apicidin and entinostat

Histone deacetylase inhibitors (HDACi) are promising antineoplastic agents, but their precise mechanisms of actions are not well understood. In particular, the relevance of p53 for HDACi-induced effects has not been fully elucidated. We investigated the anticancer effects of four structurally distinct HDACi, vorinostat, entinostat, apicidin and valproic acid, using isogenic HCT-116 colon cancer cell lines differing in p53 status.. Effects were assessed by MTT assay, flow-cytometric analyses of propidium iodide uptake, mitochondrial depolarisation and cell-cycle distribution, as well as by gene expression profiling.. Vorinostat was equally effective in p53 wild-type and null cells, whereas entinostat was less effective in p53 null cells. Histone deacetylase inhibitors treatment suppressed the expression of MDM2 and increased the abundance of p53. Combination treatments showed that vorinostat enhanced the cytotoxic activity of TRAIL and bortezomib, independent of the cellular p53 status. Investigations into the effects of an inhibitor of the sirtuin class of HDAC, tenovin-1, revealed that tenovin-1-mediated cell death hinged on p53.. These results demonstrate that vorinostat activates p53, but does not require p53 for inducing its anticancer action. Yet they also demonstrate that entinostat-induced cytotoxic effects partially depend on p53, indicating that different HDACi have a different requirement for p53.

Topics: Benzamides; Cell Proliferation; Colonic Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Peptides, Cyclic; Proto-Oncogene Proteins c-mdm2; Pyridines; Tumor Suppressor Protein p53; Valproic Acid; Vorinostat

The current paradigm stipulates that inhibition of histone deacetylase (HDAC) 6 is essential for the combinatorial effect of proteasome and HDAC inhibitors for the treatment of cancers. Our study aims to investigate the effect of combining different class I HDAC inhibitors (without HDAC6 action) with a proteasome inhibitor on apoptosis of nasopharyngeal carcinoma (NPC). We found that combination of a proteasome inhibitor, bortezomib, and several class I HDAC inhibitors, including MS-275, apicidin and romidepsin, potently induced killing of NPC cells both in vitro and in vivo. Among the drug pairs, combination of bortezomib and romidepsin (bort/romidepsin) was the most potent and could induce apoptosis at low nanomolar concentrations. The apoptosis of NPC cells was reactive oxygen species (ROS)- and caspase-dependent but was independent of HDAC6 inhibition. Of note, bort/romidepsin might directly suppress the formation of aggresome through the downregulation of c-myc. In addition, two markers of endoplasmic reticulum (ER) stress-induced apoptosis, ATF-4 and CHOP/GADD153, were upregulated, whereas a specific inhibitor of caspase-4 (an initiator of ER stress-induced apoptosis) could suppress the apoptosis. When ROS level in the NPC cells was reduced to the untreated level, ER stress-induced caspase activation was abrogated. Collectively, our data demonstrate a model of synergism between proteasome and class I HDAC inhibitors in the induction of ROS-dependent ER stress-induced apoptosis of NPC cells, independent of HDAC6 inhibition, and provide the rationale to combine the more specific and potent class I HDAC inhibitors with proteasome inhibitors for the treatment of cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Boronic Acids; Bortezomib; Carcinoma; Caspases; Cell Proliferation; Depsipeptides; DNA Damage; Endoplasmic Reticulum Stress; Enzyme Activation; Female; Fluoresceins; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Peptides, Cyclic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Pyridines; Reactive Oxygen Species

Nutlin-3, a small-molecule MDM2 inhibitor, restores p53 function and is, thus, an appealing candidate for the treatment of cancers retaining wild-type p53. However, nutlin-3 applied as single agent may be insufficient for cancer therapy. Therefore, we explored whether the anticancer activity of nutlin-3 could be enhanced by combination with histone deacetylase inhibitors (HDACi), i.e. vorinostat, sodium butyrate, MS-275 and apicidin. We found that nutlin-3 and HDACi cooperated to induce cell death in the p53 wild-type cell lines A549 and A2780, but not in the p53 null cell line PC-3, as assessed by Alamar Blue assay and flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization. Combination index analysis showed that the effect was synergistic. For comparison, we tested nutlin-3 in combination with paclitaxel, revealing that nutlin-3 antagonized the cytotoxic activity of paclitaxel. To shed light on the underlying mechanism of the synergistic action of nutlin-3 and HDACi, we determined the acetylation status of p53 by immunoblotting and the mRNA levels of MDM2 and MDM4 by real-time RT-PCR. We observed vorinostat to induce p53 hyperacetylation, to reduce the constitutive gene expression of MDM2 and MDM4, and to counteract the nutlin-3-induced upregulation of MDM2 gene expression. In conclusion, our study shows that HDACi amplify the antitumor activity of nutlin-3-possibly by inducing p53 hyperacetylation and/or MDM2 and/or MDM4 downregulation-suggesting that treatment with a combination of nutlin-3 and HDACi may be an effective strategy for treating tumors with wild-type p53.

Topics: Acetylation; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Blotting, Western; Butyrates; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Imidazoles; Neoplasms; Nuclear Proteins; Paclitaxel; Peptides, Cyclic; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyridines; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tumor Suppressor Protein p53; Vorinostat

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.

Topics: Acetylation; Arachidonate 5-Lipoxygenase; Benzamides; Chromatin Immunoprecipitation; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Peptides, Cyclic; Promoter Regions, Genetic; Pyridines; RNA, Messenger; Transcription Factors; Transcriptional Activation; U937 Cells; Up-Regulation

It has been widely debated whether class IIa HDACs have catalytic deacetylase activity, and whether this plays any part in controlling gene expression. Herein, it has been demonstrated that class IIa HDACs isolated from mammalian cells are contaminated with other deacetylases, but can be prepared cleanly in Escherichia coli. These bacteria preparations have weak but measurable deacetylase activity. The low efficiency can be restored either by: mutation of an active site histidine to tyrosine, or by the use of a non-acetylated lysine substrate, allowing the development of assays to identify class IIa HDAC inhibitors.

Topics: Acetylation; Benzamides; Catalysis; Cells, Cultured; Escherichia coli; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney; Molecular Structure; Mutation; Peptides, Cyclic; Pyridines; Pyrimidines; Sulfonamides

Some histone deacetylase inhibitors (HDACi) have recently been related to teratogenic effects in rodents. Skeletal defects have been directly associated with embryonic hyperacetylation of somitic nuclei after valproic acid or trichostatin A exposure in vivo. Albeit the antitumoral activity of HDACi has been classically related to chromatin condensation due to histonic lysine hyperacetylation, nonhistonic proteins have also been suggested as an HDACi target. The aim of this work was the study of the effects of three HDACi (apicidin, API; MS-275; sodium butyrate, BUT) on mouse development and their activity on embryonic histonic and nonhistonic proteins. Pregnant mice were ip treated with 10 mg/kg body weight API, 25 mg/kg MS-275, 2000 mg/kg BUT or with the vehicle alone on day 8 post coitum. Embryos were extracted 1, 2, or 3 h after treatment and Western blotting (using antibodies antihyperacetylated histone H4, antiacetylated lysine, or antitubulin) and immunohistochemistry (using the antibody antihyperacetylated histone H4) were performed. Fetuses, explanted at term of gestation, were double stained for bone and cartilage to detect skeletal abnormalities. The studied HDACi were teratogenic. The specific axial skeletal malformations were fusions or homeotic respecifications. These molecules induced hyperacetylation restricted to somitic histones. The hyperacetylation index of histone H4 as well as immunohistochemical and skeletal analyses indicated BUT as the less active molecule. These new data on effects of API, MS-275, and BUT on development suggest histonic hyperacetylation as the mechanism for the induction of the observed skeletal abnormalities.

Topics: Acetylation; Animals; Benzamides; Butyrates; Embryonic Development; Female; Histone Deacetylase Inhibitors; Histones; Male; Mice; Mice, Inbred Strains; Peptides, Cyclic; Pregnancy; Pyridines; Ribs; Spine; Teratogens

Pharmacologic stimulation of fetal hemoglobin (HbF) expression may be a promising approach for the treatment of beta-thalassemia. In this study, we have investigated the HbF-inducing activity and molecular mechanisms of specific histone deacetylase (HDAC) inhibitors in human K562 erythroleukemia cells. Apicidin was the most potent agent compared with other HDAC inhibitors (trichostatin A, MS-275, HC-toxin, suberoylanilide hydroxamic acid [SAHA]) and previously tested compounds (butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine), leading to a 10-fold stimulation of HbF expression at nanomolar to micromolar concentrations. Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate HbF synthesis. Furthermore, analysis of different mitogen-activated protein (MAP) kinase signaling pathways revealed that p38 signaling was activated following apicidin treatment of cells and that inhibition of this pathway abolished the HbF-inducing effect of apicidin. Additionally, activation of the Agamma-globin promoter by apicidin could be inhibited by p38 inhibitor SB203580. In summary, the novel HDAC inhibitor apicidin was found to be a potent inducer of HbF synthesis in K562 cells. The present data outline the role of histone hyperacetylation and p38 MAP kinase signaling as molecular targets for pharmacologic stimulation of HbF production in erythroid cells.

Topics: Amides; Azacitidine; Benzamides; Butyrates; Enzyme Inhibitors; Fetal Hemoglobin; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Globins; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Hydroxyurea; Imidazoles; K562 Cells; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Peptides, Cyclic; Phenylbutyrates; Pyridines; Vorinostat