Histone deacetylase inhibitor

Libraries of compounds are increasingly becoming commercially available for the use of individual academic laboratories. A high-throughput system based on a stably integrated transcriptional reporter was used to screen a library of random compounds to identify agents that conferred robust augmentation of a signal transduction pathway. A novel histone deacetylase (HDAC) inhibitor, termed scriptaid, conferred the greatest effect, a 12- to 18-fold augmentation. This facilitation of transcriptional events was generally applicable to exogenous gene constructs, including viral and cellular promoters, different cell lines and reporter genes, and stably integrated and transiently introduced sequences. Scriptaid did not interfere with a further induction provided by stimulation of the cognate signal transduction pathway (transforming growth factor beta/Smad4), which implied the functional independence of ligand-stimulated transcriptional activation and histone acetylation states in this syst...

Background: DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells.

Purpose: The expression of survivin, a member of the inhibitor-of-apoptosis protein family, is elevated in many types of human cancer. High survivin expression has been associated with poor patient prognosis and tumor resistance to chemotherapy and radiotherapy. The purpose of this study was to compare the radiosensitizing effects of five agents that target survivin on their relative ability to downregulate survivin expression. Methods and Materials: The human epidermoid carcinoma cell line A431 was treated with adenoviral-mediated wild-type p53, antisense to survivin, the mitogen-activated protein kinase inhibitor PD98059, the cyclin-dependent kinase inhibitor Purvalanol A, or the histone deacetylase inhibitor trichostatin A. The radiosensitizing effects of these treatments were determined by clonogenic survival curve analysis and their abilities to suppress survivin expression by Western blot analysis. Results: All the strategies were shown to radiosensitize A431 cells. This effect correlated with their abilities to downregulate survivin. Conclusion: Expression of survivin appears to confer a radioresistant phenotype that can be overcome using several clinically achievable strategies that target survivin either specifically or nonspecifically. © 2006 Elsevier Inc.

Trifluoroacetylthiophene carboxamides have recently been reported to be class II HDAC inhibitors, with moderate selectivity. Exploration of replacements for the carboxamide with bioisosteric pentatomic heteroaromatic like 1,3,4-oxadiazoles, 1,2,4-oxadiazoles and 1,3-thiazoles, led to the discovery that 2-trifluoroacetylthiophene 1,3,4-oxadiazole derivatives are very potent low nanomolar HDAC4 inhibitors, highly selective over class I HDACs , and moderately stable in HCT116 cell culture.

We analysed the antiproliferative activity of various histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) on human breast cancer cells. We observed a lower sensitivity to HDAC inhibition for oestrogen receptor negative (ER ) versus positive (ER+) cell lines. This differential response was associated neither with a modification of drug efflux via the multidrug resistance system nor with a global modification of histone acetyltransferase (HAT)/HDAC activities. In contrast, we demonstrated that in ER+ breast cancer cells the p21 WAF1/CIP1 gene was more sensitive to TSA regulation and was expressed at higher levels. These differences were observed both in transient transfection experiments and on the endogenous p21 WAF1/CIP1 gene. The Sp1 transcription factor, which was shown to interact in vitro with both class I and class II HDACs, is sufficient to confer the differential sensitivity to TSA and participated in the control of p21 WAF1/CIP1 basal expression. Finally, re-expression of ER following adenoviral infection of ER breast cancer cells increased both p21 WAF1/CIP1 protein accumulation and the growth inhibitory activity of TSA. Altogether, our results highlight the key role of ER and p21 WAF1/CIP1 gene expression in the sensitivity of breast cancer cells to hyperacetylating agents. 179, 41-53 R MARGUERON and others · ER increases sensitivity to HDAC inhibitors 42

NMR-based metabolic profiling was used to assess the effect of Trichostatin A (TSA) treatment on the endogenous metabolome of cultured primary hepatocytes. TSA-treated cultures were metabolically distinct from nontreated cells and the time (44 and 92 h) related effect on metabolism was also reduced. Our data suggest that TSA treatment improves the model for the study of hepatic metabolism and as an in vitro carcinogenicity screening assay.

Remodelling of the extracellular matrix (ECM) is an essential physiological process in, e.g. development, wound healing and angiogenesis. Aberrant ECM turnover is also associated with a number of pathological processes such as joint destruction in the arthritides, tumour metastasis, and fibrosis . Central to the turnover of ECM is the matrix metalloproteinase (MMP) family; these number 23 enzymes in man which, between them, have the capability of degrading the majority of ECM proteins . Four tissue inhibitors of metalloproteinases (TIMPs) safeguard ECM integrity by virtue of their ability to inhibit the MMPs [3]. The TIMPs display a high degree of functional overlap, but show dramatic differences in their Abbreviations AP-1, activating protein-1; EMSA, electrophoretic mobility-shift assay; Ets, avian erythroblastosis virus E26 (v-ets) oncogene homologue; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; MMP, matrix metalloproteinase; NaB, sodium butyrate; PMA, phorbol myristate acetate; TIMP, tissue inhibitor of metalloproteinases; TGF, transforming growth factor; TSA, trichostatin A; VPA, valproic acid. 1912 FEBS Journal 272 (2005) 1912-1926 ª 2005 FEBS D. A. Young et al. HDAC inhibitors and Timp-1 expression FEBS Journal 272 (2005) 1912-1926 ª 2005 FEBS HDAC inhibitors and Timp-1 expression D. A. Young et al. 1926 FEBS Journal 272 (2005) 1912-1926 ª 2005 FEBS HDAC inhibitors and Timp-1 expression D. A. Young et al.

Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible.

Histone deacetylase inhibitors (HDACi) are endowed with plethora of biological functions including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. Parsing the structure-activity relationship (SAR) for each disease condition is vital for long-term therapeutic applications of HDACi. We report in the present study specific cap group substitution patterns and spacer-group chain lengths that enhance the antimalarial and antileishmanial activity of aryltriazolylhydroxamates-based HDACi. We identified many compounds that are several folds selectively cytotoxic to the plasmodium parasites compared to standard HDACi. Also, a few of these compounds have antileishmanial activity that rivals that of miltefosine, the only currently available oral agent against visceral leishmaniasis. The anti-parasite properties of several of these compounds tracked well with their anti-HDAC activities. The results presented here provide further evidence on the suitability of HDAC inhibition as a viable therapeutic option to curb infections caused by apicomplexan protozoans and trypanosomatids.

Neurodegenerative disorders (NDs) such as Huntington's disease, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Friedreich's ataxia, and others are multi-factorial illnesses, in which many pathways (still poorly understood) act serially and in parallel to give a determined pathologic phenotype. Thus, presently there are no effective cures for these diseases. Some phenotypic as well as mechanistic features, common to the most of NDs, can be linked to epigenetic defects, that can lead to alteration of acetylation homeostasis and impairment of the histone acetyltransferase (HAT): histone deacetylase (HDAC) balance. Here we survey most of the recent applications of HDAC inhibitors in the cited NDs, and we make the point of our (up to now) knowledge about the involvement of singular HDAC/SIRT isoform in NDs and other CNS pathologies.

Purpose: To evaluate the potential utility of histone deacetylase inhibitors (HDACi) for treatment of retinoblastoma (RB). Experimental Design: Growth-inhibitory effects of HDACi [trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), or MS-275] were assessed in human and transgenic murine RB cells. Effects of TSA and MS-275 were also assessed in combination with standard therapeutic agents for RB. Proapoptotic effects of MS-275 and TSA were evaluated by caspase-3/7 activity, Annexin V translocation, and/or Bim expression analyses. Effects of MS-275 on cell cycle distribution and reactive oxygen species levels were determined by flow cytometry. Retinal tissue morphology was evaluated in mice after local administration of MS-275. Analysis of retinal acetyl-histone levels was used to assess MS-275 delivery after systemic administration. Therapeutic effects of MS-275 were determined in transgenic mouse and rat ocular xenograft models of RB after i.p. injection of 20 mg/kg every other day for 21or 13 days, respectively. Results:TSA, SAHA, and MS-275 dose dependently reduced RB cell survival.TSA and MS-275 showed additive growth-inhibitory effects in combination with carboplatin, etoposide, or vincristine. TSA and MS-275 increased caspase-3/7 activity. MS-275 increased Annexin V membrane translocation and induced G 1 arrest. Cytotoxicity of MS-275 was dependent on increased reactive oxygen species levels and was reversed by antioxidant pretreatment. Intraocular administration of 1 AL of 10 Amol/L MS-275 did not alter ocular tissue morphology. Increased acetyl-histone levels confirmed MS-275 delivery to retinal tissue after systemic administration. MS-275 significantly reduced tumor burden in both mouse and rat models of RB. Conclusions: HDACi should be considered for clinical trials in children with RB.

Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos.

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza R (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.

Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http:// clinicaltrials.gov/as no. NCT00519181.

Histone Deacetylases (HDACs) are intimately involved in the epigenetic regulation, and thus are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin have been recently approved for the cancer treatment. Since the screening and detailed characterization of HDAC inhibitors has been time consuming, we synthesized Coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K d ) and the dissociation off-rates (k off ) of the enzyme-inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ ex = 325 nm, λ em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme's tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented herein is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K d and k off values.

Aryloxopropenyl)pyrrolyl hydroxamates were recently reported by us as first examples of class II-selective HDAC inhibitors and can be useful tools to probe the biology of such enzymes. Molecular modelling and 3-D QSAR studies have been performed on a series of 25 (aryloxopropenyl)pyrrolyl hydroxamates to gain insights about their activity and selectivity against both maize HD1-B and HD1-A, two enzymes homologous of mammalian class I and class II HDACs, respectively. The studies have been accomplished by calculating alignment-independent descriptors (GRIND descriptors) using the ALMOND software. Highly descriptive and predictive 3-D QSAR models were obtained using either class I or class II inhibitory activity displaying r 2 /q 2 values of 0.96/0.81 and 0.98/0.85 for HD1-B and HD1-A, respectively. A deeper inspection revealed that in general a bent molecular shape structure is a prerequisite for HD1-A-selective inhibitory activity, while straight shape molecular skeleton leads to selective HD1-B compounds. The same conclusions could be achieved by molecular docking studies of the most selective inhibitors.

Chromatin modifications have gained immense significance in the past few decades as key regulators of gene expression. The enzymes responsible for these modifications along with the other non-histone proteins, remodeling factors and small RNAs modulate the chromatin dynamicity, which in turn directs the chromatin function. A concerted action of different modifying enzymes catalyzes these modifications, which are read by effector modules and converted to functional outcomes by various protein complexes. Several small molecules in the physiological system such as acetyl CoA, NAD+, and ATP are actively involved in regulating these functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of the existence of a network, ‘the epigenetic language’ involving cross talk among different modifications that could regulate cellular processes like transcription, replication and repair. Hence, these modifications are essential for the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition or disease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymes could be an attractive therapeutic strategy, popularly referred to as ‘Epigenetic therapy.’ Although this field is currently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights the modulators of the other modifications namely histone acetylation, lysine methylation and arginine methylation and argues in favor of their therapeutic potential.► Histone modifications are important regulators of gene expression in both physiological and pathophysiological states. ► The histone modifications mainly acetylation, lysine methylation and arginine methylation together establish an epigenetic network. ► Histone acetylation aberrations have been reported in diseases such as cancer, diabetes, inflammatory disorders as well as neurodegenerative states. ► Acetylation inhibitors are showing great promise as potential therapeutic agents due to their anti-inflammatory, anti-cancer as well as anti-HIV properties. ► Since the epigenetic marks exist as a network, and there are evidences implicating their role in different diseases, potential therapeutic strategy should be towards combinatorial targeting of these modifications.

The effects of histone deacetylase inhibitor Trichostatin A (TSA) on connexin (Cx) expression and gap junctional intercellular communication (GJIC) were investigated in primary cultures of adult rat hepatocytes. GJIC was monitored by using the scrape-loading/ dye transfer method. Immunoblotting and immunocytochemistry were used to investigate Cx protein levels and localization. Cx gene expression was studied by means of quantitative reverse transcriptase-polymerase chain reaction. TSA increased Cx32 protein levels and affected negatively the Cx26 protein levels. The latter was preferentially located in the cytosol of cultured cells. TSA also promoted the appearance of Cx43 in the nuclear compartment of primary cultured hepatocytes. Overall, this resulted in enhanced GJIC activity. It is important to note that the time of onset of TSA treatment was crucial for the extent of its outcome and that the effects of TSA on Cx protein levels occurred independently of transcriptional changes. TSA differentially affects Cx proteins in primary rat hepatocyte cultures, suggesting distinct regulation and/or distinct roles of the different Cx species in the control of hepatic homeostasis. TSA enhances GJIC between primary cultured rat hepatocytes, an interesting finding supporting its use to further optimize liver-based in vitro models for pharmacotoxicological purposes.

Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1 ؉ , a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1 ؉ is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1 ؉ in modulating the nuclear import of Tf1 virus-like particles. 2351 on December 12, 2014 by guest http://mcb.asm.org/ Downloaded from have been shown to use distinct domains to form histone deacetylase complexes and to interact with specific DNA-binding proteins (8, 79). Thus, Sin3p has been proposed to play an important role in recruiting the histone deacetylase activity to specific regions of chromosomes, causing targeted changes in chromatin structure and expression .

Sixty-one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and 13 of their tetrahydro-2-oxopyrimidin-1(2H)-yl analogues (PPB-SOs) were prepared and biologically evaluated. The antiproliferative activities of PIB-SOs on 16 cancer cell lines are in the nanomolar range and unaffected in cancer cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the P-glycoprotein. None of the PPB-SOs exhibit significant antiproliferative activity. PIB-SOs block the cell cycle progression in the G 2 /M phase and bind to the colchicine-binding site on β-tubulin leading to cytoskeleton disruption and cell death. Chick chorioallantoic membrane tumor assays show that compounds 36, 44, and 45 efficiently block angiogenesis and tumor growth at least at similar levels as combretastatin A-4 (CA-4) and exhibit low to very low toxicity on the chick embryos. PIB-SOs were subjected to CoMFA and CoMSIA analyses to establish quantitative structureÀactivity relationships.

Histone deacetylase inhibitors (HDACIs) are a class of antineoplastic agents previously demonstrating preclinical chemosensitizing activity against drug-resistant cancer cells and mouse xenografts. However, whereas clinical studies have shown efficacy ...

The transforming growth factor-B (TGF-B) superfamily members, TGF-B, activin, and inhibin, all have prominent roles in regulating normal ovarian function. Betaglycan, or the type III TGF-B receptor, is a coreceptor that regulates TGF-B, activin, and inhibin signaling. Here, we show that betaglycan expression is frequently decreased or lost in epithelial derived ovarian cancer at both the mRNA and protein level, with the degree of loss correlating with tumor grade. Treatment of ovarian cancer cell lines with the methyltransferase inhibitor 5-aza-2-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in significant synergistic induction of betaglycan message levels and increased betaglycan protein expression, indicating that epigenetic silencing may play a role in the loss of betaglycan expression observed in ovarian cancer. Although restoring betaglycan expression in Ovca429 ovarian cancer cells is not sufficient to restore TGF-B-mediated inhibition of proliferation, betaglycan significantly inhibits ovarian cancer cell motility and invasiveness. Furthermore, betaglycan specifically enhances the antimigratory effects of inhibin and the ability of inhibin to repress matrix metalloproteinase levels in these cells. These results show, for the first time, epigenetic regulation of betaglycan expression in ovarian cancer, and a novel role for betaglycan in regulating ovarian cancer motility and invasiveness. [Cancer Res 2007;67(11):5231-8]

Purpose The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) enhances taxolinduced antitumor eVects against some human cancer cells. The aim of this study is to investigate whether SAHA can enhance taxol-induced cell death against human breast cancer cells and to illustrate the mechanism in detail. Methods A panel of eight human breast cancer cell lines and an immortalized human breast epithelial cell line were used to determine the inhibitory eVects of SAHA, taxol, or their combination by MTT assay. The eVects of SAHA with or without taxol on cell cycle distributions, apoptosis, and protein expressions were also examined. The inhibitory eVects on tumor growth were characterized in vivo in BALB/c nude mice bearing a breast cancer xenograft model. Results Taxol-resistant and multi-resistant breast cancer cells were as sensitive to SAHA as taxol-sensitive breast cancer cells. A dose-dependent synergistic growth inhibition was found in all the tested breast cancer cell lines treated with the SAHA/taxol combinations. The synergetic eVect was also observed in the in vivo xenograft tumor model. The cell cycle analysis and apoptosis assay showed that the synergistic eVects resulted from enhanced G2/M arrest and apoptosis. Conclusions SAHA increased the anti-tumor eVects of taxol in breast cancer in vitro and in vivo. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.

Background Breast cancer is the second leading cause of cancer related deaths among females worldwide. Berbamine (BER), a kind of bis-benzylisoquinoline alkaloid, has been used to treat clinical patients with inflammation and cancer for many years in China. The purpose of this study is to investigate the activity of BER against highly-metastatic human breast cancer and its molecular mechanisms of action. Results In our study, we found that BER inhibits growth of highly-metastatic human breast cancer cell lines MDA-MB-231 and MDA-MB-435S cells dose-dependently and time-dependently. The sera from BER-treated rats suppress the growth of MDA-MB-231 cells. BER shows synergistic effects with some existing anticancer agents such as trichostatin A (TSA, the histone deacetylase inhibitor), celecoxib (the inhibitor of COX-2), and carmofur against the growth of MDA-MB-231 cells. BER also displays the strong activity of inducing apoptosis in both estrogen receptor-negative MDA-MB-231 cells and estrogen receptor-alpha-positive MCF-7 breast cancer cells, but not in normal human mammary epithelial cell line MCF10A. BER down-regulates anti-apoptotic protein Bcl-2 levels and up-regulates pro-apoptotic protein Bax expressions in MDA-MB-231 and MDA-MB-435S cells. BER also has synergistic effects with anticancer agents trichostatin A, celecoxib and/or carmofur on reducing Bcl-2/Bax ratios and VEGF secretions in MDA-MB-231 cells. In addition, BER significantly suppresses cell migration and invasion, as well as decreases pro-MMP-9/pro-MMP-2 activation in breast cancer cells. Furthermore, BER suppresses Akt and nuclear factor κB signaling by reducing the phosphorylation of c-Met and Akt, and inhibiting their downstream targets such as nuclear factor κB p-65, Bcl-2/Bax, osteopontin, VEGF, MMP-9 and MMP-2 on protein and/or mRNA levels in breast cancer cells. Conclusion Our findings have showed that BER suppresses the growth, migration and invasion in highly-metastatic human breast cancer cells by possibly inhibiting Akt and NF-κB signaling with their upstream target c-Met and downstream targets Bcl-2/Bax, osteopontin, VEGF, MMP-9 and MMP-2. BER has synergistic effects with anticancer agents trichostatin A, celecoxib and carmofur on inhibiting the growth of MDA-MB-231 cells and reducing the ratio of Bcl-2/Bax and/or VEGF expressions in the cancer cells. These findings suggest that BER may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human breast cancer and other cancers.

Candida albicans is able to establish mucosal and invasive diseases by means of different virulence factors that are frequently regulated by epigenetic mechanisms, including the acetylation-deacetylation of histones and of other regulatory proteins. The focus of our work was on understanding the possible effects of several histone deacetylase inhibitors (HDACi) on the expression of phenotypes that are associated with virulence and pathogenicity in C. albicans, such as adhesion to epithelial cells and the yeast to hypha transition. Some of the HDACi used for experiments caused a 90% reduction in the adherence of C. albicans to human cultured pneumocytes and significantly inhibited serum-induced germination. Inhibition of germination was correlated with a significant reduction in transcription of EFG1. Inhibition appeared less evident when an HDA1-deficient strain was tested. These results suggest that selective and specific HDACi could prove to be a valid approach for selected at-risk patients in the combined treatment of infections caused by C. albicans.

Cortical GABAergic dysfunction, a hallmark of both schizophrenia (SZ) and bipolar (BP) disorder pathophysiologies may relate to the hypermethylation of GABAergic gene promoters (i.e., reelin and GAD67). Benefits elicited by a combination of atypical antipsychotics with valproate (VPA) (a histone deacetylase inhibitor that may also activate brain DNA demethylation) in SZ or BP disorder treatment prompted us to investigate whether the beneficial action of this association depends on induction of a putative DNA demethylase activity. To monitor this activity, we measured the ratio of 5-methyl cytosine to unmethylated cytosine in reelin and GAD67 promoters in the mouse frontal cortex and striatum. We compared normal mice with mice pretreated with L-methionine (5.2 mmol/kg s.c. twice a day for 7 days) to hypermethylate promoters, including reelin and GAD67. Clinically relevant doses of clozapine (CLZ) (3.8 to 15 mol/kg twice a day s.c. for 3 days) and sulpiride (SULP) (12.5 to 50 mol/kg twice a day for 3 days) but not clinically relevant doses of haloperidol (HAL) (1.3 to 4 mol/kg twice a day s.c. for 3 days) or olanzapine (OLZ) (4 to 15 mol/kg twice a day for 3 days) exhibited dose-related increases in the cortical and striatal demethylation of hypermethylated reelin and GAD67 promoters. These effects of CLZ and SULP were dramatically potentiated by a clinically relevant VPA dose (0.5 mmol/kg twice a day for 3 days). By activating a DNA demethylase, the association of CLZ or SULP with VPA may facilitate a chromatin remodeling that normalizes the GABAergic gene expression down-regulation detected in the telencephalic regions of SZ and BP patients.

We analysed the antiproliferative activity of various his- tone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) on human breast cancer cells. We observed a lower sensitivity to HDAC inhibition for oestrogen receptor negative (ER) versus positive (ER+) cell lines. This differential response was associated neither with a modifi- cation of drug efflux via the multidrug resistance system nor with

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 mumol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, an...

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFIIH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 6-4PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some TTD/XPD mutations affect the recruitment of TFIIH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFIIH complexes carrying an NH 2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFIIH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFIIH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

Histone deacetylases (HDACs) are enzymes involved in tumor genesis and development. Herein, we report a novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as HDACs inhibitors. The preliminary biological screening showed that most of our compounds exhibited potent inhibitory activity against HDACs. Within this series, five compounds, 13a (IC 50 = 0.58 ± 0.10 lM), 7d (IC 50 = 1.00 ± 0.16 lM), 8l (IC 50 = 1.06 ± 0.14 lM), 7i (IC 50 = 1.17 ± 0.19 lM) and 7a (IC 50 = 1.29 ± 0.15 lM) possessed better HDACs inhibitory activity than Vorinostat (IC 50 = 1.48 ± 0.20 lM). So these five compounds could be used as novel lead compounds for further design of HDACs inhibitors. The anti-proliferative activities of a few compounds and the structure-activity relationships are also briefly discussed.

Bradyzoite-to-tachyzoite conversion plays a role in the pathogenesis of recrudescence of ocular toxoplasmosis and disease in immunocompromised persons. The currently available medicines are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. A previous study showed that the histone deacetylase inhibitor FR235222 has a dramatic effect on tachyzoite growth and induces tachyzoite-to-bradyzoite conversion in vitro. The present study shows that FR235222 can target in vitro-converted cysts and bradyzoites. Moreover, the compound is active on ex vivo T. gondii cysts. Free bradyzoites isolated after lysis of the cell wall did not proliferate in vitro when the cyst was treated with FR235222. The results imply that this compound is able to cross the T. gondii cystic cell wall. Fluorescent labeling shows that the compound impairs the capacity of the bradyzoites to convert without damaging the cyst wall integrity. In vivo inoculation of formerly treated cysts fails to infect mice when these cysts were treated with FR235222. We used our structural knowledge of FR235222 and its target, T. gondii HDAC3, to synthesize new FR235222 derivative compounds. We identified two new molecules that are highly active against tachyzoites. They harbor a better selectivity index that is more suitable for a future in vivo approach. These results identify FR235222 and its derivatives as new lead compounds in the range of therapeutics available for acute and chronic toxoplasmosis.

Effects of the PI-3 kinase inhibitor LY294002 (LY) have been examined in relation to responses of human leukemia cells to histone deacetylase inhibitors (HDIs). Coexposure of U937 cells for 24 h to marginally toxic concentrations of LY294002 (e.g., 30 lm) and sodium butyrate (SB; 1 mm) resulted in a marked increase in mitochondrial damage (e.g., cytochrome c and Smac/DIABLO release, loss of DW m), caspase activation, and apoptosis. Similar results were observed in Jurkat, HL-60, and K562 leukemic cells and with other HDIs (e.g., SAHA, MS-275). Exposure of cells to SB/LY was associated with Bcl-2 and Bid cleavage, XIAP and Mcl-1 downregulation, and diminished CD11b expression. While LY blocked SBmediated Akt activation, enforced expression of a constitutively active (myristolated) Akt failed to attenuate SB/LY-mediated lethality. Unexpectedly, treatment of cells with SB7LY resulted in a marked reduction in phosphorylation (activation) of p44/42 mitogen-activated protein (MAP) kinase. Moreover, enforced expression of a constitutively active MEK1 construct partially but significantly attenuated SB/LY-induced apoptosis. Lastly, cotreatment with LY blocked SB-mediated induction of p21 CIP1/WAF1 ; moreover, enforced expression of p21 CIP1/WAF1 significantly reduced SB/LY-mediated apoptosis. Together, these findings indicate that LY promotes SBmediated apoptosis through an AKT-independent process that involves MEK/MAP kinase inactivation and interference with p21 CIP1/WAF1 induction.

Spinal and bulbar muscular atrophy (SBMA) is a hereditary neurodegenerative disease characterized by slowly progressive muscle weakness and atrophy of bulbar, facial, and limb muscles. The cause of SBMA is expansion of a trinucleotide CAG repeat, which encodes the polyglutamine tract, in the first exon of the androgen receptor (AR) gene. SBMA chiefly occurs in adult males, whereas neurological symptoms are rarely detected in females having mutant AR gene. The cardinal histopathological finding of SBMA is loss of lower motor neurons in the anterior horn of spinal cord as well as in brainstem motor nuclei. Animal models carrying human mutant AR gene recapitulate polyglutamine-mediated motor neuron degeneration, providing clues to the pathogenesis of SBMA. There is increasing evidence that testosterone, the ligand of AR, plays a pivotal role in the pathogenesis of neurodegeneration in SBMA. The striking success of androgen deprivation therapy in SBMA mouse models has been translated into clinical trials. In addition, elucidation of pathophysiology using animal models leads to emergence of candidate drugs to treat this devastating disease: HSP inducer, Hsp90 inhibitor, and histone deacetylase inhibitor. Utilizing biomarkers such as scrotal skin biopsy would improve efficacy of clinical trials to verify the results from animal studies. Advances in basic and clinical researches on SBMA are now paving the way for clinical application of potential therapeutics.

Recent evidence demonstrates that epigenetic silencing of genes is associated with myelodysplasia and that a worse prognosis may be correlated with hypermethylation of certain genes, such as the cyclin-dependent kinase inhibitor p15. 5-Aza-2′-deoxycytidine (decitabine, DAC) is a nucleoside analog, which, at low doses, acts as a hypomethylating agent and is fivefold to tenfold more active than 5-azacytidine (azacitidine, Vidaza)—currently the only approved drug for treatment of myelodysplastic syndrome (MDS). Clinical studies have demonstrated that decitabine has activity in patients with MDS. Preliminary results of a phase III multicenter North American trial comparing low-dose decitabine to supportive care verified that therapy with decitabine resulted in higher response rates, improved quality of life, and prolonged time to leukemic transformation and/or death. However, further elucidation of its mechanism of action is required, as clinical response to decitabine does not correlate with demethylation of the p15 gene promoter or the repetitive DNA element LINE. Decitabine appears to upregulate both hypermethylated and nonmethylated genes. Ongoing studies aim to determine the optimal dose, schedule, and route of administration of decitabine, and to evaluate whether efficacy can be improved by using it in combination with other agents, such as histone deacetylase inhibitors.

Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell (SC) disorders that mainly affect the elderly population. They are characterized by ineffective hematopoiesis which results in quantitative and qualitative cellular defects and high incidence of leukemic transformation. Recent advances in MDS research have led to the development of novel agents which appears to improve remission rates and survival when compared to best supportive care. Currently azacitidine, decitabine, and lenalidomide are approved by the US FDA for the treatment of MDS, while the activity of other novel agents such as histone deacetylase inhibitors, farnesyl-transferase inhibitors, novel thrombopoietic agents, and anti-angiogenesis molecules is under evaluation. Erythropoietin-stimulating agents, iron chelating therapy and thrombopoietin receptor ligands may also improve quality of life and possibly prolong survival in MDS patients. The only treatment modality that can achieve long-term survival is the allogeneic SC transplantation which is given only in selected patients. Moreover the heterogeneity of MDS and the patient's advanced age and co-morbidity are significant factors besides cytogenetics, IPSS and WPSS that should be taken into account during the decision-making process. Therefore clinicians should treat patients with MDS on an individual basis aiming the increase of the response rates and the decrease of treatmentassociated toxicities. This can only be achieved through the better understanding of the MDS subgroups. If we can better define MDS subgroups we will be able to identify patients who will benefit from the incorporation of the novel agents, as monotherapy or in combinations regimens along with supportive care.

Purpose: LBH589 is a novel histone deacetylase inhibitor that inhibits proliferation and induces apoptosis in tumor cell lines. In this phase I study, LBH589 was administered i.v. as a 30-minute infusion on days 1to 7 of a 21-day cycle. Experimental Design: Fifteen patients (median age, 63 years; range, 42-87 years) with acute myeloid leukemia (13 patients), acute lymphocytic leukemia (1 patient), or myelodysplastic syndrome (1 patient) were treated with LBH589 at the following dose levels (mg/m 2 ): 4.8 (3 patients), 7.2 (3 patients), 9.0 (1patient), 11.5 (3 patient), and 14.0 (5 patients). The levels of histone acetylation were measured using quantitative flow cytometry and plasma LBH589 concentrations were assayed. Results: Four dose-limiting toxicities (grade 3 QTcF prolongation) were observed, four at 14.0 mg/m 2 and one at 11.5 mg/m 2 . QTcF prolongation was asymptomatic and reversed on LBH589 discontinuation. Other potentially LBH589-related toxicities included nausea (40%), diarrhea (33%), vomiting (33%), hypokalemia (27%), loss of appetite (13%), and thrombocytopenia (13%). In 8 of 11 patients with peripheral blasts, transient reductions occurred with a rebound following the 7-day treatment period. H3 acetylation increase was significant in B-cells (CD19 + ; P = 0.02) and blasts (CD34 + ; P = 0.04). The increase in H2B acetylation was highest in CD19 + and CD34 + cells [3.8-fold (P = 0.01) and 4.4-fold (P = 0.03), respectively]. The median acetylation of histones H2B and H3 in CD34 + and CD19 + cells significantly increased on therapy as did apoptosis in CD14 + cells. Area under the curve increased proportionally with dose with a terminal half-life of f11 hours.

Background: The intestinal microbiota plays an important role in immune development and homeostasis. A disturbed microbiota during early infancy is associated with an increased risk of developing inflammatory and allergic diseases later in life. The mechanisms underlying these effects are poorly understood but are likely to involve alterations in microbial production of fermentation-derived metabolites, which have potent immune modulating properties and are required for maintenance of healthy mucosal immune responses. Probiotics are beneficial bacteria that have the capacity to alter the composition of bacterial species in the intestine that can in turn influence the production of fermentation-derived metabolites. Principal among these metabolites are the short-chain fatty acids butyrate and acetate that have potent anti-inflammatory activities important in regulating immune function at the intestinal mucosal surface. Therefore strategies aimed at restoring the microbiota profile may be effective in the prevention or treatment of allergic and inflammatory diseases.