this, it remains unknown whether a common or unique transcriptional profile of HDAC and HAT genes exist during the progression of muscle atrophy. Muscles were harvested from cast immobilized, denervated, or nutrient deprived animals for quantitative reverse transcriptase-polymerase chain reaction analysis of HDAC and HAT gene expression. The mRNA levels of Hdac2, Hdac4, Hdac6, Sirt1, p300, Cbp, and Pcaf increased, and Hdac7 decreased in skeletal muscle in each experimental model of muscle atrophy. Hdac1 and Hdac3 were increased only in cast immobilized and denervated muscles. While specific HDACs and HATs are increased in multiple models of muscle atrophy, increased expression of class I HDACs was unique to muscle disuse, reinforcing that specific HDAC inhibitors may be more effective than pan-HDAC inhibitors at countering muscle atrophy.

Histone Deacetylase Gene Expression Following Binge Alcohol Consumption in Rats and Humans.

Alcohol binge drinking is one of the most common patterns of excessive alcohol use and recent data would suggest that histone deacetylases (HDACs) gene expression profiling could be useful as a biomarker for psychiatric disorders. This study aimed to characterize the gene expression patterns of Hdac 1-11 in samples of rat peripheral blood, liver, heart, prefrontal cortex, and amygdala following repeated binge alcohol consumption and to determine the parallelism of Hdac gene expression between rats and humans in peripheral blood. To accomplish this goal, we examined Hdac gene expression following 1, 4, or 8 alcohol binges (3g/kg, orally) in the rat, in patients who were admitted to the hospital emergency department for acute alcohol intoxication, and in rats trained in daily operant alcohol self-administration. We primarily found that acute alcohol binging reduced gene expression (Hdac1-10) in the peripheral blood of alcohol-naïve rats and that this effect was attenuated following repeated alcohol binges. There was also a reduction of Hdac gene expression in the liver (Hdac2,4,5), whereas there was increased expression in the heart (Hdac1,7,8) and amygdala (Hdac1,2,5). Additionally, increased blood alcohol concentrations were measured in rat blood at 1 to 4hours following repeated alcohol binging, and the only group that developed hepatic steotosis (fatty liver) were those animals exposed to 8 alcohol binge events. Finally, both binge consumption of alcohol in humans and daily operant alcohol self-administration in rats increased Hdac gene expression in peripheral blood. Our results suggest that increases in HDAC gene expression within the peripheral blood are associated with chronic alcohol consumption, whereas HDAC gene expression is reduced following initial exposure to alcohol.

Analysis of class I and II histone deacetylase gene expression in human leukemia

Histone deacetylase (HDAC) inhibitors are well-characterized anti-leukemia agents and HDAC gene expression deregulation has been reported in various types of cancers. This study sought to characterize HDAC gene expression patterns in several types of leukemia. To do so, a systematic study was performed of the mRNA expression of all drug-targetable HDACs for which reagents were available. This was done by real-time PCR in 24 leukemia cell lines and 39 leukemia patients, which included AML, MDS and CLL patients, some of whom received HDAC inhibitor treatment. Among the samples analyzed, there was no discernible pattern in HDAC expression. HDAC expression was generally increased in CLL patients, except for HDAC2 and HDAC4. HDAC expression was also generally increased in VPA-treated MOLT4 cells. However, this increased expression was not seen in AML patients treated with vorinostat. In summary, increased HDAC expression was noted in CLL patients in general, but the HDAC expression patterns in myeloid malignancies appear to be heterogeneous, which implies that the role of HDACs in leukemia may be related to global expression or protein function rather than specific expression patterns.

Further characterization of HDAC and SIRT gene expression patterns in pancreatic cancer and their relation to disease outcome.

Ductal adenocarcinoma of the pancreas is ranking 4 for patient' death from malignant disease in Western countries, with no satisfactory treatment. We re-examined more precisely the histone deacetylases (HDAC) and Sirtuin (SIRT) gene expression patterns in pancreatic cancer with more pancreatic tumors and normal tissues. We also examinedthe possible relationship between HDAC gene expression levels and long term disease outcome. Moreover, we have evaluated by using an in vitro model system of human pancreatic tumor cell line whether HDAC7 knockdown may affect the cell behavior. We analyzed 29 pancreatic adenocarcinoma (PA), 9 chronic pancreatitis (CP), 8 benign pancreatic (BP) and 11 normal pancreatic tissues. Concerning pancreatic adenocarcinoma, we were able to collect biopsies at the tumor periphery. To assess the possible involvement of HDAC7 in cell proliferation capacity, we have generated recombinant human Panc-1 tumor which underexpressed or overexpressed HDAC7. The expression of HDAC1,2,3,4,7 and Nur77 increased in PA samples at levels significantly higher than those observed in the CP group (p = 0.0160; 0.0114; 0.0227; 0.0440; 0.0136; 0.0004, respectively). The expression of HDAC7, was significantly greater in the PA compared with BP tissue samples (p = 0.05). Mean mRNA transcription levels of PA for HDAC7 and HDAC2 were higher when compared to their counterpart biopsies taken at the tumor periphery (p = 0.0346, 0.0053, respectively). Moreover, the data obtained using confocal microscopy and a quantitative method of immunofluorescence staining strongly support the HDAC7 overexpression in PA surgical specimens. The number of deaths and recurrences at the end of follow up were significantly greater in patients with overexpression of HDAC7. Interestingly, the rate of growth was significantly reduced in the case of cell carrying shRNA construct targeting HDAC7 encoding gene when compared to the parental Panc-1 tumor cells (p = 0.0015) at 48 h and 96 h (p = 0.0021). This study strongly support the notion that HDAC7play a role in pancreatic adenocarcinoma progression.

Activity and expression of histone acetylases and deacetylases in inflammatory phenotypes of asthma

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate gene expression, yet differences in the activity of these enzymes in the inflammatory phenotypes of asthma are unknown. We hypothesized that neutrophilic asthma (NA) would be associated with increased HAT and decreased HDAC activity. To investigate total HAT/HDAC activity and gene expression in isolated blood monocytes and sputum macrophages from healthy and patients with asthma. Peripheral blood and induced sputum were collected from adults with asthma (n = 52) and healthy controls (n = 9). Sputum inflammatory cell counts were performed and asthma inflammatory phenotypes were classified according to sputum eosinophil and neutrophil cut-off's of > 3% and > 61% respectively. Peripheral blood monocytes were isolated (n = 61) and sputum macrophages were isolated from a subgroup of patients with asthma (n = 14), using immunomagnetic cell separation. RNA and nuclear proteins were extracted and quantified. Enzyme activity was assessed using fluorescent assays and gene expression of EP300, KAT2B, CREBBP, and HDACs 1, 2 and 3 were measured by qPCR. There was a significant inverse association between blood monocyte HAT and HDAC activity (r = -0.58, P < 0.001). NA was associated with increased blood monocyte HAT enzyme activity (P = 0.02), decreased HDAC activity (P = 0.03), and increased HAT: HDAC ratio (P < 0.01) compared with eosinophilic asthma. There were no differences in gene expression of EP300, KAT2B, CREBBP, or HDACs 1, 2 and 3 in blood monocytes from subjects with asthma or inflammatory phenotypes of asthma. There was no effect of inhaled corticosteroid use, poor asthma control, or asthma severity on HAT/HDAC activities. Sputum macrophages had increased expression of KAT2B in eosinophilic compared with paucigranulocytic asthma. Neutrophilic airway inflammation is associated with increased HAT and reduced HDAC activity in blood monocytes, demonstrating further systemic manifestations relating to the altered inflammatory gene transcription profile of neutrophilic asthma.

Rapid changes in histone deacetylases and inflammatory gene expression in expert meditators

A growing body of research shows that mindfulness meditation can alter neural, behavioral and biochemical processes. However, the mechanisms responsible for such clinically relevant effects remain elusive. Here we explored the impact of a day of intensive practice of mindfulness meditation in experienced subjects (n=19) on the expression of circadian, chromatin modulatory and inflammatory genes in peripheral blood mononuclear cells (PBMC). In parallel, we analyzed a control group of subjects with no meditation experience who engaged in leisure activities in the same environment (n=21). PBMC from all participants were obtained before (t1) and after (t2) the intervention (t2-t1=8h) and gene expression was analyzed using custom pathway focused quantitative-real time PCR assays. Both groups were also presented with the Trier Social Stress Test (TSST). Core clock gene expression at baseline (t1) was similar between groups and their rhythmicity was not influenced in meditators by the intensive day of practice. Similarly, we found that all the epigenetic regulatory enzymes and inflammatory genes analyzed exhibited similar basal expression levels in the two groups. In contrast, after the brief intervention we detected reduced expression of histone deacetylase genes (HDAC 2, 3 and 9), alterations in global modification of histones (H4ac; H3K4me3) and decreased expression of pro-inflammatory genes (RIPK2 and COX2) in meditators compared with controls. We found that the expression of RIPK2 and HDAC2 genes was associated with a faster cortisol recovery to the TSST in both groups. The regulation of HDACs and inflammatory pathways may represent some of the mechanisms underlying the therapeutic potential of mindfulness-based interventions. Our findings set the foundation for future studies to further assess meditation strategies for the treatment of chronic inflammatory conditions.

Valproic acid, but not levetiracetam, selectively decreases HDAC7 and HDAC2 expression in human ovarian cancer cells

Histone deacetylases (HDACs) are often overexpressed in cancer cells, leading to altered expression and activity of numerous proteins involved in carcinogenesis. Recent evidence suggests that expression of class I HDACs is increased in ovarian carcinomas and plays a significant role in carcinogenesis and resistance to chemotherapeutic agents. Two compounds, valproic acid (VPA) and levetiracetam (LEV), exhibit HDAC inhibitor (HDACI) activity in various cell types, but data concerning their activity in ovarian cancer are lacking. Here we compared the effects of VPA and LEV as HDACIs, using a human ovarian cancer cell line, OVCAR-3. Cells were cultured with VPA or LEV at concentrations between 1 and 10mM for 1–24h. HDAC activity was determined by fluorometric assay and confirmed by western blotting. Expression of HDAC genes was determined by real-time PCR and HDAC proteins expression was evaluated by western blotting. Additionally, we used high-performance liquid chromatography to determine whether OVCAR-3 cells can metabolize LEV to its major metabolite, 2-pyrrolidinone-n-butyric acid (PBA), which might exert HDACI activity. LEV, however, had no apparent effect on HDAC activity, or gene and protein expression. The OVCAR-3 cell line was able to metabolize LEV to PBA, but the effect was small. Our observations suggest that VPA should be considered as a possible adjunctive drug in ovarian cancer treatment.

Class II HDAC Inhibition Hampers Hepatic Stellate Cell Activation by Induction of MicroRNA-29

BackgroundThe conversion of a quiescent vitamin A storing hepatic stellate cell (HSC) to a matrix producing, contractile myofibroblast-like activated HSC is a key event in the onset of liver disease following injury of any aetiology. Previous studies have shown that class I histone deacetylases (HDACs) are involved in the phenotypical changes occurring during stellate cell activation in liver and pancreas.AimsIn the current study we investigate the role of class II HDACs during HSC activation.MethodsWe characterized the expression of the class II HDACs freshly isolated mouse HSCs. We inhibited HDAC activity by selective pharmacological inhibition with MC1568, and by repressing class II HDAC gene expression using specific siRNAs.ResultsInhibition of HDAC activity leads to a strong reduction of HSC activation markers α-SMA, lysyl oxidase and collagens as well as an inhibition of cell proliferation. Knock down experiments showed that HDAC4 contributes to HSC activation by regulating lysyl oxidase expression. In addition, we observed a strong up regulation of miR-29, a well-known anti-fibrotic miR, upon treatment with MC1568. Our in vivo work suggests that a successful inhibition of class II HDACs could be promising for development of future anti-fibrotic compounds.ConclusionsIn conclusion, the use of MC1568 has enabled us to identify a role for class II HDACs regulating miR-29 during HSC activation.