Clinical and experimental applications of sodium phenylbutyrate

Histone acetyltransferase and histone deacetylase are enzymes responsible for histone acetylation and deacetylation, respectively, in which the histones are acetylated and deacetylated on lysine residues in the N-terminal tail and on the surface of the nucleosome core. These processes are considered the most important epigenetic mechanisms for remodeling the chromatin structure and controlling the gene expression. Histone acetylation is associated with gene activation. Sodium phenylbutyrate is a histone deacetylase inhibitor that has been approved for treatement of urea cycle disorders and is under investigation in cancer, hemoglobinopathies, motor neuron diseases, and cystic fibrosis clinical trials. Due to its characteristics, not only of histone deacetylase inhibitor, but also of ammonia sink and chemical chaperone, the interest towards this molecule is growing worldwide. This review aims to update the current literature, involving the use of sodium phenylbutyrate in experimental studies and clinical trials

Inhibition of histone deacetylase as a treatment for cardiac hypertrophy

The present invention provides for methods of treating and preventing cardiac hypertrophy. Class II HDACs, which are known to participate in regulation of chromatin structure and gene expression, have been shown to have beneficial effects on cardiac hypertrophy. Surprisingly, the present invention demonstrates that HDAC inhibitors inhibit cardiac hypertrophy by inhibiting fetal cardiac gene expression and interfering with sarcomeric organization.Board of Regents, University of Texas Syste

The nature of the GRE influences the screening for GR-activity enhancing modulators

Glucocorticoid resistance (GCR), i.e. unresponsiveness to the beneficial anti-inflammatory activities of the glucocorticoid receptor (GR), poses a serious problem in the treatment of inflammatory diseases. One possible solution to try and overcome GCR, is to identify molecules that prevent or revert GCR by hyper-stimulating the biological activity of the GR. To this purpose, we screened for compounds that potentiate the dexamethasone (Dex)induced transcriptional activity of GR. To monitor GR transcriptional activity, the screen was performed using the lung epithelial cell line A549 in which a glucocorticoid responsive element (GRE) coupled to a luciferase reporter gene construct was stably integrated. Histone deacetylase inhibitors (HDACi) such as Vorinostat and Belinostat are two broad-spectrum HDACi that strongly increased the Dex-induced luciferase expression in our screening system. In sharp contrast herewith, results from a genome-wide transcriptome analysis of Dexinduced transcripts using RNAseq, revealed that Belinostat impairs the ability of GR to transactivate target genes. The stimulatory effect of Belinostat in the luciferase screen further depends on the nature of the reporter construct. In conclusion, a profound discrepancy was observed between HDACi effects on two different synthetic promoter-luciferase reporter systems. The favorable effect of HDACi on gene expression should be evaluated with care, when considering them as potential therapeutic agents. GEO accession number GSE96649

The Broad Spectrum HDAC Inhibitor PCI-24781 Induces Caspase- and ROS-Dependent Apoptosis and is Synergistic with Bortezomib in Lymphoma

We investigated the cytotoxicity and biology of the novel broad-spectrum hydroxamic acid-based histone deacetylase inhibitor (HDACi), PCI-24781. PCI-24781 was studied alone and combined with bortezomib in Hodgkin lymphoma (L428) and non-Hodgkin's lymphoma cell lines (Ramos, HF1, SUDHL4). PCI-24781 induced dose-dependent apoptosis that was associated with prominent G0/G1 arrest, decreased S-phase, increased p21 protein expression, and production of reactive oxygen species (ROS). Furthermore, PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781 and bortezomib exposure resulted in strong synergistic apoptosis in all cell lines (combination indices 0.19-0.6). Furthermore, compared to either agent alone, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone hyperacetylation. Microarray analyses showed that PCI-24781 alone significantly downregulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects which were enhanced further with bortezomib. RT-PCR confirmed downregulation of NF-kappaB targets NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, while gel-shift showed decreased NF-kappaB DNA-binding activity. Taken together, these results suggest that increased oxidative stress and NF-kappaB inhibition, leading to caspase activation and apoptosis, are likely responsible for the activity of PCI-24781 as well as the observed synergy with bortezomib. These data indicate that PCI-24781 has potential therapeutic value in lymphoma as a single-agent and combined with bortezomib

Epigenetic treatment of solid tumours. A review of clinical trials

Epigenetic treatment has been approved by regulatory agencies for haematological malignancies. The success observe in cutaneous lymphomas represents a proof of principle that similar results may be obtained in solid tumours. Several agents that interfere with DNA methylation-demethylation and histones acetylation/deacetylation have been studied, and some (such as azacytidine, decitabine, valproic acid and vorinostat) are already in clinical use. The aim of this review is to provide a brief overview of the molecular events underlying the antitumour effects of epigenetic treatments and to summarise data available on clinical trials that tested the use of epigenetic agents against solid tumours. We not only list results but also try to indicate how the proper evaluation of this treatment might result in a better selection of effective agents and in a more rapid development. We divided compounds in demethylating agents and HDAC inhibitors. For each class, we report the antitumour activity and the toxic side effects. When available, we describe plasma pharmacokinetics and pharmacodynamic evaluation in tumours and in surrogate tissues (generally white blood cells). Epigenetic treatment is a reality in haematological malignancies and deserves adequate attention in solid tumours. A careful consideration of available clinical data however is required for faster drug development and possibly to re-evaluate some molecules that were perhaps discarded too early

Histone deacetylase inhibitors induce invasion of human melanoma cells in vitro via differential regulation of N-cadherin expression and RhoA activity

Background: Histone deacetylase inhibitors (HDACi) exert multiple cytotoxic actions on cancer cells. Currently, different synthetic HDACi are in clinical use or clinical trials; nevertheless, since both pro-invasive and anti-invasive activities have been described, there is some controversy about the effect of HDACi on melanoma cells. Methods: Matrigel and Collagen invasion assays were performed to evaluate the effect of several HDACi (Butyrate, Trichostatin A, Valproic acid and Vorinostat) on two human melanoma cell line invasion (A375 and HT-144). The expression of N- and E-Cadherin and the activity of the RhoA GTPase were analyzed to elucidate the mechanisms involved in the HDACi activity. Results: HDACi showed a pro-invasive effect on melanoma cells in vitro. This effect was accompanied by an up-regulation of N-cadherin expression and an inhibition of RhoA activity. Moreover, the down-regulation of N-cadherin through blocking antibodies or siRNA abrogated the pro-invasive effect of the HDACi and, additionally, the inhibition of the Rho/ROCK pathway led to an increase of melanoma cell invasion similar to that observed with the HDACi treatments. Conclusion: These results suggest a role of N-cadherin and RhoA in HDACi induced invasion and call into question the suitability of some HDACi as antitumor agents for melanoma patients

Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3

The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters

Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation

AbstractCurrent antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs), such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators, provides a promising strategy to reduce if not eradicate the viral reservoir. Here, we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly, these isoform-targeted HDAC inhibitors synergize with PKC modulators, namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells.</jats:p

Resistance after chronic application of the HDAC-inhibitor valproic acid is associated with elevated Akt activation in renal cell carcinoma in vivo

Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC

Histone deacetylase 2-mediated deacetylation of the Ribonuclease 1 promoter in inflamed human endothelial cells

Endothelial cells (ECs) function as protective barrier to separate the blood from the surrounding tissue by conducting crucial roles in regulation and maintenance of vascular homeostasis, such as control of vessel permeability or coagulation. Therefore, dysfunction of the EC barrier due to inflammation, infection or injury can cause a variety of vascular pathologies, such as thrombosis or atherosclerosis. In this context, the circulating extracellular endonuclease Ribonuclease 1 (RNase1) was identified as a vessel- and tissue-protective enzyme and a potent regulator of vascular homeostasis. Upon acute inflammation, RNase1 functions as a natural counterpart to extracellular RNA (eRNA), a damage-associated molecular pattern, via degradation to protect the EC cell layer from excessive inflammation. However, long-term inflammation disrupts the RNase1-eRNA system. Thereby, eRNA accumulates in the extracellular space to induce massive proinflammatory cytokine release from circulating inflammatory cells, such as tumor necrosis factor alpha (TNF-α) or interleukin 1 beta (IL-1β). These cytokines negatively affect the EC layer by downregulation of RNase1 presumably through activation of histone deacetylases (HDACs). In this regard, this study investigated whether inflammation-mediated deacetylase function of HDACs suppresses RNase1 expression in human ECs through modulation of chromatin modifications. Proinflammatory stimulation with TNF-α or IL-1β of human umbilical vein endothelial cells significantly reduced RNase1 expression. Thus, identification of the RNASE1 promoter region and analysis of its chromatin state revealed the association of RNASE1 repression with deacetylation of histone 3 at lysine 27 and histone 4. The important role of HDACs in this process was further confirmed by administration of the specific class I HDAC1-3 inhibitor MS275 that successfully restored RNASE1 promoter acetylation and mRNA abundance upon TNF-α or IL-1β treatment. These results indicate an essential impact of HDAC1-3 in RNase1 regulation. Additionally, identification of specific HDACs involved in RNase1 regulation was obtained by chromatin immunoprecipitation kinetics confirming significant accumulation of HDAC2 at the RNASE1 promoter upon TNF-α stimulation. These findings were further validated by siRNA double knockdown of HDAC2 and its redundant enzyme HDAC1, which also recovered RNase1 mRNA abundance upon proinflammatory stimulation. In conclusion, our data identified HDAC2 as a crucial factor in RNase1 regulation in human ECs. HDAC2 is recruited to the RNASE1 promoter site to attenuate histone acetylation and suppress subsequent gene repression. This effect can be blocked by the specific HDAC inhibitor MS275 implicating the potential of HDAC inhibitors as novel therapeutic strategy to promote vascular integrity by preventing RNase1 downregulation in EC inflammation