trichostatin-a and romidepsin

Among the natural histone deacetylase inhibitors (HDACi), the bicyclic depsipeptide macrolactone FK228 stands out for its unique chemical structure and mechanism of action. In order to expand the chemical diversity, exploiting the FK228 peculiar structure, we have synthesized a collection of 24 simplified novel analogs. A first series consists of bicyclic macrolactones, where the carboxy terminus of the natural compound was substituted by peptidomimetic aminomethylphenylacetic acid derivatives. These analogs, 7a-i, showed submicromolar cytotoxic activity, even though very low inhibitory activity against HDAC enzymes, suggesting that most probably they behave with a mechanism different from the natural compound. One of the most active members in the group, 7g, was evaluated in vivo and exhibited significant antitumor activity. This evidence supports that the activity is unrelated to HDAC inhibition and these compounds represent a novel series of promising active agents. Another analog series consists of monocyclic macrolactones, 9a-c and 10a-d which lack the disulfide bridge and bear the protected sulfur on the linear external chain; they showed similar cytotoxic activities compared to the natural compound, but proved to be very sensitive to the nature of the sulfur protection. In fact, when the sulfur was protected by an 1-octanoyl residue, like in 9b, the product displayed a one digit nanomolar activity. The results provide evidence that our approach may be followed to develop novel series of FK228 analogs.

Topics: Cell Survival; Depsipeptides; Drug Design; Histone Deacetylase Inhibitors; Humans; Lactones; Microwaves; Solid-Phase Synthesis Techniques

Epigenetic mechanisms are increasingly recognized as a major factor contributing to pathogenesis of cancer including glioblastoma, the most common and most malignant primary brain tumour in adults. Enzymatic modifications of histone proteins regulating gene expression are being exploited for therapeutic drug targeting. Over the last decade, numerous studies have shown promising results with histone deacetylase (HDAC) inhibitors in various malignancies. This article provides a brief overview of mechanism of anti-cancer effect and pharmacology of HDAC inhibitors and summarizes results from pre-clinical and clinical studies in glioblastoma. It analyses experience with HDAC inhibitors as single agents as well as in combination with targeted agents, cytotoxic chemotherapy and radiotherapy. Hallmark features of glioblastoma, such as uncontrolled cellular proliferation, invasion, angiogenesis and resistance to apoptosis, have been shown to be targeted by HDAC inhibitors in experiments with glioblastoma cell lines. Vorinostat is the most advanced HDAC inhibitor that entered clinical trials in glioblastoma, showing activity in recurrent disease. Multiple phase II trials with vorinostat in combination with targeted agents, temozolomide and radiotherapy are currently recruiting. While the results from pre-clinical studies are encouraging, early clinical trials showed only modest benefit and the value of HDAC inhibitors for clinical practice will need to be confirmed in larger prospective trials. Further research in epigenetic mechanisms driving glioblastoma pathogenesis and identification of molecular subtypes of glioblastoma is needed. This will hopefully lead to better selection of patients who will benefit from treatment with HDAC inhibitors.

Topics: Animals; Brain Neoplasms; Clinical Trials as Topic; Depsipeptides; Epigenesis, Genetic; Glioblastoma; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Valproic Acid; Vorinostat

Inhibitors of histone deacetylase (HDAC) are an emerging class of anticancer agents. They induce hyperacetylation in chromatin usually resulting in activation of certain genes. They induce terminal cell differentiation and/or apoptosis in cancer cells. Histone deacetylase activity is recruited by co-repressor proteins to certain regions of the chromatin and aberrant histone acetylation caused by that recruitment is responsible for the pathogenesis of certain cancers on a molecular level. Inhibitors of HDAC have been identified in natural sources and also synthetic inhibitors are available. The best studied inhibitor is trichostatin A, a hydroxamic acid that exerts its activity by complexation of a zinc ion that is supposed to mediate the acetamide cleavage at the catalytic site. There are several synthetic hydroxamic acids that bear resemblance to trichostatin. Another class of potent inhibitors are naturally occurring and synthetic cyclotetrapeptides that all contain an unusual amino acid with an epoxyketone, ketone or hydroxamic acid function in the side chain. Phenylacetate, phenylbutyrate, butyrate and similar short chain fatty acids are also weak inhibitors. Further inhibitors from natural sources are the epoxide depudecin and depsipeptide FR 901228. The benzamide MS-275 belongs to a new class of synthetic HDAC inhibitors and displays oral activity in animal models. First clinical studies have shown that histone hyperacetylation can be achieved safely in humans and that treatment of cancer is possible. Thus, inhibitors of HDAC are one of the most promising class of new anticancer agents. New screening assays are useful tools that will facilitate identification of further inhibitors.

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Antineoplastic Agents; Benzamides; Depsipeptides; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Molecular Structure; Neoplasms; Peptides, Cyclic

Topics: Acetyltransferases; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Depsipeptides; Drug Design; Enzyme Inhibitors; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Peptides; Peptides, Cyclic; Saccharomyces cerevisiae Proteins; Transcription, Genetic; Tumor Suppressor Protein p53

Cushing's disease is mainly caused by autonomous production of adrenocorticotropic hormone (ACTH) from pituitary adenomas. In our previous study, a histone deacetylase (HDAC) inhibitor, trichostatin A, inhibited cell proliferation and ACTH production via decreased pituitary tumor-transforming gene 1 (PTTG1) in AtT-20 mouse corticotroph tumor cells. In the present study, we examined the effects of romidepsin, a potent and selective HDAC1/2 inhibitor, on cell proliferation and ACTH synthesis. To elucidate further potential mechanisms of romidepsin, we examined the effects of HDAC1/2 on proopiomelanocortin (Pomc) and Pttg1 mRNA levels and cell proliferation. Small interfering RNA-mediated knockdown was used to decrease HDAC1 or 2. Romidepsin treatment decreased Pomc and Pttg1 mRNA levels, and cell proliferation. The drug also increased Hdac1 and decreased Hdac2 mRNA levels. Hdac1 knockdown decreased basal Pttg1 mRNA levels and cell proliferation, but not Pomc mRNA levels. Romidepsin treatment decreases ACTH synthesis in corticotroph tumor cells. Romidepsin suppresses cell proliferation via PTTG1. HDAC1 is also involved in the proliferation of corticotroph cells via PTTG1.

Topics: Adrenocorticotropic Hormone; Animals; Cell Line, Tumor; Cell Proliferation; Depsipeptides; Disease Models, Animal; Histone Deacetylase 1; Histone Deacetylase 2; Humans; Hydroxamic Acids; Mice; Pituitary ACTH Hypersecretion; Pituitary Neoplasms; Pro-Opiomelanocortin; RNA, Messenger; RNA, Small Interfering; Securin

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

Topics: Chromatography, High Pressure Liquid; Depsipeptides; Drug Evaluation, Preclinical; HeLa Cells; Histone Deacetylase 1; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mass Spectrometry

The Cancer Genome Atlas (TCGA) project recently identified the importance of mutations in chromatin remodeling genes in human carcinomas. These findings imply that epigenetic modulators might have a therapeutic role in urothelial cancers. To exploit histone deacetylases (HDACs) as targets for cancer therapy, we investigated the HDAC inhibitors (HDACIs) romidepsin, trichostatin A, and vorinostat as potential chemotherapeutic agents for bladder cancer. We demonstrate that the three HDACIs suppressed cell growth and induced cell death in the bladder cancer cell line 5637. To identify potential mechanisms associated with the anti-proliferative and cytotoxic effects of the HDACIs, we used quantitative proteomics to determine the proteins potentially involved in these processes. Our proteome studies identified a total of 6003 unique proteins. Of these, 2472 proteins were upregulated and 2049 proteins were downregulated in response to HDACI exposure compared to the untreated controls (P<0.05). Bioinformatic analysis further revealed that those differentially expressed proteins were involved in multiple biological functions and enzyme-regulated pathways, including cell cycle progression, apoptosis, autophagy, free radical generation and DNA damage repair. HDACIs also altered the acetylation status of histones and non-histone proteins, as well as the levels of chromatin modification proteins, suggesting that HDACIs exert multiple cytotoxic actions in bladder cancer cells by inhibiting HDAC activity or altering the structure of chromatin. We conclude that HDACIs are effective in the inhibition of cell proliferation and the induction of apoptosis in the 5637 bladder cancer cells through multiple cell death-associated pathways. These observations support the notion that HDACIs provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Depsipeptides; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Proteome; Proteomics; Signal Transduction; Urinary Bladder Neoplasms; Vorinostat

Histone deacetylase (HDAC) inhibitors are small molecule anticancer therapeutics that exhibit limiting cardiotoxicities including QT interval prolongation and life-threatening cardiac arrhythmias. Because the molecular mechanisms for HDAC inhibitor-induced cardiotoxicity are poorly understood, we performed whole cell patch voltage-clamp experiments to measure cardiac sodium currents (I

Topics: Animals; Animals, Newborn; Arrhythmias, Cardiac; Blotting, Western; Depsipeptides; Gap Junctions; Heart Ventricles; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoprecipitation; Induced Pluripotent Stem Cells; Long QT Syndrome; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; NAV1.5 Voltage-Gated Sodium Channel; Patch-Clamp Techniques; Real-Time Polymerase Chain Reaction; Vorinostat

Soluble ULBP2 is a marker for poor prognosis in several types of cancer. In this study we demonstrate that both soluble and cell surface-bound ULBP2 is transported via a so far unrecognized endosomal pathway. ULBP2 surface expression, but not MICA/B, could specifically be targeted and retained by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties. The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest that the endosomal transport pathway constitutes a novel therapeutic target for ULBP2-producing tumors.

Topics: Antigens, Differentiation, B-Lymphocyte; Antigens, Surface; Biological Transport; Carbazoles; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Depsipeptides; Endosomes; Enzyme Inhibitors; GPI-Linked Proteins; HEK293 Cells; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Jurkat Cells; Lymphocyte Activation; Melanoma; Neoplasms; Organoselenium Compounds; Protein Kinase C; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Vorinostat

Romidepsin (FK228) is a potent histone deacetylase (HDAC) inhibitor, which has a potent anticancer activity, but its molecular mechanism is unknown. We investigated the mechanism of FK228-induced apoptosis in the human leukemia cell line HL-60 and its hydrogen peroxide (H(2)O(2))-resistant sub-clone, HP100, and the human colon cancer cell line Caco-2. Cytotoxicity and DNA ladder formation induced by FK228 could be detected in HL-60 cells after a 24-h incubation, whereas they could not be detected in HP100 cells. Trichostatin A (TSA), an HDAC inhibitor, induced DNA ladder formation in both HL-60 and HP100 cells. In contrast, FK228 inhibited HDAC activity in both HL-60 and HP100 cells to a similar extent. These findings suggest that FK228-induced apoptosis involves H(2)O(2)-mediated pathways and that TSA-induced apoptosis does not. Flow cytometry revealed H(2)O(2) formation and a change in mitochondrial membrane potential (Δψm) in FK228-treated cells. FK228 also induced apoptosis in Caco-2 cells, which was prevented by N-acetyl-cysteine, suggesting that reactive oxygen species participate in apoptosis in various types of tumor cells. Interestingly, in a cell-free system, FK228 generated superoxide (O(2)(-)) in the presence of glutathione, suggesting that H(2)O(2) is derived from dismutation of O(2)(-) produced through redox-cycle of FK228. Therefore, in addition to HDAC inhibition, H(2)O(2) generated from FK228 may participate in its apoptotic effect.

Topics: Acetylcysteine; Apoptosis; Cell Line, Tumor; Depsipeptides; Glutathione; Histone Deacetylase Inhibitors; Humans; Hydrogen Peroxide; Hydroxamic Acids

Analysis of biopsies from a recent clinical trial suggested that Depsipeptide FK228 (DP) inhibits Aurora kinase expression in lung cancer cells. The present study was undertaken to confirm and extend these observations.. Aurora A and B mRNA levels in lung cancer cells were considerably higher than levels in normal pulmonary epithelia. DP, TSA and SAHA inhibited Aurora A, Aurora B and survivin expression with kinetics that were remarkably similar within individual cell lines, and appeared to coincide with p53 expression status. These effects were not observed following treatment with geldanamycins. Inhibition of Aurora B transcription coincided with decreased H3K9Ac and H3K4Me2 activation marks, and accumulation of H3K9Me3, as well as MBD1, MBD2 and MBD3 repression marks within the minimal Aurora B promoter. Knockdown of MBD1, -2 or -3 did not reproducibly abrogate inhibition of Aurora or survivin expression by DP or TSA. DP and TSA decreased expression and altered localization of Aurora kinases and survivin, resulting in mitotic catastrophe in lung cancer cells.. Aurora A, and Aurora B levels in lung cancer cells and normal respiratory epithelia were assessed using quantitative RT-PCR techniques. These methods, as well as as Western blots were used to examine expression of Auroras A/B, and several related genes/proteins in lung cancer cells exposed to DP, TSA, SAHA and geldanamycins. Transient transfection promoter-reporter assays, and chromatin immunoprecipitation (ChIP) techniques were used to examine DP-mediated changes in activity and chromatin structure of the Aurora B promoter. Confocal imaging techniques were used to examine the effects of DP and TSA on mitotic progression in lung cancer cells.. Novel transcriptional regulatory mechanisms involving Aurora kinase and survivin appear to contribute to cytotoxicity mediated by HDAC inhibitors in lung cancer cells.

Topics: Aurora Kinase B; Aurora Kinases; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Clinical Trials, Phase II as Topic; Depsipeptides; Drug Delivery Systems; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Kinetics; Lung Neoplasms; Microtubule-Associated Proteins; Neoplasm Proteins; Protein Serine-Threonine Kinases; RNA, Messenger; Survivin; Transcription, Genetic; Vorinostat

Histone acetylation and deacetylation play fundamental roles in transcriptional regulation. We investigated the role of histone deacetylases (HDACs) in human adult haematopoiesis, using the structurally distinct HDAC inhibitors FK228 (depsipeptide) and Trichostatin A. When CD34+ cells were cultured with interleukin (IL)-3 or stem cell factor (SCF) + IL-3, FK228 (0.5 ng/ml) specifically enhanced the generation of immature erythroid cells with a CD36+ glycophorin A (GPA)low phenotype. In semisolid cultures, FK228 promoted the formation of erythroid colonies by CD34+ cells with IL-3 and SCF + IL-3. Furthermore, upon exposure to FK228, CD34+ cell-derived CD36+ GPA- cells were induced to form erythroid colonies with IL-3 alone. Conversely, FK228 inhibited the generation of CD36+ GPAhigh relatively mature erythroid cells from CD34+ cells in the presence of erythropoietin (EPO) and SCF + EPO. FK228 suppressed the EPO-mediated survival of CD36+ GPAlow/- and CD36+ GPAhigh cells and induced their apoptosis. Similar effects were observed for trichostatin A in the generation of erythroid cells in IL-3- and EPO-containing cultures. These data suggest that HDACs negatively regulate the IL-3-mediated growth of early erythroid precursors by suppressing their responsiveness to IL-3, while playing an important role in EPO-mediated differentiation and survival of erythroid precursors. Our data revealed that HDACs have diverse functions in human adult erythropoiesis.

Topics: Adult; Antigens, CD34; Cell Survival; Cells, Cultured; Colony-Forming Units Assay; Depsipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoiesis; Erythropoietin; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Interleukin-3; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Stem Cell Factor

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-beta on pericentromeric heterochromatin in S-phase and G(2), decreased pericentromeric targeting of Aurora B kinase, resulting in decreased premitotic phosphorylation of pericentromeric histone H3(S10) in G(2), followed by assembly of deficient kinetochores in M-phase. HP1-beta, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.

Topics: Acetylation; Aurora Kinase B; Aurora Kinases; Cell Division; Cell Line, Tumor; Centromere; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Depsipeptides; G2 Phase; Heterochromatin; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Indoles; Kinetochores; Marine Toxins; Methotrexate; Microcystins; Microfilament Proteins; Mitosis; Peptides, Cyclic; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; S Phase; Spindle Apparatus

Mutant p53 is a cancer-specific target for pharmacologic intervention. We show that histone deacetylase inhibitors such as FR901228 and trichostatin A completely depleted mutant p53 in cancer cell lines. This depletion was preceded by induction of p53-regulated transcription. In cells with mutant p53 pretreated with histone deacetylase inhibitors, DNA damage further enhanced the p53 trans-function. Furthermore, histone deacetylase inhibitors were preferentially cytotoxic to cells with mutant p53 rather than to cells lacking wild-type p53. We suggest that, by either restoring or mimicking p53 trans-functions, histone deacetylase inhibitors initiate degradation of mutant p53. Because mutant p53 is highly expressed, a sudden restoration of p53-like functions is highly cytotoxic to cells with mutant p53. In a broader perspective, this shows how selectivity may be achieved by targeting a non-cancer-specific target, such as histone deacetylases, in the presence of a cancer-specific alteration, such as mutant p53.

Topics: Acetylation; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Depsipeptides; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mutation; Nuclear Proteins; Protein Conformation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transcription, Genetic; Tumor Suppressor Protein p53

Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents with potential for disruption of critical cellular processes in cancer cells. Transcriptional regulation, differentiation, cell cycle arrest, radiation sensitization, and apoptosis have been observed in response to exposure to HDAC inhibitors. In the present study, we observed that several potent HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, M344 (an analogue of hydroxamic acid), and the cyclic tetrapeptide, depsipeptide (FR90228), modulate cellular responses to ionizing radiation in cells of two human squamous carcinoma lines (SQ-20B and SCC-35), previously characterized as intrinsically resistant to radiation. Also exposure to IC(50) concentrations of these inhibitors, radiation sensitivities were enhanced in both cell lines. Depsipeptide exhibited the greatest effect on SQ-20B cells, decreasing D(0) values from 2.62 Gy to 1.64 Gy. M344 was the most active drug in sensitizing SCC-35 cells, decreasing D(0) values from 1.91 Gy to 1.21 Gy. The mechanisms underlying HDAC inhibitor-induced radiosensitization were further investigated by extending trichostatin A studies to assess cell cycle distributions and levels of apoptosis. Treatment of SQ-20B cells with radiosensitizing concentrations of trichostatin A resulted in cell cycle arrest in G(1) phase (>70%) and inhibition of DNA synthesis. Contrary to previous reports, induction of apoptosis was very low and caspase 3 and 9 were not activated. Taken together, these results implicate G(1) arrest and inhibition of DNA synthesis in the mechanisms underlying radiation sensitization by trichostatin A and support the use of HDAC inhibitors for targeting radioresistant cancers.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Depsipeptides; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Head and Neck Neoplasms; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Peptides, Cyclic; Radiation Tolerance; Radiation-Sensitizing Agents; Vorinostat

Androgens are critical in the development and maintenance of the male reproductive system and important in the progression of prostate cancer. The effects of androgens are mediated by the androgen receptor (AR), which is a ligand-modulated transcription factor that belongs to the nuclear receptor superfamily. We and others have previously shown that CREB-binding protein (CBP) can function as a coactivator for AR. Similar to some other nuclear receptor coactivators and/or the proteins that they interact with, CBP has histone acetyl transferase (HAT) activity that is thought to contribute to transcriptional activation by nuclear receptors. We have therefore assessed whether an increase in the histone acetylation status in the cell can influence AR transcriptional activity, by using the histone deacetylase (HDAC) inhibitors (HDACIs) trichostatin A (TSA), sodium butyrate (Na-But) and depsipeptide (FR901228). We found that inhibition of HDAC activity significantly increased the ability of endogenous AR in LNCaP cells, or ectopically expressed AR in HeLa cells, to activate transcription from AR-dependent reporter constructs. In addition, HDACIs increased the androgen-dependent activation of the prostate-specific antigen (PSA) gene in LNCaP cells, an increase that was not due to an increase in nuclear AR protein levels. Moreover, the viral oncoprotein E1A that inhibits CBP HAT activity fully repressed the ability of HDACIs to stimulate AR-mediated transcription, indicating that CBP is involved in this process. Deletional mutagenesis of AR indicated that whereas the AF-2 domain in the C-terminus is dispensable, the AF-1 domain in the N-terminus is required for augmentation of AR action by HDACIs, an observation which is in concordance with the reduced ability of CBP to activate AR N-terminal deletion mutants. Furthermore, HDACI treatment rescued the deficiency in the transactivation potential of AF-2 mutants. Taken together, our findings suggest that a change in the level of histone acetylation of target genes is an important determinant of AR action, possibly mediated by CBP.

Topics: Acetylation; Animals; Blotting, Northern; Blotting, Western; Butyrates; Depression, Chemical; Depsipeptides; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Mutation; Peptides, Cyclic; Prostatic Neoplasms; Receptors, Androgen; Transcription, Genetic

Topics: Apoptosis; Caspase 3; Caspases; Depsipeptides; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, p53; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Potentials; Mitochondria; RNA, Messenger; Tumor Cells, Cultured

During the development of leukemia, genes that suppress growth and induce differentiation can be silenced by aberrant DNA methylation and by changes in chromatin structure that involve histone deacetylation. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. Based on this observation, our working hypothesis was that a combination of inhibitors of these processes should produce an enhancement of their antineoplastic activity on leukemic cells. The cytosine nucleoside analog, 5-aza-2'-deoxycytidine (5AZA), is a potent inhibitor of DNA methylation, which can activate tumor suppressor genes in leukemic cells that have been silenced by aberrant methylation. In clinical trials, 5AZA was demonstrated to be an active antileukemic agent. Histone deacetylase inhibitors (HDI) can also activate gene expression in leukemic cell lines by producing changes in chromatin configuration, and show antineoplastic activity in preclinical studies. In this report, we investigated the in vitro antineoplastic activity of 5AZA, alone and in combination with the HDI, trichostatin A (TSA) and depsipeptide (FR901228, depsi), on the human myeloid leukemic cell lines, HL-60 and KG1a. The results showed that the combination of 5AZA with TSA or depsi produced a greater inhibition of growth and DNA synthesis and a greater loss of clonogenicity than either agent alone. These results suggest that 5AZA used in combination with HDI may be an interesting chemotherapeutic regimen to investigate in patients with acute myeloid leukemia that is resistant to conventional chemotherapy.

Topics: Acetylation; Acute Disease; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Azacitidine; Cell Division; Decitabine; Depsipeptides; DNA Methylation; DNA Replication; DNA, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Leukemia, Myeloid; Neoplasm Proteins; Peptides, Cyclic; Protein Processing, Post-Translational; Tumor Cells, Cultured; Tumor Stem Cell Assay

Uveal melanoma (UM) is the most common primary malignant ocular tumor in adults. No effective chemotherapy regimens are available for either intraocular or metastatic uveal melanoma. Therefore, the ability of the histone deacetylase inhibitors (HDACIs), depsipeptide, sodium butyrate (NaB) and trichostatin A (TSA), to induce apoptosis and inhibit cell growth of UM cell lines in vitro was examined.. Three primary and two metastatic UM cell lines were treated in vitro with different concentrations of histone deacetylase inhibitors (HDACIs). Cell proliferation was studied in 24-well plates. Induction of apoptosis was studied by flow cytometry. Changes in gene expression of Fas/FasL, p21(Waf/Cip1), and p27(Kip1) were studied by RT-PCR. Western blot analysis was used to study histone acetylation, Fas/FasL, p21(Waf/Cip1), p27(Kip1) and caspase-3 protein levels. Real-time PCR was used to study changes in bcl-2/bax gene expression.. A dose-dependent increase in histone acetylation was observed in all cell lines. This corresponded to significant inhibition of cell growth and induction of apoptosis in all melanoma cell lines in a concentration-dependent manner. Western blot analysis revealed dose-dependent increases in the amount of caspase-3, Fas/FasL, p21(Waf/Cip1), and p27(Kip1) proteins. However, no changes in bcl-2/bax gene expression were detected by real-time PCR.. HDACIs are potent inhibitors of primary and metastatic UM cell growth in vitro. The apoptosis is probably mediated through the Fas/FasL signaling pathway, whereas bcl-2 appears not to be involved. These data support further clinical evaluation of depsipeptide and other HDACIs in patients with primary and metastatic UM.

Topics: Antibiotics, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Butyrates; Caspase 3; Caspases; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Depsipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Melanoma; Membrane Glycoproteins; Peptides, Cyclic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins; Uveal Neoplasms

Histone deacetylase (HDAC) inhibitors are known to induce expression of genes such as p21(WAF1), thereby, leading to cell cycle arrest. In this work, we show that p21(WAF1) induction by HDAC inhibitors (depsipeptide and trichostatin A) is defective in Ataxia telangiectasia (AT) cells but normal in matched wild-type (WT) cells (human diploid fibroblasts). To verify the role of ATM in this effect, we show that ectopic expression of the WT ATM gene in an AT cell line fully restores p21(WAF1) induction by the HDAC inhibitors. Furthermore, because caffeine and wortmannin attenuate p21(WAF1) induction in WT cells, it is probable that the phosphatidylinositol 3'-kinase activity is essential for this process. Besides the p21(WAF1) promoter, activation of topoisomerase IIIalpha and SV40 promoters by the HDAC inhibitors are also decreased in the AT cell lines relative to WT cells; thus, these findings pertain to other promoters. Finally, despite the obvious induction deficiency of gene expression, the overall levels of H3 and H4 histone acetylation appear to be the same between AT and normal cells in response to HDAC inhibitor treatments. Taken together, the data indicate that ATM is involved in histone acetylation-mediated gene regulation.

Topics: Acetylation; Ataxia Telangiectasia; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Depsipeptides; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Peptides, Cyclic; Phosphatidylinositol 3-Kinases; Phosphorylation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Transfection; Tumor Suppressor Proteins

By preventing deacetylation of histones, histone deacetylase inhibitors (HDIs) transcriptionally induce p21. Here we show that the HDIs sodium butyrate (Bu), trichostatin A (TSA) and depsipeptide (FR901228) all induced p21, but only TSA and FR901228 caused mitotic arrest (in addition to arrest in G1 and G2). The ability to cause mitotic arrest correlated with the higher cytotoxicity of these compounds. Although causing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR9012208, TSA caused acetylation of tubulin at lysine 40; both soluble tubulin and microtubules were acetylated. Whereas the induction of p21 reached a maximum by 8 h, tubulin was maximally acetylated after only 1 h of TSA treatment. Tubulin acetylation was detectable after treatment with 12-25 ng/ml TSA although acetylation plateaued at 50 ng/ml TSA, coinciding with G2-M arrest, appearance of cells with a sub-2N DNA content, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We conclude that HDIs have differential effects on non-histone deacetylases and that rapid acetylation of tubulin caused by TSA is a marker of nontranscriptional effects of TSA.

Topics: Anesthetics, Intravenous; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoptosis; Butyrates; Cell Cycle; Cell Line; Coloring Agents; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Depsipeptides; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; G2 Phase; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Immunoblotting; Jurkat Cells; Mitosis; Peptides; Peptides, Cyclic; Protein Synthesis Inhibitors; Sodium Oxybate; Tetrazolium Salts; Thiazoles; Time Factors; Transcription, Genetic; Tubulin; Tumor Cells, Cultured

Histone acetylation and deacetylation are closely linked to transcriptional activation and repression, respectively. In acute promyelocytic leukemia (APL), histone deacetylase inhibitors (HDACIs) have a synergistic effect with all-trans retinoic acid (ATRA) in vitro to induce differentiation. Here we report in vitro and in vivo effects of a HDACI, FK228 (formerly FR901228 or depsipeptide), on the human APL cell line NB4. FK228 had a strong and irreversible cytotoxicity compared with another HDACI, trichostatin A. In vivo administration of ATRA or FK228 alone partly inhibited the growth of established tumors of NB4 subcutaneously transplanted in NOD / Shi-scid / scid mice, and the combination was synergistically effective. Histopathological examination revealed that the combination induced apoptosis and differentiation as well as histone acetylation. Intravenous injection of NB4 in NOD / Shi-scid / scid mice followed by combination treatment significantly prevented leukemia death, whereas single administration did not. These findings suggest that FK228 is a promising agent to enhance ATRA-sensitivity in the treatment of APL.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cell Differentiation; Depsipeptides; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Kinetics; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Peptides, Cyclic; Tretinoin; Tumor Cells, Cultured

Screening for microbial metabolites that induce transcriptional activation of the SV40 promoter resulted in the identification of two known compounds, FR901228 and trichostatin A (TSA). FR901228 is a potent antitumor drug that is currently under clinical investigation. TSA is a specific inhibitor of histone deacetylase. Despite structural unrelatedness, both FR901228 and TSA greatly enhanced the transcriptional activity of the SV40 promoter in an enhancer-dependent manner. The effects of FR901228 on the cell cycle, chromatin structure, and histone acetylation were examined and compared with those of TSA. Both compounds caused arrest of the cell cycle at both G1 and G2/M phases and induction of internucleosomal breakdown of chromatin. FR901228, like TSA, inhibited intracellular histone deacetylase activity, as a result of which marked amounts of acetylated histone species accumulated. FR901228 is therefore a new type of histone deacetylase inhibitor, whose chemical structure is unrelated to known inhibitors such as trichostatins and trapoxins.

Topics: Acetylation; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Cell Division; Depsipeptides; DNA Fragmentation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Peptides, Cyclic; Promoter Regions, Genetic; Simian virus 40; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured