trichostatin-a and Neoplasms

The histone lysine demethylase 4 (KDM4) family plays an important role in regulating gene transcription, DNA repair, and metabolism. The dysregulation of KDM4 functions is associated with many human disorders, including cancer, obesity, and cardiovascular diseases. Selective and potent KDM4 inhibitors may help not only to understand the role of KDM4 in these disorders but also to provide potential therapeutic opportunities. Here, we provide an overview of the field and discuss current status, challenges, and opportunities lying ahead in the development of KDM4-based anticancer therapeutics.

Topics: Enzyme Inhibitors; Histone Demethylases; Humans; Jumonji Domain-Containing Histone Demethylases; Neoplasms

Even though one is moving towards the success in the discovery of efficient anti-cancer molecules, the drugs used in the treatment of various malignancies are found to possess toxicity and adverse reactivity in the human body that limit their use. The scientists all over the world are engaged in bringing up strategies that aim to develop small molecules that target the abnormal epigenetic factors. The discovery of the role of Histone deacetylases (HDACs) has promised to be a turning point in the treatment of various malignancies. Thus, the invention of potent and safe anticancer therapeutics agents with minimal adverse and side effects are still a major topic of concern and a huge number of research works have been reported in the past few years. This review has been written to discuss on the influence of Histone Deacetylases in cancer malignancies. We have tried to embrace majority of the developments made till date in the field of HDAC and its inhibitors herein. The drugs that are clinically applied, synthesis and SAR study that highlight the chemical groups responsible for evoking the HDAC inhibition and potential of various new classes of HDAC inhibitors (synthetic, hybrid and natural) have also been included.

Topics: Animals; Antineoplastic Agents; Biological Products; Chemistry Techniques, Synthetic; Drug Discovery; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Models, Molecular; Molecular Targeted Therapy; Neoplasms; Structure-Activity Relationship

Topics: Animals; Cytidine; DNA Methylation; DNA Modification Methylases; Gene Knockout Techniques; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasms; RNA Interference; Valproic Acid

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Molecular Structure; Neoplasms

Topics: Acetylation; Animals; Antigens, Polyomavirus Transforming; Antineoplastic Agents; Cortactin; Enzyme Inhibitors; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lysine; mRNA Cleavage and Polyadenylation Factors; Neoplasms; Tubulin; Tumor Suppressor Protein p53

Histone acetylation regulates many chromosome functions, such as gene expression and chromosome segregation. Histone deacetylase inhibitors (HDACIs) induce growth arrest, differentiation and apoptosis of cancer cells ex vivo, as well as in vivo in tumour-bearing animal models, and are now undergoing clinical trials as anti-tumour agents. However, little attention has been paid to how HDACIs function in these biological settings and why different cells respond in different ways. Here, we discuss the consequences of inhibiting histone deacetylases in cycling versus non-cycling cells, in light of the dynamics of histone acetylation patterns with a specific emphasis on heterochromatic regions of the genome.

Topics: Cell Cycle; Heterochromatin; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Neoplasms

Topics: Acetylation; Animals; Apoptosis; Benzamides; Drug Delivery Systems; Enzyme Inhibitors; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Neoplasms; Peptides; Pyridines; Valproic Acid

HDAC inhibitor has been focused as a molecular target agent for cancer treatment because of unique pharmacological activity. HDAC inhibitors induce cancer cells to undergo growth arrest, differentiation or induce apoptosis in vitro, and also inhibit the growth of transplanted tumors in vivo, but the enhancement effect or interaction of HDAC inhibitors with other chemotherapeutic agents or radiation has not yet been fully investigated. Biade et al examined the effect of trichostatin A (TSA), and reported synergistic enhancement of radiosensitivity in human cancer cell lines. We also confirmed the similar results in vitro by using TSA in combination with radiation. The main purpose of this paper is to review the current status of HDAC inhibitor in clinical use, and evaluate the potential possibility for the potentiation of radiation-induced cell killing including the results of our research.

Topics: Antineoplastic Agents; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasms; Radiation-Sensitizing Agents

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

Numerous studies have shown that histone deacetylase inhibitors (HDACis) improve cellular acetylation while also enhancing the radiation sensitivity. In this work, however, we confirmed that low-dose trichostatin A (TSA) as a typical HDACi could reduce rather than increase the radiosensitivity of cancer cells, while the cellular acetylation was also increased with TSA-induced epigenetic modification. The surviving fraction of HeLa/HepG2 cells pretreated with 25 nM TSA for 24 h was higher at 1 Gy/2 Gy of γ-ray radiation than that of the cells with the same radiation dose but without TSA pretreatment. To understand the underlying mechanism, we investigated the effect of low-dose TSA on HO-1, SOD and CAT induction and activating Akt together with its downstream Nrf2 signaling pathway. Our results indicated that TSA activated HO-1, SOD and CAT expression by increasing the phosphorylation level of Nrf2 in an Akt-dependent manner. In addition, we also observed that the 25-nM-TSA-pretreated group showed a significant increase in the antioxidant capacity in terms of SOD and CAT activities. Therefore, our results suggest that low-dose TSA can activate the Akt/Nrf2 pathway and upregulate expression of HO-1, SOD and CAT to stimulate the cellular defense mechanism. This work demonstrates that low-dose TSA treatment may activate the adaptation mechanism against the oxidative stress induced by ionizing radiation, and application of HDACi treatment should be undertaken with caution to avoid its possible radioresistance in radiotherapy.

Topics: Antioxidants; Cell Proliferation; Gene Expression Regulation, Neoplastic; HeLa Cells; Hep G2 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasms; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Signal Transduction

Quercetin, a flavonol, displays anti-inflammatory and anti-cancer properties. This study aimed to investigate whether a diet containing 0.1% or 1% quercetin (LQ and HQ, respectively) enhances the anti-tumor effects of trichostatin A (TSA) and prevents muscle wasting induced by TSA. The positive control group received quercetin intraperitoneally (IQ). Three weeks after injecting A549 cells, nude mice were given TSA alone or in combination with quercetin administered orally or intraperitoneally for 16 weeks. Tumor volumes as well as body, muscle and epididymal fat weights were determined during or after the experiment. Quercetin given as a diet supplement dose-dependently enhanced the anti-tumor potency of TSA (p < 0.05). The enhancing effect of HQ was similar to that of IQ. HQ also significantly increased the expression of p53, a tumor suppressor, in tumor tissues compared with the TSA alone group. In addition, TSA-induced loss of gastrocnemius muscle weight was inhibited by oral quercetin in a dose dependent manner; the efficiencies of LQ and HQ were similar to or better than IQ. Moreover, both LQ and HQ decreased TSA-induced activation of Forkhead box O1 (FOXO1), a crucial transcription factor that regulates muscle wasting associated genes. Consistently, LQ and HQ suppressed muscle wasting associated proteins atrophy gene-1 and muscle ring-finger protein-1 expression as well as increased the myosin heavy chain level in the gastrocnemius muscles. Besides, quercetin attenuated TSA-increased oxidative damage and proinflammatory cytokines (p < 0.05). These findings demonstrate that a diet containing 0.1% or 1% quercetin enhances the antitumor effect of TSA and prevents TSA-induced muscle wasting.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Therapy, Combination; Forkhead Box Protein O1; Humans; Hydroxamic Acids; Male; Mice; Mice, Nude; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Neoplasms; Quercetin

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the

Topics: Antineoplastic Agents; Cell Line; Cell Line, Tumor; Cell Survival; Chromatin; Chromosomes; Chromosomes, Artificial, Human; DNA Damage; Drug Design; HCT116 Cells; Humans; Hydroxamic Acids; Mitosis; Neoplasms; Telomerase; Telomere; Transgenes

The number of potential applications of non-thermal atmospheric pressure plasma (NTAPP) discharges in medicine, particularly in cancer therapy, has increased in recent years. NTAPP has been shown to affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated medium (PAM). Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation. The aim of the present study was to demonstrate the advantage of the combined application of PAM and HDAC inhibitors on A549 cancer cell survival and elucidate the underlying mechanisms. Cell death with DNA breaks in the nucleus was greater using combined regimens of PAM and HDAC inhibitors such as trichostatin A (TSA) and valproic acid (VPA) than a single PAM treatment and was accompanied by the activation of poly (ADP-ribose) polymerase-1 (PARP-1), depletion of ATP, and elevations in intracellular calcium levels. Moreover, the expression of Rad 51, a DNA repair factor in homologous recombination pathways, was significantly suppressed by the treatment with HDAC inhibitors. These results demonstrate that HDAC inhibitors may synergistically induce the sensitivity of cancer cells to PAM components.

Topics: A549 Cells; Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Calcium; Cell Death; Cell Survival; Culture Media; DNA Damage; DNA Repair; Down-Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Neoplasms; Plasma Gases; Poly (ADP-Ribose) Polymerase-1; Rad51 Recombinase; Valproic Acid

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

Topics: Acetylation; Animals; Antineoplastic Agents; Azacitidine; Cell Death; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Docetaxel; Epigenesis, Genetic; HEK293 Cells; Histones; Humans; Hydroxamic Acids; Kidney; Male; Methylation; Neoplasms; Oligopeptides; Prostatic Neoplasms; Rats; Taxoids

The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors.. The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression.. The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation.. We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

Topics: Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Code; Humans; Hydroxamic Acids; Neoplasms; Palatine Tonsil; Promoter Regions, Genetic; SOXC Transcription Factors; Vorinostat

We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity.. HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency.. HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival.

Topics: Animals; Antigens, Nuclear; B-Lymphocytes; Blotting, Western; Cell Survival; Cells, Cultured; Chickens; DNA Damage; DNA End-Joining Repair; DNA Repair; DNA-Binding Proteins; Electrophoresis, Gel, Pulsed-Field; Fluorescent Antibody Technique; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ku Autoantigen; Luciferases; Mutation; Neoplasms; Rad51 Recombinase; RecQ Helicases; RNA, Small Interfering; Vorinostat

Our previous study showed that quercetin enhances the anticancer effect of trichostatin A (TSA) in xenograft mice given quercetin intraperitoneally (10 mg/kg, 3 times/week). Herein, we investigate whether quercetin administered orally exerts such an effect and prevents the cytotoxic side effects of TSA. We found that quercetin given orally (20 and 100 mg/kg, 3 times/week) failed to enhance the antitumor effect of TSA although it increased the total quercetin concentration more than quercetin administered intraperitoneally in the plasma. The compound quercetin-3-glucuronide (Q3G) increased the most. However, quercetin administered intraperitoneally increased the total quercetin level in tumor tissues more than oral quercetin. Oral and intraperitoneal administration of quercetin similarly decreased lymphocyte DNA damage and plasma lipid peroxidation level induced by TSA. Furthermore, we found that the enhancing effect of Q3G on the antitumor effect of TSA and the incorporation of Q3G was less than that of quercetin in A549 cells. However, we found that A549 cells possessed the ability to convert Q3G to quercetin. In conclusion, different from quercetin administered intraperitoneally, quercetin administered orally failed to enhance the antitumor effect of TSA because of its metabolic conversion. However, it prevented TSA-induced DNA damage and lipid peroxidation.

Topics: Administration, Oral; Animals; Cell Line, Tumor; Cell Proliferation; DNA Damage; Humans; Hydroxamic Acids; Infusions, Parenteral; Lipid Peroxidation; Lymphocytes; Male; Mice; Neoplasm Transplantation; Neoplasms; Quercetin

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

EBV-transformed lymphoblastoid cell lines (LCL) are potent antigen-presenting cells. To investigate their potential use as cancer testis antigen (CTA) vaccines, we studied the expression of 12 cancer testis (CT) genes in 20 LCL by RT-PCR. The most frequently expressed CT genes were SSX4 (50 %), followed by GAGE (45 %), SSX1 (40 %), MAGE-A3 and SSX2 (25 %), SCP1, HOM-TES-85, MAGE-C1, and MAGE-C2 (15 %). NY-ESO-1 and MAGE-A4 were found in 1/20 LCL and BORIS was not detected at all. Fifteen of 20 LCL expressed at least one antigen, 9 LCL expressed ≥2 CT genes, and 7 of the 20 LCL expressed ≥4 CT genes. The expression of CT genes did not correlate with the length of in vitro culture, telomerase activity, aneuploidy, or proliferation state. While spontaneous expression of CT genes determined by real-time PCR and Western blot was rather weak in most LCL, treatment with DNA methyltransferase 1 inhibitor alone or in combination with histone deacetylase inhibitors increased CTA expression considerably thus enabling LCL to induce CTA-specific T cell responses. The stability of the CT gene expression over prolonged culture periods makes LCL attractive candidates for CT vaccines both in hematological neoplasias and solid tumors.

Topics: Antigen-Presenting Cells; Antigens, Neoplasm; Azacitidine; B-Lymphocytes; Cancer Vaccines; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Transformed; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Interferon-gamma; Melanoma; Neoplasms; RNA, Messenger; Telomerase; Telomere; Tumor Necrosis Factor-alpha; Valproic Acid; Vorinostat

Over the last decade, several drugs that inhibit class I and/or class II histone deacetylases (HDACs) have been identified, including trichostatin A, the cyclic depsipeptide FR901228 and the antibiotic apicidin. These compounds have had immediate application in cancer research because of their ability to reactivate aberrantly silenced tumour suppressor genes and/or block tumour cell growth. Although a number of HDAC inhibitors are being evaluated in preclinical cancer models and in clinical trials, little is known about the differences in their specific mechanism of action and about the unique determinants of cancer cell sensitivity to each of these inhibitors.. Using a combination of cell viability assays, HDAC enzyme activity measurements, western blots for histone modifications, microarray gene expression analysis and qRT-PCR, we have characterised differences in trichostatin A vs depsipeptide-induced phenotypes in lung cancer, breast cancer and skin cancer cells and in normal cells and have then expanded these studies to other HDAC inhibitors.. Cell viability profiles across panels of lung cancer, breast cancer and melanoma cell lines showed distinct sensitivities to the pan-inhibitor TSA compared with the class 1 selective inhibitor depsipeptide. In several instances, the cell lines most sensitive to one inhibitor were most resistant to the other inhibitor, demonstrating these drugs act on at least some non-overlapping cellular targets. These differences were not explained by the HDAC selectivity of these inhibitors alone since apicidin, which is a class 1 selective compound similar to depsipeptide, also showed a unique drug sensitivity profile of its own. TSA had greater specificity for cancer vs normal cells compared with other HDAC inhibitors. In addition, at concentrations that blocked cancer cell viability, TSA effectively inhibited purified recombinant HDACs 1, 2 and 5 and moderately inhibited HDAC8, while depsipeptide did not inhibit the activity of purified HDACs in vitro but did in cellular extracts, suggesting a potentially indirect action of this drug. Although both depsipeptide and TSA increased levels of histone acetylation in cancer cells, only depsipeptide decreased global levels of transcriptionally repressive histone methylation marks. Analysis of gene expression profiles of an isogenic cell line pair that showed discrepant sensitivity to depsipeptide, suggested that resistance to this inhibitor may be mediated by increased expression of multidrug resistance genes triggered by exposure to chemotherapy as was confirmed by verapamil studies.. Although generally thought to have similar activities, the HDAC modulators trichostatin A and depsipeptide demonstrated distinct phenotypes in the inhibition of cancer cell viability and of HDAC activity, in their selectivity for cancer vs normal cells, and in their effects on histone modifications. These differences in mode of action may bear on the future therapeutic and research application of these inhibitors.

Topics: Base Sequence; Blotting, Western; Cell Line, Tumor; DNA Methylation; DNA Primers; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Neoplasms; Oligopeptides; Real-Time Polymerase Chain Reaction

Histone deacetylase inhibitors (HDACi) are actively explored as new-generation epigenetic drugs but have low efficacy in cancer monotherapy. To reveal new mechanism for combination therapy, we show that HDACi induce cell death but simultaneously activate tumor-progressive genes to ruin therapeutic efficacy. Combined treatments to target tumorigenesis and HDACi-activated metastasis with low toxic modalities could develop new strategies for long-term cancer therapy.. Because metastasis is the major cause of cancer mortality, we measured cell migration activity and profiled metastasis-related gene expressions in HDACi-treated cancer cells. We developed low toxic combination modalities targeting tumorigenesis and HDACi-activated metastasis for preclinical therapies in mice.. We showed that cell migration activity was dramatically and dose dependently enhanced by various classes of HDACi treatments in 13 of 30 examined human breast, gastric, liver, and lung cancer cell lines. Tumor metastasis was also enhanced in HDACi-treated mice. HDACi treatments activated multiple PKCs and downstream substrates along with upregulated proapoptotic p21. For targeting tumorigenesis and metastasis with immediate clinical impact, we showed that new modalities of HDACi combined drugs with PKC inhibitory agent, curcumin or tamoxifen, not only suppressed HDACi-activated tumor progressive proteins and cell migration in vitro but also inhibited tumor growth and metastasis in vivo.. Treatments of different structural classes of HDACi simultaneously induced cell death and promoted cell migration and metastasis in multiple cancer cell types. Suppression of HDACi-induced PKCs leads to development of low toxic and long-term therapeutic strategies to potentially treat cancer as a chronic disease.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Combined Modality Therapy; Curcumin; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Neoplasm Metastasis; Neoplasms; Protein Kinase C; Tamoxifen

Histone deacetylases (HDACs) are involved in post-translational modification and gene expression. Cancer cells recruited amounts of HDACs for their survival by epi-genetic down regulation of tumor suppressor genes. HDACs have been the promising targets for treatment of cancer, and many HDAC inhibitors have been investigated nowadays. In previous study, we synthesized δ-lactam core HDAC inhibitors which showed potent HDAC inhibitory activities as well as cancer cell growth inhibitory activities. Through QSAR study of the δ-lactam based inhibitors, the smaller core is suggested as more active than larger one because it fits better in narrow hydrophobic tunnel of the active pocket of HDAC enzyme. The smaller γ-lactam core HDAC inhibitors were designed and synthesized for biological and property optimization. Phenyl, naphthyl and thiophenyl groups were introduced as the cap groups. Hydrophobic and bulky cap groups increase potency of HDAC inhibition because of hydrophobic interaction between HDAC and inhibitors. In overall, γ-lactam based HDAC inhibitors showed more potent than δ-lactam analogues.

Topics: Animals; Antineoplastic Agents; Binding Sites; Cell Line, Tumor; Cell Membrane Permeability; Computer Simulation; Drug Design; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Lactams; Mice; Microsomes, Liver; Neoplasms; Quantitative Structure-Activity Relationship; Transplantation, Heterologous

Previously, we reported a click-chemistry based approach to the synthesis of a novel class of histone deacetylase (HDAC) inhibitors [1]. The lead compound NSC746457 was found to be as potent as SAHA (Vorinostat). Further optimization of NSC746457 by using the HDAC2-TSA crystal structure is described herein. Docking of NSC746457 into HDAC2 binding domain suggested that the hydrophobic residue Phe210 flanking the cap-group binding-motif could be exploited for structural optimization. Substitution on the methylene group of cinnamic cap region led to identification of more potent HDAC inhibitors: isopropyl derivative 5 and tert-butyl derivative 6, with an IC(50) value of 22 nM and 18 nM, respectively.

Topics: Antineoplastic Agents; Binding Sites; Cell Line, Tumor; Cell Proliferation; Click Chemistry; Crystallography, X-Ray; Drug Design; Drug Screening Assays, Antitumor; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydrophobic and Hydrophilic Interactions; Hydroxamic Acids; Models, Molecular; Neoplasms; Phenylalanine; Protein Binding; Protein Structure, Secondary; Structure-Activity Relationship; Triazoles; Vorinostat

The enzymatic inhibition of histone deacetylase activity has come out as a novel and effectual means for the treatment of cancer. Two novel series of 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-ylamino]-pyrimidine-5-carboxylic acid (tetrahydro-pyran-2-yloxy)-amides were designed and synthesized as novel hydroxamic acid based histone deacetylase inhibitors. The antiproliferative activities of the compounds were investigated in vitro using histone deacetylase inhibitory assay and MTT assay. The synthesized compounds were also tested for antitumor activity against Ehrlich ascites carcinoma cells in Swiss albino mice. The efforts were also made to establish structure-activity relationships among synthesized compounds. The results of the present studying indicates 2,5-disubstituted 1,3,4-oxadiazole/thiadiazole as promising surface recognition moiety for development of newer hydroxamic acid based histone deacetylase inhibitor.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Proliferation; Drug Design; Drug Evaluation, Preclinical; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Hydroxamic Acids; Mice; Neoplasms; Oxadiazoles; Structure-Activity Relationship; Surface Properties; Thiadiazoles; Zinc

Histone deacetylase inhibitors (HDACIs) have shown promising anti-tumor effects for a variety of malignancies, however, many tumors are reportedly resistant to them. In this study, we made a novel discovery that co-administration of HDACIs (Trichostatin A (TSA) and others) and exogenous cell-permeable short-chain ceramide (C6) results in striking increase in cancer cell death and apoptosis in multiple cancer cells. These events are associated with perturbations in diverse cell signaling pathways, including inactivation of Akt/mTOR and increase in α-tubulin acetylation (both in vivo and in vitro). TSA interacts in a highly synergistic manner with C6-ceramide to disrupt HDAC6/protein phosphatase 1 (PP1)/tubulin complex, to induce α-tubulin hyperacetylation, and to release and activate PP1, which then leads to AKT dephosphorylation and eventually causes cancer cell death. Interestingly, TSA itself results in short-term ceramide accumulation, which as a result of metabolic (glycosylation) removal, does not result in evident increase of cancer cell death. However, adding C6-ceramide led to a very pronounced increase in ceramide level and marked increase in cell death. Importantly, the effective synergistic anti-tumor activity of TSA plus C6-ceramide is also seen in in vivo mice xenograft pancreatic and ovarian cancer models, indicating that this regimen (HDACI plus C6-ceramide) may represent a more effective form of therapy against pancreatic and ovarian carcinoma.

Topics: Acetylation; Animals; Antineoplastic Agents; Apoptosis; Ceramides; Drug Synergism; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mice; Mice, Nude; Neoplasms; Oncogene Protein v-akt; Phosphorylation; Tubulin

Epigenetic modifications, such as DNA methylation or histone deacetylation, are early events in cell tumorigenesis. The consequences of these modifications are repression of gene transcription and, notably, of tumor suppressor gene transcription. New therapeutic strategies aim to 'normalize' the epigenetic status of cancer cells. Histone deacetylase inhibitors (HDACi) have shown promising effects against proliferation and resistance to apoptosis of a large number of cancer cells. Vorinostat (SAHA), a hydroxamate HDACi, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory cutaneous T-cell lymphoma (CTCL). However, HDACi are poorly specific, present toxicities and many have very low half-lives in the plasma. Thus, the development of new compounds is necessary in order to increase the potential of HDACi in cancer treatment. We designed an assay, based on bioluminescence resonance energy transfer (BRET) technology, to screen and characterize HDACi activity in living cells. Using our specific and reproducible BRET assay, we characterized the pharmacological properties of benzofuranone HDACi compounds for the induction of histone acetylation and performed a comparison with the properties of suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA). We defined a benzofuranone HDACi compound that induced histone acetylation at nanomolar concentrations and showed an increased duration of histone acetylation. These properties correlated with the pharmacological properties of this HDACi for the growth inhibition of cancer cells. We, thus, demonstrated the applicability of BRET technology for the screening and characterization of new HDACi compounds in living cells, and identified an interesting benzofuranone HDACi.

Topics: Acetylation; Animals; Antineoplastic Agents; Benzofurans; Bioluminescence Resonance Energy Transfer Techniques; Cell Line, Tumor; Cell Proliferation; Drug Design; Drug Resistance, Neoplasm; High-Throughput Screening Assays; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Kinetics; Molecular Targeted Therapy; Neoplasms; Rats; Recombinant Fusion Proteins

Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression.. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay.. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters.. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.

Topics: Ataxia Telangiectasia Mutated Proteins; Azacitidine; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Decitabine; DNA Breaks; DNA Methylation; DNA-Binding Proteins; Gamma Rays; Genes, p16; Humans; Hydroxamic Acids; Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Radiation Tolerance; Tumor Suppressor Proteins

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

Topics: Activin Receptors, Type II; Animals; Cachexia; Carcinoma, Lewis Lung; Disease Models, Animal; Follistatin; Histone Deacetylase Inhibitors; Hydroxamic Acids; Immunoglobulin Fc Fragments; Ligands; Melanoma, Experimental; Mice; Mice, Knockout; Muscle, Skeletal; Muscular Dystrophies; Myostatin; Neoplasms; Recombinant Fusion Proteins

The psiTPTE22 gene has been designated as a TPTE pseudogene. Our study found that the 5' part of psiTPTE22 has no sequence similarity to TPTE and contains a 3.8-kb human endogenous retrovirus (HERV) element. Because of the HERV element, the 5' part of psiTPTE22 (psiTPTE22-HERV) expresses independently as a gene. Comparison between the DNA sequences of humans and chimps indicated that psiTPTE22-HERV is human specific. We identified 3 alternatively spliced transcript variants from psiTPTE22-HERV by a PCR-based strategy, which use the transcriptional termination signal contained in the HERV element. A 402-nt ORF was contained in the 2 longer transcripts. Western blotting using antibodies produced with chemically synthesized peptide confirmed that a 15-kDa protein was translated from this ORF. RT-PCR results indicated that the ORF-containing transcripts were mainly expressed in psiTPTE22-HERV-expressing samples. Real-time quantitative RT-PCR results showed that expression of the 402-nt ORF was upregulated in normal tissues of kidney, liver, stomach, and lung but downregulated in corresponding tumor tissues. This gene is located near the centromere of chromosome 22 and has a high GC content around the promoter region. Bisulfite sequencing PCR results indicated that it is silenced in cancers by DNA methylation. The expression of psiTPTE22-HERV can be recovered in cancer cells using DNA methylation and histone deacetylase inhibitors. These results suggest psiTPTE22-HERV is regulated epigenetically by DNA methylation. Our study paved the way for further study on an interesting HERV-related human-specific gene, which is silenced in cancers by DNA methylation.

Topics: Azacitidine; Blotting, Western; Chromosomes, Human, Pair 22; Cytoplasm; Decitabine; DNA Methylation; DNA Modification Methylases; Endogenous Retroviruses; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Proteins; Neoplasms; Open Reading Frames; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

Various inhibitors of histone deacetylase (HDAC) activity can sensitize drug resistant cancer cells to chemotherapeutic agents. However, the mechanisms underlying such effects of distinct HDAC inhibitors (HDACi) remain poorly understood. Here we show that both the HDACi trichostatin A and valproic acid induced a sensitization of multidrug-resistant cancer cells to the topoisomerase II inhibitor etoposide/VP16. This effect was associated with increased acetylation of certain lysines on histones H3 and H4, including lysine 16 on histone H4 (H4K16). Overexpression of the histone acetyltransferase hMOF, known to target H4K16, was sufficient to mimic HDACi treatment on sensitization and H4K16 acetylation, and importantly, small-interfering RNA (siRNA)-mediated knockdown of hMOF abolished the HDACi-mediated sensitizing effects as well as the increase in H4K16 acetylation. Conversely, siRNA-mediated knockdown of the H4K16 deacetylase SIRT1 mimicked HDACi treatment whereas overexpression of SIRT1 abolished H4K16 acetylation and significantly reduced the sensitizing effects of HDACi. Interestingly, the effects of hMOF on H4K16 acetylation and sensitization to the topoisomerase II inhibitor could be directly counteracted by exogenous expression of increasing amounts of SIRT1 and vice versa. Our study results suggest that hMOF and SIRT1 activities are critical parameters in HDACi-mediated sensitization of multidrug-resistant cancer cells to topoisomerase II inhibitor and increased H4K16 acetylation.

Topics: Acetylation; Animals; Cell Death; Cell Line, Tumor; DNA Damage; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Etoposide; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lysine; Male; Neoplasms; Sirtuin 1; Topoisomerase II Inhibitors; Valproic Acid

Topors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain.. Mice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1alpha and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A.. These results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.

Topics: Animals; Chromobox Protein Homolog 5; Fibroblasts; Genomic Instability; Heterozygote; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Neoplasms; Small Ubiquitin-Related Modifier Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

Hypoxia is a strong signal for cell migration and invasion in cancer. The reversion-inducing cysteine-rich protein with Kazal motif (RECK), a tumor suppressor, inhibits cancer cell migration and invasion and is frequently silenced in aggressive tumor cells by histone deacetylases (HDAC). However, the effect of RECK silencing in several cancer cells in a hypoxic microenvironment has not been fully delineated. In this report, we investigated whether hypoxia suppressed RECK expression and used HDAC inhibitor (HDACI) inhibition to restore RECK expression to inhibit cancer cell migration and invasion. HDACIs, including trichostatin A (TSA), completely rescued RECK expression, which was suppressed by hypoxia, in the H-Ras-transformed human breast MCF10A and the HT1080 cell lines (human fibrosarcoma). TSA suppressed the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9, induced by hypoxia, and significantly inhibited hypoxia-stimulated migration and invasion of both cancer cells. RECK overexpression significantly inhibited the migration and invasion of cancer cells induced by hypoxia. The hypoxic effect on the migration and invasion of cells was equivalent to the effect seen using the small interfering RNA (siRNA) of RECK under normoxia, suggesting an inhibitory role for RECK in hypoxic conditions. We also showed that siRNA silencing of HDAC1 suppressed hypoxia-induced RECK downregulation and inhibited the migration and invasion of cancer cells. In conclusion, the inhibition of HDAC successfully restored the expression of RECK under hypoxic conditions. This resulted in the inhibition of cancer cell migration and invasion through the repression of MMP-2 and MMP-9 activity.

Topics: Antineoplastic Agents; Cell Hypoxia; Cell Movement; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasms; RNA, Small Interfering; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immune System; Interferons; Melanoma, Experimental; Mice; Neoplasm Transplantation; Neoplasms; Oncolytic Virotherapy; Oncolytic Viruses; Vaccinia virus

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

Topics: Acetylation; Antigens, Neoplasm; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Silencing; Histones; Humans; Hydroxamic Acids; Immunotherapy; Minor Histocompatibility Antigens; Neoplasm Proteins; Neoplasms; Oligopeptides; Promoter Regions, Genetic; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; T-Lymphocytes, Cytotoxic; Transcription, Genetic

Mucin 4 (MUC4) is a high molecular weight transmembrane mucin that is overexpressed in many carcinomas and is a risk factor associated with a poor prognosis. In this study, we show that the DNA methylation pattern is intimately correlated with MUC4 expression in breast, lung, pancreas and colon cancer cell lines. We mapped the DNA methylation status of 94 CpG sites from -3622 to +29 using MassARRAY analysis that utilises base-specific cleavage of nucleic acids. MUC4-negative cancer cell lines and those with low MUC4 expression (eg, A427) were highly methylated near the transcriptional start site, whereas MUC4-positive cell lines (eg, NCI-H292) had low methylation levels. Moreover, 5-aza-2'-deoxycytidine and trichostatin A treatment of MUC4-negative cells or those with low MUC4 expression caused elevation of MUC4 mRNA. Our results suggest that DNA methylation in the 5' flanking region play an important role in MUC4 gene expression in carcinomas of various organs. An understanding of epigenetic changes in MUC4 may contribute to the diagnosis of carcinogenic risk and prediction of outcome in patients with cancer.

Topics: Acetylation; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Enzyme Inhibitors; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Mucin-4; Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The identification and characterization of the first potent histone deacetylase inhibitors constituted a major step, which not only gave a key to researchers worldwide to tackle the unknown universe of cell signalling by protein lysine acetylation, but also opened the way for the development of large series of molecules with important therapeutic properties. Minoru Yoshida, who played a critical role in characterizing the fist potent and specific histone deacetylase inhibitor, trichostatin A, reveals here how and why his work led to this discovery. He also comments on his other outstanding contributions and gives his opinion on the impact of chemical genomics as a key to open the mysteries of the epigenome.

Topics: Acetylation; Enzyme Inhibitors; Epigenesis, Genetic; Genomics; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Models, Biological; Molecular Structure; Neoplasms

The expression of the NKG2D ligands on cancer cells leads to their recognition and elimination by host immune responses mediated by natural killer and T cells. UL16-binding proteins (ULBPs) are NKG2D ligands, which are scarcely expressed in epithelial tumours, favouring their evasion from the immune system. Herein, we investigated the epigenetic mechanisms underlying the repression of ULBPs in epithelial cancer cells. We show that ULBP1-3 expression is increased in tumour cells after exposure to the inhibitor of histone deacetylases (HDACs) trichostatin A (TSA), which enhances the natural killer cell-mediated cytotoxicity of HeLa cells. Our experiments showed that the transcription factor Sp3 is crucial in the activation of the ULBP1 promoter by TSA. Furthermore, by small interfering RNA-mediated knockdown and overexpression of HDAC1-3, we showed that HDAC3 is a repressor of ULBPs expression in epithelial cancer cells. Remarkably, TSA treatment caused the complete release of HDAC3 from the ULBP1-3 promoters. HDAC3 is recruited to the ULBP1 promoter through its interaction with Sp3 and TSA treatment interfered with this association. Together, we describe a new mechanism by which cancer cells may evade the immune response through the epigenetic modulation of the ULBPs expression and provide a model in which HDAC inhibitors may favour the elimination of transformed cells by increasing the immunogenicity of epithelial tumours.

Topics: Apoptosis; Blotting, Western; Cell Proliferation; Chromatin Immunoprecipitation; Cytotoxicity, Immunologic; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Immunoprecipitation; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Killer Cells, Natural; Ligands; Membrane Proteins; Neoplasms; NK Cell Lectin-Like Receptor Subfamily K; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sp3 Transcription Factor; Tumor Cells, Cultured

Suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) is the lead compound of a new class of histone deacetylase (HDAC) inhibitors used as anticancer drugs that have been shown to affect multiple proteins associated with gene expression, cell proliferation, and migration. Studies have also demonstrated the essential role of the hydroxamate moiety of SAHA in HDAC inhibition. The ability of SAHA and its structural analog trichostatin A (TSA) to generate NO upon oxidation was tested directly, by spin trapping of NO using electron paramagnetic resonance spectroscopy, and also indirectly, via the determination of nitrite using the Griess assay. H2O2/metmyoglobin was used to oxidize SAHA and TSA. These studies demonstrate, for the first time, the release of NO from SAHA and its structural analog TSA. We tested the protective effects of SAHA, TSA, and valproic acid (VPA) in mammalian Chinese hamster V79 cells exposed to a bolus of H2O2 for 1 h and monitored the clonogenic cell survival. Both SAHA and TSA afforded significant cytoprotection when co-incubated with H2O2, whereas VPA was ineffective. These studies provide evidence for the release of NO by hydroxamate-containing HDAC inhibitors and their antioxidant effects. Such roles may be an added advantage of this class of HDAC agents used for epigenetic therapies in cancer.

Topics: Animals; Antineoplastic Agents; Antioxidants; Cells, Cultured; Cricetinae; Electron Spin Resonance Spectroscopy; Fibroblasts; Histone Deacetylases; Hydrogen Peroxide; Hydroxamic Acids; Metmyoglobin; Neoplasms; Nitric Oxide; Oxidation-Reduction; Vorinostat

SIMCA classification can be applied to 2D-PAGE maps to identify changes occurring in cellular protein contents as a consequence of illnesses or therapies. These data sets are complex to treat due to the large number of proteins detected. A method for identifying relevant proteins from SIMCA discriminating powers is proposed, based on the Box-Cox transformation coupled to probability papers. The method successfully allowed the identification of the relevant spots from 2D maps.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Endothelium; Humans; Hydroxamic Acids; Mice; Neoplasms; Neuroblastoma; Pancreas; Pancreatic Neoplasms; Probability; Proteins; Proteomics; Sirolimus; Statistical Distributions; Vinblastine

Histone deacetylases (HDAC) modify the architecture of chromatin, leading to decreased gene expression, an effect that is reversed by HDAC inhibition. The balance between deacetylation and acetylation is central to many biological events including the regulation of cell proliferation and cancer but also the differentiation of immune T cells. The effects of HDAC inhibition on the interaction between antitumor effector T cells and tumor cells are not known. Here, we studied presentation of a universal self-tumor antigen, telomerase reverse transcriptase, in human tumor cells during HDAC inhibition. We found that HDAC inhibition with trichostatin A was associated with a decreased presentation and diminished killing of tumor cells by CTLs. Using gene array analysis, we found that HDAC inhibition resulted in a decrease of genes coding for proteasome catalytic proteins and for tapasin, an endoplasmic reticulum resident protein involved in the MHC class I pathway of endogenous antigen presentation. Our findings indicate that epigenetic changes in tumor cells decrease self-tumor antigen presentation and contribute to reduced recognition and killing of tumor cells by cytotoxic T lymphocytes. This mechanism could contribute to tumor escape from immune surveillance.

Topics: Antigen Presentation; Antigens, Neoplasm; Autoantigens; CD8-Positive T-Lymphocytes; Cell Death; Down-Regulation; Epigenesis, Genetic; Gene Expression Profiling; Histone Deacetylase Inhibitors; HLA-A2 Antigen; Humans; Hydroxamic Acids; Membrane Transport Proteins; Neoplasms; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Telomerase; Tumor Cells, Cultured

A possible epigenetic regulation of the two isoenzymes of fructose 1,6-bisphosphatase (FBPase) was studied in liver, muscle, mamma, breast cancer and in different cancer cell lines. Results obtained after bisulfite sequencing revealed a different CpG methylation of both promoters in liver, muscle and breast tissue which is putatively involved in the cell-type specific gene expression of the two enzymes. In tumor cell lines, demethylation with 5-aza-deoxycytidine activated the expression of both isoenzymes. Additional inhibition of histone deacetylase with trichostatin A further increased FBPase mRNA concentrations. Since cancers typically have an abnormal energy metabolism and exhibit a low gluconeogenic phenotype, it was studied whether promoter methylation contributes to the decreased expression of FBPase in breast cancer. When non-malignant and malignant tissue samples from the same patient were compared a correlation between an increase of FBPase promoter methylation and a decrease of FBPase mRNA levels was observed.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Female; Fructose-Bisphosphatase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Middle Aged; Neoplasms; Promoter Regions, Genetic

Cellular populations have been widely observed to respond heterogeneously to perturbation. However, interpreting the observed heterogeneity is an extremely challenging problem because of the complexity of possible cellular phenotypes, the large dimension of potential perturbations, and the lack of methods for separating meaningful biological information from noise. Here, we develop an image-based approach to characterize cellular phenotypes based on patterns of signaling marker colocalization. Heterogeneous cellular populations are characterized as mixtures of phenotypically distinct subpopulations, and responses to perturbations are summarized succinctly as probabilistic redistributions of these mixtures. We apply our method to characterize the heterogeneous responses of cancer cells to a panel of drugs. We find that cells treated with drugs of (dis-)similar mechanism exhibit (dis-)similar patterns of heterogeneity. Despite the observed phenotypic diversity of cells observed within our data, low-complexity models of heterogeneity were sufficient to distinguish most classes of drug mechanism. Our approach offers a computational framework for assessing the complexity of cellular heterogeneity, investigating the degree to which perturbations induce redistributions of a limited, but nontrivial, repertoire of underlying states and revealing functional significance contained within distinct patterns of heterogeneous responses.

Topics: Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Artifacts; Biomarkers, Tumor; Dexamethasone; DNA Replication; Dose-Response Relationship, Drug; Doxorubicin; Genetic Heterogeneity; Glucocorticoids; HeLa Cells; Humans; Hydroxamic Acids; Methotrexate; Microscopy, Fluorescence; Models, Biological; Neoplasms; Paclitaxel; Phenotype; Protein Synthesis Inhibitors; Tubulin Modulators

In order to investigate the epigenetic component of Aiolos regulation, we analyzed the methylation status of its 5' CpG island in relation to histone modifications. Inhibition of CpG methylation restores Aiolos expression, as well as euchromatin-associated markers, in U937 and 1106 mel cell lines. DNA methylation and low levels of euchromatin-associated signatures are observed in U937 and 1106 mel cell lines, while the opposite characterizes Daudi, Jurkat, T and B cells. CpG methylation is not necessary to repress transcription in monocytes and melanocytes where silencing mechanism involves heterochromatin-associated signature. We show that DNA methylation directs Aiolos silencing and chromatin status in tumor cell lines, while in primary cells is mainly regulated by histone modifications.

Topics: Acetylation; Antineoplastic Agents; Azacitidine; Cell Culture Techniques; Cell Line, Tumor; Chromatin; CpG Islands; Decitabine; DNA Methylation; Drug Combinations; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Histones; Humans; Hydroxamic Acids; Ikaros Transcription Factor; Jurkat Cells; Neoplasms; Promoter Regions, Genetic; Transcription Factors; Transcriptional Activation; U937 Cells

Previous studies from our laboratory have demonstrated that thyroid hormones play a key role in cancer progression. In addition, a deaminated form, tetraiodothyroacetic acid (tetrac), that antagonizes the proliferative action of these hormones was found to possess anti-cancer functions through its ability to inhibit cellular proliferation and angiogenesis. The present study was undertaken to investigate whether tetrac could also suppress the development of drug resistance, known as a causative factor of disease relapse. Tetrac was shown to enhance cellular response in vitro to doxorubicin, etoposide, cisplatin, and trichostatin A in resistant tumor cell lines derived from neuroblastoma, osteosarcoma, and breast cancer. The mechanism of action of tetrac did not involve expression of classical drug resistance genes. However, radiolabeled doxorubicin uptake in cells was enhanced by tetrac, suggesting that one or more export mechanisms for chemotherapeutic agents are inhibited. Tetrac was also found to enhance cellular susceptibility to senescence and apoptosis, suggesting that the agent may target multiple drug resistance mechanisms. Tetrac has previously been shown to inhibit tumor cell proliferation in vitro. In vivo studies reported here revealed that tetrac in a pulsed-dose regimen was effective in suppressing the growth of a doxorubicin-resistant human breast tumor in the nude mouse. In this paradigm, doxorubicin-sensitivity was not restored, indicating that (1) the in vitro restoration of drug sensitivity by tetrac may not correlate with in vivo resistance phenomena and (2) tetrac is an effective chemotherapeutic agent in doxorubicin-resistant cells.

Topics: Animals; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Cisplatin; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Etoposide; Humans; Hydroxamic Acids; Mice; Mice, Nude; Neoplasms; Thyroid Hormones; Thyroxine; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Tumour-derived p53 mutants are thought to have acquired 'gain-of-function' properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.

Topics: Apoptosis; Binding Sites; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, p53; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mutation; Neoplasms; Promoter Regions, Genetic; RNA Interference; Tumor Suppressor Protein p53

The human genes MUC2, MUC5AC, MUC5B and MUC6 are clustered on chromosome 11 and encode large secreted gel-forming mucins. The frequent occurrence of their silencing in cancers and the GC-rich structure of their promoters led us to study the influence of epigenetics on their expression. Pre- and post-confluent cells were treated with demethylating agent 5-aza-2'-deoxycytidine and histone deacetylase (HDAC) inhibitor, trichostatin A. Mapping of methylated cytosines was performed by bisulfite-treated genomic DNA sequencing. Histone modification status at the promoters was assessed by chromatin immunoprecipitation assays. Our results indicate that MUC2 was regulated by site-specific DNA methylation associated with establishment of a repressive histone code, whereas hypermethylation of MUC5B promoter was the major mechanism responsible for its silencing. DNA methyltransferase 1 was identified by small interfering RNA approach as a regulator of MUC2 and MUC5B endogenous expression that was potentiated by HDAC2. MUC2 and MUC5B epigenetic regulation was cell-specific, depended on cell differentiation status and inhibited their activation by Sp1. The expression of MUC5AC was rarely influenced by epigenetic mechanisms and methylation of MUC6 promoter was not correlated to its silencing. In conclusion, this study demonstrates the important role for methylation and/or histone modifications in regulating the 11p15 mucin genes in epithelial cancer cells.

Topics: Acylation; Azacitidine; Chromosomes, Human, Pair 11; Decitabine; DNA Methylation; Epigenesis, Genetic; Epithelial Cells; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Molecular Sequence Data; Mucin 5AC; Mucin-2; Mucin-5B; Mucin-6; Mucins; Neoplasms; Tumor Cells, Cultured

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA.

Topics: Butyrates; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Promoter Regions, Genetic; Protein Processing, Post-Translational; RNA, Small Interfering; Urokinase-Type Plasminogen Activator

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.

Topics: Base Sequence; Cell Line, Tumor; CpG Islands; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Neoplasm; Genome, Human; Humans; Hydroxamic Acids; Lymphocytes; Male; Molecular Sequence Data; Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Sequence Analysis, DNA; Transcription, Genetic; Transcriptional Activation

The histone deacetylase inhibitor trichostatin A (TSA) is a promising agent for the treatment of certain types of cancers alone or in synergistic combination with other anticancer agents. One of the advantages of the use of histone deacetylase inhibitors, such as TSA, is that its effects have been found to be more potent toward cancer cells compared to normal cells. The effect of anticancer agents on the immune system, and on lymphocytes in particular, is of major importance to the success of anticancer regimens. In this respect, information documenting the effect of such agents on normal lymphocytes compared to malignant cells may be of significant value for the successful designing of clinical protocols. Moreover, the parameter of age may be a factor in the differential effects of such protocols. Histone deacetylase inhibitors lead to the accumulation of acetylated histones and, depending on the cell type, may induce either apoptosis, cell cycle arrest, or differentiation. Previous work from our lab has shown that TSA induces the accumulation of histone H4 acetylation and apoptosis in human peripheral blood lymphocytes. In light of the above, we have extended our investigation of the effects of TSA on human lymphocytes to include the parameter of age, which has not been previously studied. Our results show that TSA induces apoptosis of lymphocytes from donors of all age groups, but no age-related changes in the levels of apoptosis are observed.

Topics: Acetylation; Adult; Aged; Aged, 80 and over; Aging; Antineoplastic Agents; Apoptosis; Blood Donors; Cell Cycle; Cell Differentiation; Drug Synergism; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Lymphocytes; Male; Middle Aged; Neoplasms

The broad range of expression of cancer-testis antigens in various tumor types makes the proteins encoded by human MAGE gene family promising targets for anticancer immunotherapy. However, a major drawback is their heterogeneous expression. In the current study, we have examined the influence of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylase inhibitor trichostatin A on the expression of MAGE-A1, -A2, -A3, and -A12 genes in different cell lines. Reverse transcription-PCR, Western blot analyses, and immunocytochemical staining show that trichostatin A was able to significantly up-regulate 5-aza-CdR-induced MAGE gene expression. Transient transfection assays with methylated reporter plasmids containing promoter fragments of the different MAGE genes show that trichostatin A was able to overcome gene silencing. In addition, the methylation status of the MAGE promoters was assessed by sodium bisulfite mapping in the various cell lines before and after stimulation with 5-aza-CdR and/or trichostatin A. In contrast to the methylation patterns, which clearly correlated with the basal MAGE RNA transcripts, up-regulation of the MAGE-A mediated by both agents only resulted in a reduction in promoter methylation ranging between 1% and 19%. In conclusion, our data show for the first time that not only hypermethylation but also histone deacetylation is responsible for the mechanism underlying MAGE gene silencing.

Topics: Acetylation; Antigens, Neoplasm; Azacitidine; Blotting, Western; Cell Line, Tumor; Corynebacterium; Decitabine; DNA Methylation; DNA Modification Methylases; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Melanoma-Specific Antigens; Neoplasm Proteins; Neoplasms; Promoter Regions, Genetic

In a genome-wide screen for putative tumor suppressor genes, the EBF3 locus on the human chromosome 10q26.3 was found to be deleted or methylated in 73% of the examined cases of brain tumors. EBF3 is expressed in normal brain but is silenced in brain tumors. Therefore, it is suggested that EBF3 is a tumor suppressor. However, it remains unknown whether inactivation of EBF3 locus also occurs in other types of tumors and what functions of EBF3 underlie EBF3-mediated tumor suppression. We show here that expression of EBF3 resulted in cell cycle arrest and apoptosis. The expression of cyclin-dependent kinase inhibitors was profoundly affected with early activation and then repression of p21(cip1/waf1) and persistent activation of both p27(kip1) and p57(kip2), whereas genes involved in cell survival and proliferation were suppressed. EBF3 bound directly to p21(cip1/waf1) promoter and regulated transcription from both p21(cip1/waf1) and p27(kip1) promoters in reporter assays. Apoptosis occurred 48 hours after EBF3 expression with caspase-3 activation. Silencing of the EBF3 locus was observed in brain, colorectal, breast, liver, and bone tumor cell lines and its reactivation was achieved on treatment with 5-aza-2'-deoxycytidine and trichostatin A in a significant portion of these tumor cells. Therefore, EBF3 regulates a transcriptional program underlying a putative tumor suppression pathway.

Topics: Apoptosis; Azacitidine; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Chromosomes, Human, Pair 10; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Neoplasms; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transcription, Genetic

Thymidylate synthase (TS) overexpression is a key determinant of 5-fluorouracil (5-FU) resistance in human cancer cells. TS is also acutely up-regulated with 5-FU treatment, and, thus, novel strategies targeting TS down-regulation seem to be promising in terms of modulating 5-FU resistance. Here, we report that histone deacetylase inhibitors can reverse 5-FU resistance by down-regulating TS. By using cDNA microarrays and validation experiments, we found that trichostatin A reduced the expression of both TS mRNA and TS protein. Cotreatment with trichostatin A and cycloheximide restored TS mRNA expression, suggesting that TS mRNA is repressed through new protein synthesis. On the other hand, TS protein expression was significantly reduced by lower doses of trichostatin A (50 nmol/L). Mechanistically, TS protein was found to interact with heat shock protein (Hsp) complex, and trichostatin A treatment induced chaperonic Hsp90 acetylation and subsequently enhanced Hsp70 binding to TS, which led to the proteasomal degradation of TS protein. Of note, combined treatment with low-dose trichostatin A and 5-FU enhanced 5-FU-mediated cytotoxicity in 5-FU-resistant cancer cells in accordance with TS protein down-regulation. We conclude that a combinatorial approach using histone deacetylase inhibitors may be useful at overcoming 5-FU resistance.

Topics: Acetylation; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cycloheximide; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Fluorouracil; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Neoplasms; Proteasome Endopeptidase Complex; RNA, Messenger; Thymidylate Synthase

Microtubule-disrupting agents such as the taxanes comprise some of the most clinically useful chemotherapeutic agents and invoke the spindle checkpoint in proliferating cells. A robust spindle checkpoint in turn may forestall mitotic catastrophe, potentially providing a mechanism that permits cancer cells to survive transient exposure to these drugs. Previous reports on G2-M cell cycle progression by histone deacetylase inhibitors suggested a potential role in modulating the therapeutic efficacy of microtubule-disrupting agents. As both classes of agents are generally administered in clinical trials as pulse treatments, we investigated in human cancer cells the effects of brief treatments with the histone deacetylase inhibitor trichostatin A (TSA) alone or with nocodazole or paclitaxel (Taxol) on cell cycle progression and the spindle checkpoint. Treatment of synchronized cells with 200 ng/ml of TSA alone for eight hours to completely block class I and II HDACs did not interfere with progression into mitosis with chromosomal condensation as confirmed by MPM-2 expression. TSA treatment at this concentration surprisingly did not interfere with formation of the mitotic spindle or centrosomal separation, but instead led to missegregation of chromosomes, suggesting effects on the spindle checkpoint. Consistent with this hypothesis, TSA abrogated the phosphorylation and kinetochore localization of the mitotic checkpoint protein BubR1 and the phosphorylation of histone H3 after paclitaxel and nocodazole treatment. These effects in turn led to rapid cell death and considerably reduced clonogenic survival. These results together suggest that by inactivating the spindle checkpoint, TSA can potentiate the lethal effects of microtubule-disrupting drugs, a strategy that might be usefully exploited for optimizing anticancer therapy.

Topics: Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cell Survival; Centrosome; Drug Therapy, Combination; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; HeLa Cells; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Kinetochores; Microtubules; Mitosis; Neoplasms; Nocodazole; Paclitaxel; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Spindle Apparatus

The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (DeltaCH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of approximately 35-50% of global HIF-1-responsive gene expression, whereas another HIF transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSA(S)) accounts for approximately 70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription.

Topics: Amino Acid Sequence; Animals; Basic Helix-Loop-Helix Transcription Factors; CREB-Binding Protein; Gene Expression Profiling; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Mice; Molecular Sequence Data; Neoplasms; p300-CBP Transcription Factors; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Sequence Alignment; Survival Rate; Transcription, Genetic; Transcriptional Activation

DNA topoisomerase II inhibitors and poisons are among the most efficacious drugs for the treatment of cancer. Sensitivity of cancer cells to the cytotoxic effects of topoisomerase II targeting agents is thought to depend on the expression of the topoisomerase IIalpha isoform, and drug resistance is often associated with loss or mutation of topoisomerase IIalpha. Histone deacetylase inhibitors (HDACi) are a novel class of compounds that potentiate the antitumor effects of topoisomerase II-targeting agents.. The interaction between HDACi and topoisomerase II-targeting agents in cancer cells was evaluated as a function of topoisomerase IIalpha and topoisomerase IIbeta expression. Topoisomerase II isoforms were selectively depleted using small interfering RNA and antisense. Drug-induced formation of cleavable complexes involving topoisomerase IIalpha and topoisomerase IIbeta was evaluated by trapped-in-agarose DNA immunostaining and band depletion assays in the presence and absence of HDACi.. Preexposure to HDACi increased the cytotoxicity of topoisomerase II poisons. This was associated with a down-regulation of topoisomerase IIalpha expression but had no effects on topoisomerase IIbeta. In the setting of HDACi-induced chromatin decondensation and topoisomerase IIalpha depletion, topoisomerase II poison cytotoxicity was mediated through topoisomerase IIbeta cleavable complex formation. The HDACi-induced sensitization was also observed in cells with target-specific resistance to topoisomerase II poisons.. The recruitment of topoisomerase IIbeta as a target may overcome primary or emergent drug resistance to topoisomerase II-targeting agents and hence may broaden the applicability of this important class of anticancer agents.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Butyrates; Chromatin; DNA Topoisomerases, Type II; DNA-Binding Proteins; DNA, Antisense; Drug Interactions; Drug Synergism; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Profiling; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mitoxantrone; Neoplasms; Oligonucleotide Array Sequence Analysis; RNA, Small Interfering; Teniposide; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Valproic Acid

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Topics: Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney Neoplasms; Liver Neoplasms; Nasopharyngeal Neoplasms; Neoplasms; Oligonucleotide Array Sequence Analysis; Tumor Cells, Cultured; Vorinostat

Radiotherapy is an effective treatment for head and neck, skin, anogenital, and breast cancers. However, radiation-induced skin morbidity limits the therapeutic benefits. A low-toxicity approach to selectively reduce skin morbidity without compromising tumor killing by radiotherapy is needed. We found that the antitumor agents known as histone deacetylase (HDAC) inhibitors (phenylbutyrate, trichostatin A, and valproic acid) could suppress cutaneous radiation syndrome. The effects of HDAC inhibitors in promoting the healing of wounds caused by radiation and in decreasing later skin fibrosis and tumorigenesis were correlated with suppression of the aberrant expression of radiation-induced transforming growth factor beta and tumor necrosis factor alpha. Our findings implicate that the inhibition of HDAC may provide a novel strategy to increase the therapeutic gain in cancer radiotherapy by not only inhibiting tumor growth but also protecting normal tissues.

Topics: Acetylation; Animals; Antineoplastic Agents; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Enzyme Inhibitors; Female; Fibrosis; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms; Phenylbutyrates; Radiation Injuries; Radiotherapy; Rats; Rats, Sprague-Dawley; Ribonucleases; Skin; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Valproic Acid; Wound Healing

We tested the hypothesis that the effects on gene expression of altered DNA methylation by 5-aza-2'-deoxycytidine (5-aza-CdR) and genetic (DNMT knockout) manipulation of DNA are similar, and distinct from Trichostatin A (TSA)-induced chromatin decondensation. Surprisingly, the effects of 5-aza-CdR were more similar to those of TSA than to DNMT1, DNMT3B, or double DNMT somatic cell knockout. Furthermore, the effects of 5-aza-CdR were similar at one and five days exposure, suggesting active demethylation or direct influence of both drugs on the stability of methylation and/or chromatin marks. Agents that induce gene activation through hypomethylation may have unintended consequences, since nearly as many genes were downregulated as upregulated after demethylation. In addition, a 75 kb cluster of metallothionein genes was coordinately regulated.

Topics: Algorithms; Apoptosis; Azacitidine; Cell Division; Cell Line, Tumor; Cluster Analysis; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Genome; Humans; Hydroxamic Acids; Metallothionein; Methyltransferases; Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reproducibility of Results; RNA, Messenger; Transcriptional Activation

Epigenetic regulation of gene expression significantly influences cell growth and differentiation. Here we show that epigenetic silencing of Fas determines tumor growth in vivo and apoptotic sensitivity in vitro. In established tumors with epigenetically repressed Fas, restoration of Fas activity either by transfection of fas or treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase, suppresses tumor growth and restores chemosensitivity. The TSA-dependent chemosensitivity and tumor growth control require both tumor Fas and the host NK (natural killer) cell functions. This work demonstrates the importance of epigenetic modification of Fas in tumor progression and immune evasion, and emphasizes the essential interplay between Fas and innate immunity in the control of chemoresistant tumors.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Disease Progression; Drug Resistance, Neoplasm; fas Receptor; Flow Cytometry; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Hydroxamic Acids; Immunity, Innate; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Mice, SCID; Neoplasms; Time Factors; Transfection; Tumor Cells, Cultured

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P < 0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.

Topics: Azacitidine; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 10; Caspase 8; Caspase 9; Caspases; Child; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Amplification; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Neoplasm Proteins; Neoplasms; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins

Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing's syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.

Topics: Adenoma; Adrenocorticotropic Hormone; Carcinoma, Small Cell; Carrier Proteins; Cell Cycle Proteins; Cell Line; CpG Islands; DNA Methylation; DNA-Binding Proteins; E2F Transcription Factors; Enzyme Inhibitors; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Neoplasms; Organ Specificity; Pituitary Neoplasms; Plasmids; Pro-Opiomelanocortin; Promoter Regions, Genetic; Retinoblastoma-Binding Protein 1; Sequence Analysis, DNA; Transcription Factor DP1; Transcription Factors; Transcription, Genetic; Transfection

Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential.. Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy.. OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms.. These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

Topics: Alkenes; Cell Survival; Chromatin; Histones; HT29 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; Neoplasms; Okadaic Acid; Polyenes; Pyrones; Radiation Tolerance

Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes, genes on the female inactive X chromosome and tumour-suppressor genes inactivated in cancer cells. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.

Topics: Adaptor Proteins, Signal Transducing; Azacitidine; Carrier Proteins; Cell Cycle Proteins; CpG Islands; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MutL Protein Homolog 1; Neoplasm Proteins; Neoplasms; Nuclear Proteins; Promoter Regions, Genetic; Tissue Inhibitor of Metalloproteinase-3; Transcriptional Activation; Tumor Cells, Cultured; Tumor Suppressor Proteins