Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.     

Histone deacetylase 3 mediates allergic skin inflammation by regulating expression of MCP1 protein

We have shown the induction of histone deacetylase 3 (HDAC3) in antigen-stimulated rat basophilic leukemia cells via NF-κB. We investigated the role of HDAC3 in allergic skin inflammation. We used a BALB/c mouse model of triphasic cutaneous anaphylaxis (triphasic cutaneous reaction; TpCR) and passive cutaneous anaphylaxis (PCA) to examine the role of HDAC3 in allergic skin inflammation. Triphasic cutaneous reaction involved induction of HDAC3 and was mediated by HDAC3. HDAC3 showed an interaction with FcεRIβ. Trichostatin A (TSA), an inhibitor of HDAC(s), disrupted this interaction. Cytokine array analysis showed that the down-regulation of HDAC3 led to the decreased secretion of monocyte chemoattractant protein 1 (MCP1). FcεRI was necessary for induction of HDAC3 and MCP1. ChIP assays showed that HDAC3, in association with Sp1 and c-Jun, was responsible for induction of MCP1 expression. TSA exerted a negative effect on induction of MCP1. HDAC3 exerted a negative regulation on expression of HDAC2 via interaction with Rac1. The down-regulation of HDAC3 or inactivation of Rac1 induced binding of HDAC2 to MCP1 promoter sequences. TSA exerted a negative effect on HDAC3-mediated TpCR. The BALB/c mouse model of PCA involved induction of HDAC3 and MCP1. HDAC3 and MCP1 were necessary for PCA that involved ear swelling, enhanced vascular permeability, and angiogenesis. Recombinant MCP1 enhanced β-hexosaminidase activity and histamine release and also showed angiogenic potential. TSA exerted a negative effect on PCA. Our data show HDAC3 as a valuable target for the development of allergic skin inflammation therapeutics.     

Dual targeting of epigenetic therapy in cancer

Aberrant epigenetic silencing of tumor suppressor genes by promoter DNA hypermethylation and histone deacetylation plays an important role in the pathogenesis of cancer. The potential reversibility of epigenetic abnormalities encouraged the development of pharmacologic inhibitors of DNA methylation and histone deacetylation as anti-cancer therapeutics. (Pre)clinical studies of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have yielded encouraging results, especially against hematologic malignancies. Recently, several studies demonstrated that DNMT and HDAC inhibitors are also potent angiostatic agents, inhibiting (tumor) endothelial cells and angiogenesis in vitro and in vivo. By reactivation of epigenetically silenced tumor suppressor genes with angiogenesis inhibiting properties, DNMT and HDAC inhibitors might indirectly - via their effects on tumor cells - decrease tumor angiogenesis in vivo. However, this does not explain the direct angiostatic effects of these agents, which can be unraveled by gene expression studies and examination of epigenetic promoter modifications in endothelial cells treated with DNMT and HDAC inhibitors. Clearly, the dual targeting of epigenetic therapy on both tumor cells and tumor vasculature makes them attractive combinatorial anti-tumor therapeutics. Here we review the therapeutic potential of DNMT and HDAC inhibitors as anti-cancer drugs, as evaluated in clinical trials, and their angiostatic activities, apart from their inhibitory effects on tumor cells.     

Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.     

Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6.PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6.PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC.phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.     

Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichostatin A and repression by HDAC3

Histone deacetylase (HDAC) inhibitors inhibit the proliferation of transformed cells in vitro, restrain tumor growth in animals, and are currently being actively exploited as potential anticancer agents. To identify gene targets of the HDAC inhibitor trichostatin A (TSA), we compared the gene expression profiles of BALB/c-3T3 cells treated with or without TSA. Our results show that TSA up-regulates the expression of the gene encoding growth-differentiation factor 11 (Gdf11), a transforming growth factor beta family member that inhibits cell proliferation. Detailed analyses indicated that TSA activates the gdf11 promoter through a conserved CCAAT box element. A comprehensive survey of human HDACs revealed that HDAC3 is necessary and sufficient for the repression of gdf11 promoter activity. Chromatin immunoprecipitation assays showed that treatment of cells with TSA or silencing of HDAC3 expression by small interfering RNA causes the hyperacetylation of Lys-9 in histone H3 on the gdf11 promoter. Together, our results provide a new model in which HDAC inhibitors reverse abnormal cell growth by inactivation of HDAC3, which in turn leads to the derepression of gdf11 expression.     

Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.     

Histone Deacetylase Classes I and II Regulate Kaposi's Sarcoma-Associated Herpesvirus Reactivation

In primary effusion lymphoma (PEL) cells infected with latent Kaposi''s sarcoma-associated herpesvirus (KSHV), the promoter of the viral lytic switch gene, Rta, is organized into bivalent chromatin, similar to cellular developmental switch genes. Histone deacetylase (HDAC) inhibitors (HDACis) reactivate latent KSHV and dramatically remodel the viral genome topology and chromatin architecture. However, reactivation is not uniform across a population of infected cells. We sought to identify an HDACi cocktail that would uniformly reactivate KSHV and reveal the regulatory HDACs. Using HDACis with various specificities, we found that class I HDACis were sufficient to reactivate the virus but differed in potency. Valproic acid (VPA) was the most effective HDACi, inducing lytic cycle gene expression in 75% of cells, while trichostatin A (TSA) induced less widespread lytic gene expression and inhibited VPA-stimulated reactivation. VPA was only slightly superior to TSA in inducing histone acetylation of Rta''s promoter, but only VPA induced significant production of infectious virus, suggesting that HDAC regulation after Rta expression has a dramatic effect on reactivation progression. Ectopic HDACs 1, 3, and 6 inhibited TPA-stimulated KSHV reactivation. Surprisingly, ectopic HDACs 1 and 6 stimulated reactivation independently, suggesting that the stoichiometries of HDAC complexes are critical for the switch. Tubacin, a specific inhibitor of the ubiquitin-binding, proautophagic HDAC6, also inhibited VPA-stimulated reactivation. Immunofluorescence indicated that HDAC6 is expressed diffusely throughout latently infected cells, but its expression level and nuclear localization is increased during reactivation. Overall, our data suggest that inhibition of HDAC classes I and IIa and maintenance of HDAC6 (IIb) activity are required for optimal KSHV reactivation.