Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT-IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH3 (methyl) stretching bands at 2872 and 2960 cm1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm1, which originates from stretching vibrations of CH2 (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH2 stretching are concentration-dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH2 groups, we hypothesized that CH2 vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre-treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH2 vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl-lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH2 stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Infrared Spectroscopic Studies to Understand the Effect of Drugs at Molecular Level

In the recent past, there have been enormous efforts to understand effect of drugs on human body. Prior to understand the effect of drugs on human body most of the experiments are carried out on cells or model organisms. Here we present our study on the effect of chemotherapeutic drugs on cancer cells and the acetaminophen (APAP) induced hepatotoxicity in mouse model. Histone deacetylase inhibitors (HDIs) have attracted attention as potential drug molecules for the treatment of cancer. These are the chemotherapeutic drugs which have indirect mechanistic action against cancer cells via acting against histone deacetylases (HDAC). It has been known that different HDAC enzymes are over-expressed in various types of cancers for example; HDAC1 is over expressed in prostate, gastric and breast carcinomas. Therefore, in order to optimise chemotherapy, it is important to determine the efficacy of various classes of HDAC inhibitor drugs against variety of over-expressed HDAC enzymes. In the present study, FTIR microspectroscopy has been employed to predict the acetylation and propionylation brought in by HDIs. The liver plays an important role in cellular metabolism and is highly susceptible to drug toxicity. APAP which is an analgesic and antipyretic drug is extensively used for therapeutic purposes and has become the most common cause of acute liver failure (ALF). In the current study, we have focused to understand APAP induced hepatotoxicity using FTIR microspectroscopy. In the IR spectrum the bands corresponding to glycogen, ester group and were found to be suitable markers to predict liver injury at early time point (0.5hr) due to APAP both in tissue and serum in comparison to standard biochemical assays. Our studies show the potential of FTIR spectroscopy as a rapid, sensitive and non invasive detection technique for future clinical diagnosis.

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4'-imidazolidine]-2',5'-dione (DFH) and 8-(3,4-dichlorobenzyl)-1'-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4'-imidazolidine]-2',5'-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8ES. Incorporation of tritiated thymidine ([H-3) thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy. (C) 2008 Elsevier Inc. All rights reserved.

Histone Deacetylases Regulate Multicellular Development in the Social Amoeba Dictyostelium discoideum

Epigenetic modifications of histones regulate gene expression and lead to the establishment and maintenance of cellular phenotypes during development. Histone acetylation depends on a balance between the activities of histone acetyltransferases and histone deacetylases (HDACs) and influences transcriptional regulation. In this study, we analyse the roles of HDACs during growth and development of one of the cellular slime moulds, the social amoeba Dictyostelium discoideum. The inhibition of HDAC activity by trichostatin A results in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations are normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes is delayed. Bioinformatic analysis indicates that there are four genes encoding putative HDACs in D. discoideum. Using biochemical, genetic and developmental approaches, we demonstrate that one of these four genes, hdaB, is dispensable for growth and development under laboratory conditions. A knockout of the hdaB gene results in a social context-dependent phenotype: hdaB- cells develop normally but sporulate less efficiently than the wild type in chimeras. We infer that HDAC activity is important for regulating the timing of gene expression during the development of D. discoideum and for defining aspects of the phenotype that mediate social behaviour in genetically heterogeneous groups.

Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

A Synthetic Model for the Inhibition of Glutathione Peroxidase by Antiarthritic Gold Compounds

In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis[2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose (1), chloro(triethylphosphine)gold(I), chloro(trimethylphosphine)gold(I), and chloro(triphenylphosphine)gold(I) is described. The inhibition is found to be competitive with respect to a peroxide (H2O2) substrate and noncompetitive with respect to a thiol (PhSH) cosubstrate. The diselenide 5 reacts with PhSH to produce the corresponding selenol (6), which upon treatment with 1 equiv of gold(I) chlorides produces the corresponding gold selenolate complexes 11−13. However, the addition of 1 equiv of selenol 6 to complexes 11−13 leads to the formation of bis-selenolate complex 14 by ligand displacement reactions involving the elimination of phosphine ligands. The phosphine ligands eliminated from these reactions are further converted to the corresponding phosphine oxides (R3PO) and selenides (R3PSe). In addition to the replacement of the phosphine ligand by selenol 6, an interchange between two different phosphine ligands is also observed. For example, the reaction of complex 11 having a trimethylphosphine ligand with triphenylphosphine produces complex 13 by phosphine interchange reactions via the formation of intermediates 15 and 16. The reactivity of selenol 6 toward gold(I) phosphines is found to be similar to that of selenocysteine.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by cytosolic proteins - real or artifact

The inhibitory effect of FeSO4-dependent cytosolic protein on microsomal HMGCoA reductase is on the enzyme activity and not an artifact of loss of the product, mevalonate, through phosphorylation, unlike that of ATP.Mg effect.

Organization and bidirectional transcription of H2A, H2B and H4 histone genes in rice embryos

Our current understanding of the evolution of the histone gene family suffers from a lack of information on plant histone genes1. With a view to gathering some much needed information on these genes, we studied a rice genomic clone in pBR322 carrying H2A, H2B and H4 histone genes on a DNA fragment2 of 6.64 kilobases (kb). A restriction map of the insert was constructed and the organization of the three genes on this insert was determined. H2A and H2B histone genes were located at one end of the insert and H4 gene at the other with a 3.1 kb spacer in between. This cluster of three histone genes was found to be transcribed in a bidirectional fashion with H2A and H2B genes being encoded by one strand and the H4 gene by the other. These results indicate that plant histone gene organization differs from that of the sea urchin, but shows many similarities to the systems in other animals.

Inhibition of avian myeloblastosis virus reverse transcriptase and virus inactivation by metal complexes of isonicotinic acid hydrazide

The cupric and ferric complexes of isonicotinic acid hydrazide (INH) inhibit the DNA synthesis catalysed by avian myeloblastosis virus (AMV) reverse transcriptase. The inhibition was to the extent of 95% by 50 μM of cupric-INH complex and 55% by 100 μM of ferric-INH complex. These complexes have been found to bind preferentially to the enzyme than to the template-primer. Kinetic analysis showed that the cupric-INH complex is a non-competitive inhibitor with respect to dTTP. The time course of inhibition has revealed that the complexes are inhibitory even after the initiation of polynucleotide synthesis. In vivo toxicity studies in 1-day-old chicks have shown that the complexes are not toxic up to a concentration of 500 μg per chick. Infection of the 1-day-old chicks with AMV pretreated with 150 μg of either of the complexes prevented symptoms of leukemia due to virus inactivation.

Inhibition of Peroxidase-Catalyzed Iodination by Cephalosporins: Metallo-beta-Lactamase-Induced Antithyroid Activity of Antibiotics

Broad-spectrum antibiotics with heterocyclic side chains strongly inhibit peroxidase-catalyzed iodination in the presence of metallo--lactamase. This suggests that antibiotic resistance due to hydrolysis of the -lactam ring in antibiotics would have negative effects on thyroid activity.

Inhibition of mitochondrial oxidative phosphorylation by adriamycin

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Sccinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.