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Sample records for apoptotic topoisomerase i-dna

  1. Structural and dynamical effects induced by the anticancer drug topotecan on the human topoisomerase I - DNA complex.

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    Giordano Mancini

    Full Text Available BACKGROUND: Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death. METHODOLOGY/PRINCIPAL FINDINGS: Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633-643 (the region connecting the linker to the core domain that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed. CONCLUSIONS/SIGNIFICANCE: Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in

  2. La ADN topoisomerasa tipo I de protozoos patógenos como Diana terapéutica de fármacos antitumorales Type I DNA topoisomerase from protozoan pathogens as a potential target for anti-tumoral drugs

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    Rosa M Reguera

    2007-12-01

    , Chagas disease or leishmaniasis, among others, are unicellular protozoan parasites with no immune-prophylactic treatment and where the chemotherapeutical treatment is still under controversy. At present, the chemotherapeutic approach to these diseases is expensive, has side or toxic effects and it does not provide economic profits to the Pharmaceuticals which then have no or scarce enthusiasm in R & D investments in this field. The identification of type I DNAtopoisomerases as promising drug targets is based on the excellent results obtained with camptothecin derivatives in anticancer therapy. The recent finding of significant structural differences between human type I DNAtopoisomerase and their counterparts in trypanosomatids has open a new field in drug discovery, the aim is to find structural insights to be targeted by new drugs. This review is an update of DNA-topoisomerases as potential chemotherapeutic targets against the most important protozoan agents of medical interest.

  3. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline;

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...... with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion...... extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors....

  4. DNA Topoisomerases in Transcription

    DEFF Research Database (Denmark)

    Rødgaard, Morten Terpager

    2015-01-01

    This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...

  5. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

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    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L.; Kim, Jong-Hoon; Kim, Jong Seung

    2016-07-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic (“phototheranostic”).

  6. Inhibition of DNA topoisomerase I activity and induction of apoptosis by thiazacridine derivatives

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    Barros, Francisco W.A. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Bezerra, Daniel P., E-mail: danielpbezerra@gmail.com [Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe (Brazil); Ferreira, Paulo M.P. [Department of Biological Sciences, Federal University of Piauí, Picos, Piauí (Brazil); Cavalcanti, Bruno C. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Silva, Teresinha G.; Pitta, Marina G.R.; Lima, Maria do C.A. de; Galdino, Suely L.; Pitta, Ivan da R. [Department of Antibiotics, Federal, University of Pernambuco, Recife, Pernembuco (Brazil); Costa-Lotufo, Letícia V.; Moraes, Manoel O. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Burbano, Rommel R. [Institute of Biological Sciences, Federal University of Pará, Belém, Pará (Brazil); Guecheva, Temenouga N.; Henriques, João A.P. [Biotechnology Center, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul (Brazil); Pessoa, Cláudia, E-mail: cpessoa@ufc.br [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil)

    2013-04-01

    Thiazacridine derivatives (ATZD) are a novel class of cytotoxic agents that combine an acridine and thiazolidine nucleus. In this study, the cytotoxic action of four ATZD were tested in human colon carcinoma HCT-8 cells: (5Z)-5-acridin-9-ylmethylene-3-(4-methylbenzyl)-thiazolidine-2,4-dione — AC-4; (5ZE)-5-acridin-9-ylmethylene-3-(4-bromo-benzyl)-thiazolidine-2,4-dione — AC-7; (5Z)-5-(acridin-9-ylmethylene)-3-(4-chloro-benzyl) -1,3-thiazolidine-2,4-dione — AC-10; and (5ZE)-5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2, 4-dione — AC-23. All of the ATZD tested reduced the proliferation of HCT-8 cells in a concentration- and time-dependent manner. There were significant increases in internucleosomal DNA fragmentation without affecting membrane integrity. For morphological analyses, hematoxylin–eosin and acridine orange/ethidium bromide were used to stain HCT-8 cells treated with ATZD, which presented the typical hallmarks of apoptosis. ATZD also induced mitochondrial depolarisation and phosphatidylserine exposure and increased the activation of caspases 3/7 in HCT-8 cells, suggesting that this apoptotic cell death was caspase-dependent. In an assay using Saccharomyces cerevisiae mutants with defects in DNA topoisomerases 1 and 3, the ATZD showed enhanced activity, suggesting an interaction between ATZD and DNA topoisomerase enzyme activity. In addition, ATZD inhibited DNA topoisomerase I action in a cell-free system. Interestingly, these ATZD did not cause genotoxicity or inhibit the telomerase activity in human lymphocyte cultures at the experimental levels tested. In conclusion, the ATZD inhibited the DNA topoisomerase I activity and induced tumour cell death through apoptotic pathways. - Highlights: ► Thiazacridine derivatives induce mitochondrial-dependent apoptotic cell death. ► Thiazacridine derivatives inhibit DNA topoisomerase I action. ► Thiazacridine derivatives failed to cause genotoxicity on human lymphocytes.

  7. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

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    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso (NWU); (SKI)

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  8. Inhibition of topoisomerase II by liriodenine.

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    Woo, S H; Reynolds, M C; Sun, N J; Cassady, J M; Snapka, R M

    1997-08-15

    The cytotoxic oxoaporphine alkaloid liriodenine, isolated from Cananga odorata, was found to be a potent inhibitor of topoisomerase II (EC 5.99.1.3) both in vivo and in vitro. Liriodenine treatment of SV40 (simian virus 40)-infected CV-1 cells caused highly catenated SV40 daughter chromosomes, a signature of topoisomerase II inhibition. Strong catalytic inhibition of topoisomerase II by liriodenine was confirmed by in vitro assays with purified human topoisomerase II and kinetoplast DNA. Liriodenine also caused low-level protein-DNA cross-links to pulse-labeled SV40 chromosomes in vivo, suggesting that it may be a weak topoisomerase II poison. This was supported by the finding that liriodenine caused topoisomerase II-DNA cross-links in an in vitro assay for topoisomerase II poisons. Verapamil did not increase either liriodenine-induced protein-DNA cross-links or catalytic inhibition of topoisomerase II in SV40-infected cells. This indicates that liriodenine is not a substrate for the verapamil-sensitive drug efflux pump in CV-1 cells. PMID:9313773

  9. Pharmacologic Modulation of Topoisomerase I Inhibitors

    OpenAIRE

    Kehrer, Diederik

    2001-01-01

    textabstractCamptothecin, a plant alkaloid isolated from Camptotheca acuminata, was identified in the late 1950's. Due to severe and unpredictable toxic side effects in early clinical studies, the clinical development of this drug was halted in the 1970's. In the early 1980's several important events occurred that resulted in renewed interest in this agent. The molecular target of camptothecin, the nuclear enzyme topoisomerase I, was identified. This topoisomerase I was described as an enzyme...

  10. Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

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    Walid Fayad

    Full Text Available BACKGROUND: Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. METHOD AND FINDINGS: A library of natural products (NCI Natural Product set was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine, an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. CONCLUSIONS: The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

  11. Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli.

    OpenAIRE

    Shuman, S

    1989-01-01

    Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Express...

  12. Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA, a eukaryotic type II topoisomerase poison.

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    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2014-04-18

    m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.

  13. Furanocoumarins: novel topoisomerase I inhibitors from Ruta graveolens L.

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    Diwan, Renuka; Malpathak, Nutan

    2009-10-01

    Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA-topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme-substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme-extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA-enzyme complex was observed after enzyme-extract preincubation. The activity could be assigned to furanocoumarins-psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.

  14. Topoisomerases, new targets in cancer chemotherapy

    NARCIS (Netherlands)

    Zijlstra, J G; de Jong, Steven; de Vries, Liesbeth; Mulder, Nanno

    1990-01-01

    The enzymes involved in the regulation of the three-dimensional structure of DNA, topoisomerase I and II, are important for the handling of DNA during vital cellular processes such as translation, transcription and mitosis. The enzymes are currently being studied intensively, they are being biochemi

  15. DNA topoisomerase inhibitors: biflavonoids from Ouratea species

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    Grynberg N.F.

    2002-01-01

    Full Text Available Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I and amentoflavone (II are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

  16. DNA damaging and cell cycle effects of the topoisomerase I poison camptothecin in irradiated human cells

    International Nuclear Information System (INIS)

    This study addressed the potential radiosensitizing and DNA-damaging actions of the DNA topoisomerase I poison camptothecin (CPT) on SV40 transformed normal (MRC5CVI) and ataxia-telangiectasia (AT5BIVA) fibroblast cell lines. In both cell lines CPT induced a dose-dependent delay of cells in S phase, followed by a dose-dependent trapping in G2/M phase. Acute X-irradiation produced patterns of G2/M arrest and S-phase delay similar to those observed for CPT in the MRC5CVI cell line, but no S phase delay was observed in the AT5BIVA cell line consistent with the ataxia-telangiectasia phenotype of this cell line. X-irradiation of CPT-treated cells resulted in additive prolongation of S phase delay in MRC5CVI cultures and additive effects for cell killing in both cell lines. The potential for topoisomerase I-DNA cross-linking by CPT was not altered by 24 h pretreatment with CPT, or by acute X-irradiation. Hypersensitivity of AT5BIVA to CPT was not attributable to elevated levels of complex trapping. (author)

  17. Differential cytotoxic pathways of topoisomerase I and II anticancer agents after overexpression of the E2F-1/DP-1 transcription factor complex

    DEFF Research Database (Denmark)

    Hofland, K; Petersen, B O; Falck, J;

    2000-01-01

    of an E2F-1/ DP-1-induced post-DNA damage pathway rather than an increase in the number of replication forks caused by the S-phase initiation. In contrast, topoisomerase IIalpha levels (but not topoisomerase IIbeta levels), together with topoisomerase IIalpha promoter activity, increased 2--3-fold in UE1...... and drug sensitivity in detail, we established human osteosarcoma U-20S-TA cells expressing full-length E2F-1/ DP-1 under the control of a tetracycline-responsive promoter, designated UE1DP-1 cells. Topoisomerase I levels and activity as well as the number of camptothecin-induced DNA single- and double......DP-1/tc-cells. Furthermore, the number of etoposide-induced DNA single- and double-strand breaks increased in UE1DP-1/tc-cells together with a rise in clonogenic sensitivity to etoposide, but an equal apoptotic sensitivity to etoposide. The increase in topoisomerase IIalpha promoter activity in UE1DP...

  18. Mechanisms Regulating Resistance to Inhibitors of Topoisomerase II

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    Ram eGanapathi

    2013-08-01

    Full Text Available Inhibitors of topoisomerase II are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topoisomerase II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug-transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However in clinical trials, modulating the multidrug resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topoisomerase II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topoisomerase IIα and topoisomerase IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topoisomerase IIα is associated with cell division, topoisomerase IIβ is involved in differentiation. In addition to site specific mutations that can affect drug induced topoisomerase II-mediated DNA damage, post-translation modification of topoisomerase II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topoisomerase II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topoisomerase II inhibitors. Overall, tumor cell resistance to inhibitors of topoisomerase II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

  19. Targeting bacterial topoisomerases: how to counter mechanisms of resistance.

    Science.gov (United States)

    Tse-Dinh, Yuk-Ching

    2016-06-01

    DNA gyrase and topoisomerase IV are type IIA bacterial topoisomerases that are targeted by highly effective antibiotics. However, resistance via multiple mechanisms arises to limit the efficacies of these drugs. Continued research on type IIA bacterial topoisomerases has provided novel approaches to counter the most common resistance mechanism for utilization of these proven targets in antibacterial therapy. Bacterial topoisomerase I is being explored as an alternative target that is not expected to show cross-resistance. Dual targeting or combination therapy could be strategies for circumventing the development of resistance to topoisomerase-targeting antibiotics. Bacterial topoisomerases are high-value bactericidal targets that could continue to be exploited for antibacterial therapy, if new tactics to counter resistance can be adopted. PMID:27285067

  20. The importance of topoisomerases for chromatin regulated genes

    DEFF Research Database (Denmark)

    Fredsøe, Jacob Christian; Pedersen, Jakob Madsen; Rødgaard, Morten Terpager;

    2013-01-01

    DNA topoisomerases are enzymes, which function to relieve torsional stress in the DNA helix by introducing transient breaks into the DNA molecule. By use of Saccharomyces cerevisiae and microarray technology we have previously shown that topoisomerases are required for the activation of chromatin...... topoisomerases for optimal activation, but in contrast to the PHO5 gene, topoisomerases are not required for chromatin remodeling of the GAL1/10 promoter region, indicating a different role of the enzymes. We are currently performing a detailed investigation of the GAL genes to elucidate the precise role...

  1. Phytochemicals as Anticancer and Chemopreventive Topoisomerase II Poisons

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    Ketron, Adam C.

    2013-01-01

    Phytochemicals are a rich source of anticancer drugs and chemopreventive agents. Several of these chemicals appear to exert at least some of their effects through interactions with topoisomerase II, an essential enzyme that regulates DNA supercoiling and removes knots and tangles from the genome. Topoisomerase II-active phytochemicals function by stabilizing covalent protein-cleaved DNA complexes that are intermediates in the catalytic cycle of the enzyme. As a result, these compounds convert topoisomerase II to a cellular toxin that fragments the genome. Because of their mode of action, they are referred to as topoisomerase II poisons as opposed to catalytic inhibitors. The first sections of this article discuss DNA topology, the catalytic cycle of topoisomerase II, and the two mechanisms (interfacial vs. covalent) by which different classes of topoisomerase II poisons alter enzyme activity. Subsequent sections discuss the effects of several phytochemicals on the type II enzyme, including demethyl-epipodophyllotoxins (semisynthetic anticancer drugs) as well as flavones, flavonols, isoflavones, catechins, isothiocyanates, and curcumin (dietary chemopreventive agents). Finally, the leukemogenic potential of topoisomerase II-targeted phytochemicals is described. PMID:24678287

  2. A Rapid Procedure to Purify E. coli DNA Topoisomerase I

    OpenAIRE

    Xu, Xiaozhou; Leng, Fenfei

    2011-01-01

    On the basis of the asymmetrical charge distribution of E. coli DNA topoisomerase I, we developed a new procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-Sepharose FF and Q-Sepharose FF columns. The E. coli DNA topoisomerase I purified here is free of DNase contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were also determined.

  3. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    Science.gov (United States)

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  4. Topoisomerase I inhibitors: camptothecins and beyond.

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    Pommier, Yves

    2006-10-01

    Nuclear DNA topoisomerase I (TOP1) is an essential human enzyme. It is the only known target of the alkaloid camptothecin, from which the potent anticancer agents irinotecan and topotecan are derived. As camptothecins bind at the interface of the TOP1-DNA complex, they represent a paradigm for interfacial inhibitors that reversibly trap macromolecular complexes. Several camptothecin and non-camptothecin derivatives are being developed to further increase anti-tumour activity and reduce side effects. The mechanisms and molecular determinants of tumour response to TOP1 inhibitors are reviewed, and rational combinations of TOP1 inhibitors with other drugs are considered based on current knowledge of repair and checkpoint pathways that are associated with TOP1-mediated DNA damage. PMID:16990856

  5. Anti-topoisomerase drugs as potent inducers of chromosomal aberrations

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    Loredana Bassi

    2000-12-01

    Full Text Available DNA topoisomerases catalyze topological changes in DNA that are essential for normal cell cycle progression and therefore they are a preferential target for the development of anticancer drugs. Anti-topoisomerase drugs can be divided into two main classes: "cleavable complex" poisons and catalytic inhibitors. The "cleavable complex" poisons are very effective as anticancer drugs but are also potent inducers of chromosome aberrations so they can cause secondary malignancies. Catalytic inhibitors are cytotoxic but they do not induce chromosome aberrations. Knowledge about the mechanism of action of topoisomerase inhibitors is important to determine the best anti-topoisomerase combinations, with a reduced risk of induction of secondary malignancies.As topoisomerases de DNA catalisam alterações topológicas no DNA que são essenciais para a progressão do ciclo celular normal e, portanto, são um alvo preferencial para o desenvolvimento de drogas anticâncer. Drogas anti-topoisomerases podem ser divididas em duas classes principais: drogas anti-"complexos cliváveis" e inibidores catalíticos. As drogas anti-"complexos cliváveis" são muito eficazes como drogas anticancerígenas, mas são também potentes indutores de aberrações cromossômicas, podendo causar neoplasias malignas secundárias. Inibidores catalíticos são citotóxicos mas não induzem aberrações cromossômicas. Conhecimento a respeito do mecanismo de ação de inibidores de topoisomerases é importante para determinar as melhores combinações anti-topoisomerases, com um reduzido risco de indução de neoplasias malignas secundárias.

  6. Topoisomerase II poisoning by indazole and imidazole complexes of ruthenium

    Indian Academy of Sciences (India)

    Y N Vashisht Gopal; Anand K Kondapi

    2001-06-01

    Trans-imidazolium (bis imidazole) tetrachloro ruthenate (RuIm) and trans-indazolium (bis indazole) tetrachloro ruthenate (RuInd) are ruthenium coordination complexes, which were first synthesized and exploited for their anticancer activity. These molecules constitute two of the few most effective anticancer ruthenium compounds. The clinical use of these compounds however was hindered due to toxic side effects on the human body. Our present study on topoisomerase II poisoning by these compounds shows that they effectively poison the activity of topoisomerase II by forming a ternary cleavage complex of DNA, drug and topoisomerase II. The thymidine incorporation assays show that the inhibition of cancer cell proliferation correlates with topoisomerase II poisoning. The present study on topoisomerase II poisoning by these two compounds opens a new avenue for renewing further research on these compounds. This is because they could be effective lead candidates for the development of more potent and less toxic ruthenium containing topoisomerase II poisons. Specificity of action on this molecular target may reduce the toxic effects of these ruthenium-containing molecules and thus improve their therapeutic index.

  7. Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions

    Energy Technology Data Exchange (ETDEWEB)

    Zwelling, L.A.; Mayes, J.; Hinds, M.; Chan, D.; Altschuler, E.; Carroll, B.; Parker, E.; Deisseroth, K.; Radcliffe, A.; Seligman, M.; Li, Li; Farquhar, D. (Univ. of Texas M.D. Anderson Cancer Center, Houston (USA))

    1991-04-23

    HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. The authors now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. These results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations. Under these conditions, these latter drugs may not produce protein-associated DNA cleavage while still inhibiting topoisomerase II function as well as the actions of other topoisomerase II reactive drugs.

  8. DNA-Binding and Topoisomerase-I-Suppressing Activities of Novel Vanadium Compound Van-7

    Directory of Open Access Journals (Sweden)

    Xiao-mei Mo

    2012-01-01

    Full Text Available Vanadium compounds were studied during recent years to be considered as a representative of a new class of nonplatinum metal anticancer agents in combination to its low toxicity. Here, we found a vanadium compound Van-7 as an inhibitor of Topo I other than Topo II using topoisomerase-mediated supercoiled DNA relaxation assay. Agarose gel electrophoresis and comet assay showed that Van-7 treatment did not produce cleavable complexes like HCPT, thereby suggesting that Topo I inhibition occurred upstream of the relegation step. Further studies revealed that Van-7 inhibited Topo I DNA binding involved in its intercalating DNA. Van-7 did not affect the catalytic activity of DNase I even up to100 μM. Van-7 significantly suppressed the growth of cancer cell lines with IC50 at nanomolar concentrations and arrested cell cycle of A549 cells at G2/M phase. All these results indicate that Van-7 is a potential selective Topo I inhibitor with anticancer activities as a kind of Topo I suppressor, not Topo I poison.

  9. DNA-Binding and Topoisomerase-I-Suppressing Activities of Novel Vanadium Compound Van-7.

    Science.gov (United States)

    Mo, Xiao-Mei; Chen, Zhan-Fang; Qi, Xin; Li, Yan-Tuan; Li, Jing

    2012-01-01

    Vanadium compounds were studied during recent years to be considered as a representative of a new class of nonplatinum metal anticancer agents in combination to its low toxicity. Here, we found a vanadium compound Van-7 as an inhibitor of Topo I other than Topo II using topoisomerase-mediated supercoiled DNA relaxation assay. Agarose gel electrophoresis and comet assay showed that Van-7 treatment did not produce cleavable complexes like HCPT, thereby suggesting that Topo I inhibition occurred upstream of the relegation step. Further studies revealed that Van-7 inhibited Topo I DNA binding involved in its intercalating DNA. Van-7 did not affect the catalytic activity of DNase I even up to100 μM. Van-7 significantly suppressed the growth of cancer cell lines with IC(50) at nanomolar concentrations and arrested cell cycle of A549 cells at G2/M phase. All these results indicate that Van-7 is a potential selective Topo I inhibitor with anticancer activities as a kind of Topo I suppressor, not Topo I poison. PMID:23055949

  10. Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA

    Directory of Open Access Journals (Sweden)

    Candace L. Minchew

    2011-06-01

    Full Text Available In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.

  11. Topoisomerase poisoning by genistein in the intestine of rats.

    Science.gov (United States)

    Baechler, Simone A; Soukup, Sebastian T; Molzberger, Almut F; Kulling, Sabine E; Diel, Patrick; Marko, Doris

    2016-01-22

    The isoflavone genistein has been shown to act as topoisomerase II poison in various cell lines. Here, we address the question whether genistein is able to affect topoisomerase II in vivo. Juvenile male Wistar rats received either a single dose of genistein subcutaneously (s.c.; 10 mg/kg BW) or a lifelong isoflavone-rich diet encompassing in utero, lactation phase and 10 days of oral consumption, whereas genistein was mainly taken up as glycosides (25-50 mg/kg BW). The effects on the level of covalent topoisomerase II-DNA-complexes in the duodenum and colon were measured using the "Isolation of in vivo complexes of enzyme to DNA" (ICE)-bioassay. Simultaneously, serum as well as tissue concentrations of genistein and its metabolites were quantified by LC-MS. Genistein (s.c.) significantly increased the amount of covalent topoisomerase IIα and β-DNA complexes in the gut, showing more persistent effects in the colon than in the duodenum. In case of a lifelong dietary isoflavone exposure, no effects on the stabilization of cleavage complexes was observed, except a slight increase of topoisomerase IIα-DNA-complexes in the colon. The differences between the exposure routes might be attributed to the higher serum concentration of the genistein aglycon after subcutaneous treatment probably due to circumvention of first-pass metabolism compared to oral consumption of an isoflavone-rich diet. These data indicate that subcutaneously administrated genistein clearly possesses topoisomerase poisoning properties in vivo, whereas an isoflavone-rich diet containing genistein only caused a slight effect which relevance has to be clarified in further studies.

  12. Cuminaldehyde from Cinnamomum verum Induces Cell Death through Targeting Topoisomerase 1 and 2 in Human Colorectal Adenocarcinoma COLO 205 Cells.

    Science.gov (United States)

    Tsai, Kuen-Daw; Liu, Yi-Heng; Chen, Ta-Wei; Yang, Shu-Mei; Wong, Ho-Yiu; Cherng, Jonathan; Chou, Kuo-Shen; Cherng, Jaw-Ming

    2016-01-01

    Cinnamomum verum, also called true cinnamon tree, is employed to make the seasoning cinnamon. Furthermore, the plant has been used as a traditional Chinese herbal medication. We explored the anticancer effect of cuminaldehyde, an ingredient of the cortex of the plant, as well as the molecular biomarkers associated with carcinogenesis in human colorectal adenocarcinoma COLO 205 cells. The results show that cuminaldehyde suppressed growth and induced apoptosis, as proved by depletion of the mitochondrial membrane potential, activation of both caspase-3 and -9, and morphological features of apoptosis. Moreover, cuminaldehyde also led to lysosomal vacuolation with an upregulated volume of acidic compartment and cytotoxicity, together with inhibitions of both topoisomerase I and II activities. Additional study shows that the anticancer activity of cuminaldehyde was observed in the model of nude mice. Our results suggest that the anticancer activity of cuminaldehyde in vitro involved the suppression of cell proliferative markers, topoisomerase I as well as II, together with increase of pro-apoptotic molecules, associated with upregulated lysosomal vacuolation. On the other hand, in vivo, cuminaldehyde diminished the tumor burden that would have a significant clinical impact. Furthermore, similar effects were observed in other tested cell lines. In short, our data suggest that cuminaldehyde could be a drug for chemopreventive or anticancer therapy. PMID:27231935

  13. A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.

    Science.gov (United States)

    Jones, Jesse A; Price, Emily; Miller, Donovan; Hevener, Kirk E

    2016-08-01

    Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity. PMID:27117979

  14. Cinnamomum verum component 2-methoxycinnamaldehyde: a novel antiproliferative drug inducing cell death through targeting both topoisomerase I and II in human colorectal adenocarcinoma COLO 205 cells

    Science.gov (United States)

    Tsai, Kuen-daw; Cherng, Jonathan; Liu, Yi-Heng; Chen, Ta-Wei; Wong, Ho-Yiu; Yang, Shu-mei; Chou, Kuo-Shen; Cherng, Jaw-Ming

    2016-01-01

    Background Cinnamomum verum is used to manufacture the spice cinnamon. In addition, the plant has been used as a Chinese herbal medication. Methods We investigated the antiproliferative effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the cortex of the plant, and the molecular biomarkers associated with tumorigenesis in human colorectal adenocarcinoma COLO 205 cells. Specifically, cell viability was evaluated by colorimetric assay; apoptosis was determined by flow cytometry and morphological analysis with bright field, acridine orange, and neutral red stainings, as well as comet assay; topoisomerase I activity was determined by assay based upon DNA relaxation and topoisomerase II by DNA relaxation plus decatentation of kinetoplast DNA; lysosomal vacuolation and volume of acidic compartments (VACs) were determined by neutral red staining. Results The results demonstrate that 2-MCA inhibited proliferation and induced apoptosis as implicated by mitochondrial membrane potential (ΔΨm) loss, activation of both caspase-3 and -9, increase of annexin V+PI+ cells, as well as morphological characteristics of apoptosis. Furthermore, 2-MCA also induced lysosomal vacuolation with elevated VAC, cytotoxicity, and inhibitions of topoisomerase I as well as II activities. Additional study demonstrated the antiproliferative effect of 2-MCA found in a nude mice model. Conclusions Our data implicate that the antiproliferative activity of 2-MCA in vitro involved downregulation of cell growth markers, both topoisomerase I and II, and upregulation of pro-apoptotic molecules, associated with increased lysosomal vacuolation. In vivo 2-MCA reduced the tumor burden that could have significant clinical impact. Indeed, similar effects were found in other tested cell lines, including human hepatocellular carcinoma SK-Hep-1 and Hep 3B, lung adenocarcinoma A549 and squamous cell carcinoma NCI-H520, and T-lymphoblastic MOLT-3 (results not shown). Our data implicate that 2-MCA could be a

  15. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    Science.gov (United States)

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2015-03-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

  16. Recent advances in topoisomerase I-targeting agents, camptothecin analogues.

    Science.gov (United States)

    Kim, Dae-Kee; Lee, Namkyu

    2002-12-01

    The present review concentrates on camptothecin (CPT) analogues, the most extensively studied topoisomerase I (topo I) inhibitors, and provides concise information on the structural features of human topo I enzyme, mechanisms of interaction of CPT with topo I, structure-activity relationship study of CPT analogues including the influence of lactone stability on antitumor activity, and recent updates of valuable CPT analogues. PMID:12370044

  17. Conversion of DNA gyrase into a conventional type II topoisomerase

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1996-01-01

    -dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme...

  18. Electrostatics of DNA–DNA juxtapositions: consequences for type II topoisomerase function

    OpenAIRE

    Randall, Graham L; Pettitt, B. Montgomery; Buck, Gregory R; Zechiedrich, E Lynn

    2006-01-01

    Type II topoisomerases resolve problematic DNA topologies such as knots, catenanes, and supercoils that arise as a consequence of DNA replication and recombination. Failure to remove problematic DNA topologies prohibits cell division and can result in cell death or genetic mutation. Such catastrophic consequences make topoisomerases an effective target for antibiotics and anticancer agents. Despite their biological and clinical importance, little is understood about how a topoisomerase differ...

  19. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

    OpenAIRE

    Weon Sup Shin; Jiyou Han; Rajesh Kumar; Gyung Gyu Lee; Sessler, Jonathan L.; Jong-Hoon Kim; Jong Seung Kim

    2016-01-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a ...

  20. SMAC mimetic Debio 1143 synergizes with taxanes, topoisomerase inhibitors and bromodomain inhibitors to impede growth of lung adenocarcinoma cells.

    Science.gov (United States)

    Langdon, Casey G; Wiedemann, Norbert; Held, Matthew A; Mamillapalli, Ramanaiah; Iyidogan, Pinar; Theodosakis, Nicholas; Platt, James T; Levy, Frederic; Vuagniaux, Gregoire; Wang, Shaomeng; Bosenberg, Marcus W; Stern, David F

    2015-11-10

    Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here.

  1. Immunosuppressive effects of apoptotic cells

    Science.gov (United States)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  2. Topoisomerase I and II inhibitors: chemical structure, mechanisms of action and role in cancer chemotherapy

    Science.gov (United States)

    Dezhenkova, L. G.; Tsvetkov, V. B.; Shtil, A. A.

    2014-01-01

    The review summarizes and analyzes recent published data on topoisomerase I and II inhibitors as potential antitumour agents. Functions and the mechanism of action of topoisomerases are considered. The molecular mechanism of interactions between low-molecular-weight compounds and these proteins is discussed. Topoisomerase inhibitors belonging to different classes of chemical compounds are systematically covered. Assays for the inhibition of topoisomerases and the possibilities of using the computer-aided modelling for the rational design of novel drugs for cancer chemotherapy are presented. The bibliography includes 127 references.

  3. Topoisomerase IIα in chromosome instability and personalized cancer therapy.

    Science.gov (United States)

    Chen, T; Sun, Y; Ji, P; Kopetz, S; Zhang, W

    2015-07-30

    Genome instability is a hallmark of cancer cells. Chromosome instability (CIN), which is often mutually exclusive from hypermutation genotypes, represents a distinct subtype of genome instability. Hypermutations in cancer cells are due to defects in DNA repair genes, but the cause of CIN is still elusive. However, because of the extensive chromosomal abnormalities associated with CIN, its cause is likely a defect in a network of genes that regulate mitotic checkpoints and chromosomal organization and segregation. Emerging evidence has shown that the chromosomal decatenation checkpoint, which is critical for chromatin untangling and packing during genetic material duplication, is defective in cancer cells with CIN. The decatenation checkpoint is known to be regulated by a family of enzymes called topoisomerases. Among them, the gene encoding topoisomerase IIα (TOP2A) is commonly altered at both gene copy number and gene expression level in cancer cells. Thus, abnormal alterations of TOP2A, its interacting proteins, and its modifications may have a critical role in CIN in human cancers. Clinically, a large arsenal of topoisomerase inhibitors has been used to suppress DNA replication in cancer. However, they often lead to the secondary development of leukemia because of their effect on the chromosomal decatenation checkpoint. Therefore, topoisomerase drugs must be used judiciously and administered on an individual basis. In this review, we highlight the biological function of TOP2A in chromosome segregation and the mechanisms that regulate this enzyme's expression and activity. We also review the roles of TOP2A and related proteins in human cancers, and raise a perspective for how to target TOP2A in personalized cancer therapy. PMID:25328138

  4. Proteins of the origin recognition complex (ORC and DNA topoisomerases on mammalian chromatin

    Directory of Open Access Journals (Sweden)

    Baack Martina

    2009-04-01

    Full Text Available Abstract Background The process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells. Results We have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5 and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin. Conclusion Topoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

  5. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    Directory of Open Access Journals (Sweden)

    Sapetto-Rebow Beata

    2011-11-01

    Full Text Available Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm, a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization. Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

  6. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    LENUS (Irish Health Repository)

    Sapetto-Rebow, Beata

    2011-11-23

    Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

  7. Human DNA topoisomerase II : biochemistry and role in chemotherapy resistance (review)

    NARCIS (Netherlands)

    Withoff, S; De Jong, S; De Vries, E G; Mulder, N H; de Jong, Steven

    1996-01-01

    Recently, it has been discovered that DNA topoisomerases are the target of many anti-cancer drugs which were already widely used in the clinic. Using the latest molecular biological techniques much has been learned about the function of DNA topoisomerases in normal cells and in cancer cells, and abo

  8. Topoisomerase I inhibitors: clinical studies on oral administration and/or combinations with cisplatin

    NARCIS (Netherlands)

    M.J.A. de Jonge (Maja)

    1999-01-01

    textabstractThe topoisomerases were discovered in 1971, but it was not until the 1980s that the significance of these enzymes as potential therapeutic targets was appreciated. Topoisomerase I plays a crucial role in the normal replication of DNA. In its physiological state in the chromosome, the DNA

  9. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager;

    2012-01-01

    To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-re...... transcriptional activation of genes with a repressible/inducible mode of regulation....

  10. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jakob Madsen Pedersen

    Full Text Available To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

  11. Identification of a minimal functional linker in human topoisomerase I by domain swapping with Cre recombinase

    DEFF Research Database (Denmark)

    Hougaard, Rikke Frøhlich; Juul, Sissel; Vinther, Maria;

    2008-01-01

    study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase. In vitro characterization of the resulting chimeras, denoted Cropos, reveals that...

  12. Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding.

    Science.gov (United States)

    Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

    2010-07-14

    Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.

  13. Apoptotic Signaling in Mouse Odontogenesis

    OpenAIRE

    Matalova, Eva; Svandova, Eva; Tucker, Abigail S.

    2012-01-01

    Apoptosis is an important morphogenetic event in embryogenesis as well as during postnatal life. In the last 2 decades, apoptosis in tooth development (odontogenesis) has been investigated with gradually increasing focus on the mechanisms and signaling pathways involved. The molecular machinery responsible for apoptosis exhibits a high degree of conservation but also organ and tissue specific patterns. This review aims to discuss recent knowledge about apoptotic signaling networks during odon...

  14. Topoisomerase IIα poisoning and DNA double-strand breaking by chiral ruthenium(ii) complexes containing 2-furanyl-imidazo[4,5-f][1,10]phenanthroline derivatives.

    Science.gov (United States)

    Qian, Chen; Wu, Jingheng; Ji, Liangnian; Chao, Hui

    2016-06-28

    Four chiral Ru(ii) complexes bearing furan ligands, Δ/Λ-[Ru(bpy)2(pocl)](2+) () and Δ/Λ-[Ru(bpy)2(poi)](2+) () (bpy = 2,2'-bipyridine, pocl = 2-(5-chlorofuran-2-yl)imidazo[4,5-f][1,10]phenanthroline, poi = 2-(5-5-iodofuran-2-yl)imidazo[4,5-f][1,10]phenanthroline), were synthesized and characterized. These Ru(ii) complexes showed antitumor activities against HeLa, A549, HepG2, HL-60 and K562 tumor cell lines, especially the HL-60 tumor cell line. Moreover, was more active than other complexes accounting for the different cellular uptakes. In addition, could accumulate in the nucleus of HL-60 cells, suggesting that nucleic acids were the cellular target of . Topoisomerase inhibition tests in vitro and in living cells confirmed that the four complexes acted as efficient topoisomerase IIα poisons, DNA double-strand breaks had also been observed from neutral single cell gel electrophoresis (comet assay). inhibited the growth of HL-60 cells through the induction of apoptotic cell death, as evidenced by the Alexa Fluor® 488 annexin V staining assays. The results demonstrated that acted as a topoisomerase IIα poison and caused DNA double-strand damage that could lead to apoptosis. PMID:27226117

  15. Topoisomerase I in Human Disease Pathogenesis and Treatments

    Institute of Scientific and Technical Information of China (English)

    Min Li; Yilun Liu

    2016-01-01

    Mammalian topoisomerase 1 (TOP1) is an essential enzyme for normal development. TOP1 relaxes supercoiled DNA to remove helical constraints that can otherwise hinder DNA repli-cation and transcription and thus block cell growth. Unfortunately, this exact activity can covalently trap TOP1 on the DNA that could lead to cell death or mutagenesis, a precursor for tumorigenesis. It is therefore important for cells to find a proper balance between the utilization of the TOP1 cat-alytic activity to maintain DNA topology and the risk of accumulating the toxic DNA damages due to TOP1 trapping that prevents normal cell growth. In an apparent contradiction to the negative attribute of the TOP1 activity to genome stability, the detrimental effect of the TOP1-induced DNA lesions on cell survival has made this enzyme a prime target for cancer therapies to kill fast-growing cancer cells. In addition, cumulative evidence supports a direct role of TOP1 in pro-moting transcriptional progression independent of its topoisomerase activity. The involvement of TOP1 in transcriptional regulation has recently become a focus in developing potential new treat-ments for a subtype of autism spectrum disorders. Clearly, the impact of TOP1 on human health is multifold. In this review, we will summarize our current understandings on how TOP1 contributes to human diseases and how its activity is targeted for disease treatments.

  16. Cinnamomum verum Component 2-Methoxycinnamaldehyde: A Novel Anticancer Agent with Both Anti-Topoisomerase I and II Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo.

    Science.gov (United States)

    Wong, Ho-Yiu; Tsai, Kuen-daw; Liu, Yi-Heng; Yang, Shu-mei; Chen, Ta-Wei; Cherng, Jonathan; Chou, Kuo-Shen; Chang, Chen-Mei; Yao, Belen T; Cherng, Jaw-Ming

    2016-02-01

    Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human lung adenocarcinoma A549 cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an upregulation of pro-apoptotic Bax and Bak genes and downregulation of anti-apoptotic Bcl-2 and Bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase-3 and -9, and morphological characteristics of apoptosis, including plasma membrane blebbing and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartment (VAC) and suppressions of nuclear transcription factors nuclear factor-κB (NF-κB) and both topoisomerase I and II activities. Further study reveals that the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against A549 cells is accompanied by downregulations of NF-κB binding activity and proliferative control involving apoptosis and both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26676220

  17. Discovery of a novel anti-cancer agent targeting both topoisomerase I and II in hepatocellular carcinoma Hep 3B cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

    Science.gov (United States)

    Perng, Daw-Shyong; Tsai, Yu-Hsin; Cherng, Jonathan; Kuo, Chih-Wei; Shiao, Chih-Chung; Cherng, Jaw-Ming

    2016-08-01

    Cinnamomum verum has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, in hepatocellular carcinoma Hep 3B cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an up-regulation of pro-apoptotic bax and bak genes and down-regulation of anti-apoptotic bcl-2 and bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase 3 and 9, increase in the DNA content in sub G1, and morphological characteristics of apoptosis. 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against Hep 3B cells is accompanied by downregulations of NF-κB binding activity, inflammatory responses involving COX-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26707867

  18. Experimental study of etoposide- and radiation-induced apoptosis and topoisomerase II α expression

    International Nuclear Information System (INIS)

    A human tumor with wild-type p53 was transplanted to nude mice to investigate the relationship between induction of apoptosis and p53 expression and topoisomerase II α immunohistochemically. In contrast to induction of p53-dependent apoptosis, topoisomerase II α positive rate decreased transiently following administration of etoposide, and then increased a little. However, the combination of etoposide and radiation therapy did not always cause an evident decrease of the II α positive rate. A correlation between the topoisomerase II α positive rate and Ki-67 labeling index was also suggested. (author)

  19. Secondary metabolites as DNA topoisomerase inhibitors: A new era towards designing of anticancer drugs

    Directory of Open Access Journals (Sweden)

    Supriya Baikar

    2010-01-01

    Full Text Available A large number of secondary metabolites like alkaloids, terpenoids, polyphenols and quinones are produced by the plants. These metabolites can be utilized as natural medicines for the reason that they inhibit the activity of DNA topoisomerase which are the clinical targets for anticancer drugs. DNA topoisomerases are the cellular enzymes that change the topological state of DNA through the breaking and rejoining of DNA strands. Synthetic drugs as inhibitors of topoisomerases have been developed and used in the clinical trials but severe side effects are a serious problem for them therefore, there is a need for the development of novel plant-derived natural drugs and their analogs which may serve as appropriate inhibitors with respect to drug designing. The theme for this review is how secondary metabolites or natural products inactivate the action of DNA topoisomerases and open new avenues towards isolation and characterization of compounds for the development of novel drugs with anticancer potential.

  20. Apoptotic engulfment pathway and schizophrenia.

    LENUS (Irish Health Repository)

    Chen, Xiangning

    2009-01-01

    BACKGROUND: Apoptosis has been speculated to be involved in schizophrenia. In a previously study, we reported the association of the MEGF10 gene with the disease. In this study, we followed the apoptotic engulfment pathway involving the MEGF10, GULP1, ABCA1 and ABCA7 genes and tested their association with the disease. METHODOLOGY\\/PRINCIPAL FINDINGS: Ten, eleven and five SNPs were genotyped in the GULP1, ABCA1 and ABCA7 genes respectively for the ISHDSF and ICCSS samples. In all 3 genes, we observed nominally significant associations. Rs2004888 at GULP1 was significant in both ISHDSF and ICCSS samples (p = 0.0083 and 0.0437 respectively). We sought replication in independent samples for this marker and found highly significant association (p = 0.0003) in 3 Caucasian replication samples. But it was not significant in the 2 Chinese replication samples. In addition, we found a significant 2-marker (rs2242436 * rs3858075) interaction between the ABCA1 and ABCA7 genes in the ISHDSF sample (p = 0.0022) and a 3-marker interaction (rs246896 * rs4522565 * rs3858075) amongst the MEGF10, GULP1 and ABCA1 genes in the ICCSS sample (p = 0.0120). Rs3858075 in the ABCA1 gene was involved in both 2- and 3-marker interactions in the two samples. CONCLUSIONS\\/SIGNIFICANCE: From these data, we concluded that the GULP1 gene and the apoptotic engulfment pathway are involved in schizophrenia in subjects of European ancestry and multiple genes in the pathway may interactively increase the risks to the disease.

  1. Synthesis, DNA binding and topoisomerase inhibition of mononaphthalimide homospermidine derivatives

    Institute of Scientific and Technical Information of China (English)

    Zhi Yong Tian; Hong Xia Ma; Song Qiang Xie; Xue Wang; Jin Zhao; Chao Jie Wang; Wen Yuan Gao

    2008-01-01

    Two novel mononaphthalimide homospermidine derivatives (2a, 2b) with three or four methylene unit as linkages weresynthesized and evaluated for cytotoxicity against human leukemia K562, murine melanoma B 16 and Chinese hamster ovary CHOcell lines. The presence of homospermidine motif could greatly elevate the potency of 1,8-naphthalimide. Conjugate 2b with longerspacer exhibited higher in vitro cytotoxicity than 2a. The DNA binding experiments indicated that conjugates 2b could bind toherring sperm DNA. The topoisomerase Ⅱ poison trials revealed that 2b could inhibit the activity of top. Ⅱ.2008 Chao Jie Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  2. DNA topoisomerase targets of the fluoroquinolones: A strategy for avoiding bacterial resistance

    OpenAIRE

    Zhao, Xilin; Xu, Chen; Domagala, John; Drlica, Karl

    1997-01-01

    Fluoroquinolones are antibacterial agents that attack DNA gyrase and topoisomerase IV on chromosomal DNA. The existence of two fluoroquinolone targets and stepwise accumulation of resistance suggested that new quinolones could be found that would require cells to obtain two topoisomerase mutations to display resistance. For wild-type cells to become resistant, the two mutations must be acquired concomitantly. That is expected to occur infrequently. To identify such compounds, fluoroquinolones...

  3. Spatial organization of topoisomerase I-mediated DNA cleavage induced by camptothecin–oligonucleotide conjugates

    OpenAIRE

    Arimondo, Paola B.; Angenault, Stéphane; Halby, Ludovic; Boutorine, Alexandre; Schmidt, Frédéric; Monneret, Claude; Garestier, Thérèse; Sun, Jian-Sheng; Bailly, Christian; Hélène, Claude

    2003-01-01

    Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3′ or the 5′ side of the triplex, and the location of the cleava...

  4. Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin; Cao, Nan; Cheng, Bokun; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

    2016-01-16

    The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 angstrom resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel beta-sheet stabilized by a crossing-over alpha-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.

  5. Use of in vitro topoisomerase II assays for studying quinolone antibacterial agents.

    OpenAIRE

    Barrett, J F; Gootz, T D; McGuirk, P R; C.A. Farrell; Sokolowski, S A

    1989-01-01

    Several quinolones and antitumor compounds were tested as inhibitors of purified calf thymus topoisomerase II in unknotting, catenation, radiolabeled DNA cleavage, and quantitative nonradiolabeled cleavage assays. The antitumor agents VP-16 (demethylepipodophyllotoxin ethylio-beta-D-glucoside) and ellipticine demonstrated drug-enhanced topoisomerase II DNA cleavage (the concentration of drug that induced 50% of the maximal DNA cleavage in the test system [CC50]) at levels of less than or equa...

  6. A naturally chimeric type IIA topoisomerase in Aquifex aeolicus highlights an evolutionary path for the emergence of functional paralogs

    OpenAIRE

    Tretter, Elsa M.; Lerman, Jeffrey C.; Berger, James M.

    2010-01-01

    Bacteria frequently possess two type IIA DNA topoisomerases, gyrase and topo IV, which maintain chromosome topology by variously supercoiling, relaxing, and disentangling DNA. DNA recognition and functional output is thought to be controlled by the C-terminal domain (CTD) of the topoisomerase DNA binding subunit (GyrA/ParC). The deeply rooted organism Aquifex aeolicus encodes one type IIA topoisomerase conflictingly categorized as either DNA gyrase or topo IV. To resolve this enzyme’s catalyt...

  7. Voreloxin is an anticancer quinolone derivative that intercalates DNA and poisons topoisomerase II.

    Directory of Open Access Journals (Sweden)

    Rachael E Hawtin

    Full Text Available BACKGROUND: Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research. METHODS/PRINCIPAL FINDINGS: Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more

  8. Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI.

    Directory of Open Access Journals (Sweden)

    James A Taylor

    Full Text Available DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.

  9. Application of a novel microtitre plate-based assay for the discovery of new inhibitors of DNA gyrase and DNA topoisomerase VI.

    Science.gov (United States)

    Taylor, James A; Mitchenall, Lesley A; Rejzek, Martin; Field, Robert A; Maxwell, Anthony

    2013-01-01

    DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.

  10. Intercellular transfer of apoptotic signals via electrofusion

    International Nuclear Information System (INIS)

    We determined whether cells that are induced to undergo anoikis by matrix detachment can initiate apoptosis in healthy cells following electroporation-induced fusion. Separate populations of MDCK cells undergoing anoikis and stained with FITC-annexin or viable MDCK cells that were labeled with spectrally discrete fluorescent beads were electroporated. Cells were analyzed by flow cytometry for enumeration of viable cells with beads, apoptotic cells or fused cells. Electroporation promoted a 49-fold increase of the percentage of viable cells that had fused with apoptotic cells. Apoptotic cell-viable cell fusions were 8-fold more likely to not attach to cell culture plastic and 2.3-fold less likely to proliferate after 24 hr incubation than viable cell fusion controls. These data demonstrate that apoptotic signals can be transferred between cells by electrofusion, possibly suggesting a novel investigative approach for optimizing targeted cell deletion in cancer treatment.

  11. Intercellular transfer of apoptotic signals via electrofusion

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin Suk; Lee, Wilson; McCulloch, Christopher A., E-mail: christopher.mcculloch@utoronto.ca

    2012-05-01

    We determined whether cells that are induced to undergo anoikis by matrix detachment can initiate apoptosis in healthy cells following electroporation-induced fusion. Separate populations of MDCK cells undergoing anoikis and stained with FITC-annexin or viable MDCK cells that were labeled with spectrally discrete fluorescent beads were electroporated. Cells were analyzed by flow cytometry for enumeration of viable cells with beads, apoptotic cells or fused cells. Electroporation promoted a 49-fold increase of the percentage of viable cells that had fused with apoptotic cells. Apoptotic cell-viable cell fusions were 8-fold more likely to not attach to cell culture plastic and 2.3-fold less likely to proliferate after 24 hr incubation than viable cell fusion controls. These data demonstrate that apoptotic signals can be transferred between cells by electrofusion, possibly suggesting a novel investigative approach for optimizing targeted cell deletion in cancer treatment.

  12. Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy.

    Science.gov (United States)

    Li, Yan; Wong, Ying Lei; Ng, Fui Mee; Liu, Boping; Wong, Yun Xuan; Poh, Zhi Ying; Liu, Shuang; Then, Siew Wen; Lee, Michelle Yueqi; Ng, Hui Qi; Huang, Qiwei; Hung, Alvin W; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Kang, CongBao

    2016-08-19

    Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of β1-α2 and residues in the loop of β3-β4 might improve the inhibitor potency. PMID:27365392

  13. The phosphoCTD-interacting domain of Topoisomerase I

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jianhong; Phatnani, Hemali P.; Hsieh, Tao-Shih [Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 (United States); Greenleaf, Arno L., E-mail: arno.greenleaf@duke.edu [Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 (United States)

    2010-06-18

    The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

  14. The phosphoCTD-interacting domain of Topoisomerase I

    International Nuclear Information System (INIS)

    The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

  15. REDUCED TOPOISOMERASE-II ACTIVITY IN MULTIDRUG-RESISTANT HUMAN NONSMALL CELL LUNG-CANCER CELL-LINES

    NARCIS (Netherlands)

    EIJDEMS, EWHM; DEHAAS, M; TIMMERMAN, AJ; VANDERSCHANS, GP; KAMST, E; DENOOIJ, J; RICOTTI, GCBA; BORST, P; BAAS, F

    1995-01-01

    Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulte

  16. Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival.

    Science.gov (United States)

    Ahmed, Wareed; Menon, Shruti; Godbole, Adwait Anand; Karthik, Pullela V D N B; Nagaraja, Valakunja

    2014-04-01

    Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M. tuberculosis topoisomerase I (TopoI(Mt) ) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M. tuberculosis growth and open up new avenues for targeting the enzyme.

  17. Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I

    Energy Technology Data Exchange (ETDEWEB)

    Trucksis, M.; Depew, R.E.

    1981-04-01

    A gene that specifies production of Escherichia coli DNA topoisomerase I (..omega.. protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli ..omega.. protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX.

  18. Novel ametantrone-amsacrine related hybrids as topoisomerase IIβ poisons and cytotoxic agents.

    Science.gov (United States)

    Zagotto, Giuseppe; Gianoncelli, Alessandra; Sissi, Claudia; Marzano, Cristina; Gandin, Valentina; Pasquale, Riccardo; Capranico, Giovanni; Ribaudo, Giovanni; Palumbo, Manlio

    2014-10-01

    The precise definition of the structural requirements for effective topoisomerase II poisoning by drug molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we prepared several conjugates combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human topoisomerase IIβ isoform, and cytotoxic activity resembling that of the parent compounds. In addition, the properties of the lateral groups linked to the anthracenedione group play an important role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19 appear to be promising for further development. PMID:25042690

  19. The Effects of Arolycoricidine and Narciprimine on Tumor Cell Killing and Topoisomerase Activity

    Directory of Open Access Journals (Sweden)

    Buket Bozkurt Sarikaya

    2012-07-01

    Full Text Available In this study, narciprimine and arolycoricidine were isolated from G. rizehensis Stern (Amaryllidaceae. The structures of the alkaloids were elucidated by spectroscopic methods (1D NMR, EI-MS. Due to the previous reports on anti-cancer activity of this group of alkaloids, we investigated their effects on DNA topoisomerase reactions, which are known as the cellular targets of a number of chemotherapeutical drugs. The results revealed that arolycoricidine and narciprimine were effective in both type I and type II DNA topoisomerase reactions in a dose-dependent manner. Topoisomerase-interfering ability of these alkaloids partially correlated with cytostaticity assays, using HeLa (cervix adenocarcinoma, MCF7 (breast adenocarcinoma and A431 (skin epidermoid carcinoma cells. Our results are discussed in relation to the potential significance of these alkaloids in the course of drug-development studies.

  20. Increased topoisomerase IIalpha expression in colorectal cancer is associated with advanced disease and chemotherapeutic resistance via inhibition of apoptosis.

    LENUS (Irish Health Repository)

    Coss, Alan

    2012-02-01

    Topoisomerase IIalpha is a nuclear enzyme that regulates the tertiary structure of DNA. The influence of topoisomerase IIalpha gene (TOP2A) or protein alterations on disease progression and treatment response in colorectal cancer (CRC) is unknown. The study investigated the clinical relevance of topoisomerase IIalpha in CRC using in vivo and in vitro models. Differentially expressed genes in early and late-stage CRC were identified by array comparative genomic hybridization (CGH). Cellular location of gene amplifications was determined by fluorescence in situ hybridization (FISH). Topoisomerase IIalpha levels, proliferation index, and HER2 expression were examined in 228 colorectal tumors by immunohistochemistry. Overexpression of topoisomerase IIalpha in vitro was achieved by liposome-based transfection. Cell growth inhibition and apoptosis were quantified using the crystal violet assay and flow cytometry, respectively, in response to drug treatment. Amplification of TOP2A was identified in 3 (7.7%) tumors using array CGH and confirmed using FISH. At the protein level, topoisomerase IIalpha staining was observed in 157 (69%) tumors, and both staining and intensity levels were associated with an aggressive tumor phenotype (p values 0.04 and 0.005, respectively). Using logistic regression analysis, topoisomerase IIalpha remained significantly associated with advanced tumor stage when corrected for tumor proliferation (p=0.007) and differentiation (p=0.001). No association was identified between topoisomerase IIalpha and HER2. In vitro, overexpression of topoisomerase IIalpha was associated with resistance to irinotecan (p=0.001) and etoposide chemotherapy (p=0.03), an effect mediated by inhibition of apoptosis. Topoisomerase IIalpha overexpression is significantly associated with alterations in tumor behavior and response to drug treatment in CRC. Our results suggest that gene amplification may represent an important mechanism underlying these changes.

  1. A systematic review on topoisomerase 1 inhibition in the treatment of metastatic breast cancer

    DEFF Research Database (Denmark)

    Kümler, Iben; Brünner, Nils; Stenvang, Jan;

    2013-01-01

    Following treatment with anthracyclines and taxanes, few established options exist for the treatment of metastatic breast cancer (MBC). Although the topoisomerase 1 inhibitors irinotecan, etirinotecan, and topotecan have been used in clinical trials on MBC, the drugs have never been introduced...... as standard treatment for the disease. We performed a systematic review on topoisomerase 1 inhibitors in MBC and found 22 prospective trials and three retrospective ones. No phase III trials were identified. Only one study was randomized, and generally studies were small. Response rates (RR) for irinotecan...

  2. Interplay between Type 1A Topoisomerases and Gyrase in Chromosome Segregation in Escherichia coli

    OpenAIRE

    Usongo, Valentine; Tanguay, Cynthia; Nolent, Flora; Bessong, Jill Egbe; Drolet, Marc

    2013-01-01

    Escherichia coli possesses two type 1A topoisomerases, Topo I (topA) and Topo III (topB). Topo I relaxes excess negative supercoiling, and topA mutants can grow only in the presence of compensatory mechanisms, such as gyrase mutations. topB mutants grow as well as wild-type cells. In vitro, Topo III, but not Topo I, can efficiently decatenate DNA during replication. However, in vivo, a chromosome segregation defect is seen only when both type 1A topoisomerases are absent. Here we present expe...

  3. Flavonoids acting on DNA topoisomerases: recent advances and future perspectives in cancer therapy.

    Science.gov (United States)

    Russo, P; Del Bufalo, A; Cesario, A

    2012-01-01

    Flavonoids, secondary metabolites ubiquitously produced in the plant kingdom, are low molecular weight polyphenolic molecules. They are characterized by variable chemical structures and show a vast array of biological activities (i.e... antiviral, antiinflammatory, antitumor, antimicrobial, estrogenic, antiestrogenic, antioxidant, mutagenic and antimutagenic) targeting different pathways. Some of these compounds such as Genistein, Daidzein or its synthetic derivative Phenoxodiol as well as Luteolin and Quercetin are able to inhibit DNA topoisomerases. This review discusses that Flavonoids targeting DNA topoisomerases may lead to novel drug development with anticancer potential. PMID:22998568

  4. Highly sensitive detection of M.SssI DNA methyltransferase activity using a personal glucose meter.

    Science.gov (United States)

    Deng, Huimin; Peng, Si Ying; Gao, Zhiqiang

    2016-08-01

    A simple method for highly sensitive and selective detection of M.SssI CpG methyltransferase (M.SssI MTase) activity is developed, leveraging on the portability and ease of use of a personal glucose meter (PGM). Briefly, DNA-invertase conjugates are hybridized with their complementary DNA strands pre-immobilized on magnetic beads. The 5'-CCGG-3' sequence present in the DNA duplexes serves as the recognition site for both Hpa II restriction enzyme and M.SssI MTase (5'-CG-3'). Hpa II restriction enzyme specifically cleaves at unmethylated 5'-CCGG-3' sequence, and the invertase that remains on the methylated DNA catalyzes the hydrolysis of sucrose to glucose and fructose. It is found that the amount of glucose is proportional to the M.SssI MTase methylation activity in the range of 0.5 to 80 U/mL with a detection limit of 0.37 U/mL. Due to the specific recognition sequence present in the DNA strands, this method also shows high selectivity for M.SssI MTase. In addition, inhibition studies with 5'-azacytidine demonstrate the capability of inhibition screening using this method. Graphical abstract Deteciton of M.SssI DNA methyltransferase activity by a personal glucose meter. PMID:27311957

  5. Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

    OpenAIRE

    Gudkov, A V; Zelnick, C R; Kazarov, A R; Thimmapaya, R; Suttle, D P; Beck, W T; Roninson, I B

    1993-01-01

    Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random ...

  6. CFS-1686 causes cell cycle arrest at intra-S phase by interference of interaction of topoisomerase 1 with DNA.

    Directory of Open Access Journals (Sweden)

    Ru-Wei Lin

    Full Text Available CFS-1686 (chemical name (E-N-(2-(diethylaminoethyl-4-(2-(2-(5-nitrofuran-2-ylvinylquinolin-4-ylaminobenzamide inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin's induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.

  7. A mouse model for studying the interaction of bisdioxopiperazines with topoisomerase IIα in vivo

    DEFF Research Database (Denmark)

    Grauslund, Morten; Vinding, Annemette; Füchtbauer, Annette C.;

    2007-01-01

    -opened hydrolysis products are strong iron chelator. The clinically approved analog ICRF-187 is a pharmacological modulator of topoisomerase II poisons such as etoposide in preclinical animal models. ICRF-187 is also used to protect against anthracycline-induced cardiomyopathy and has recently been approved...

  8. Quantum dot based DNA nanosensors for amplification-free detection of human topoisomerase I

    DEFF Research Database (Denmark)

    Jepsen, Morten Leth; Ottaviani, Alessio; Knudsen, Birgitta R.;

    2014-01-01

    We develop a quantum dot based DNA nanosensor specifically targeting the cleavage–religation activity of an essential DNA-modifying enzyme, human topoisomerase I. The assay has shown great promise in biological crude samples and thus is expected to contribute to clinical diagnostics and anti...

  9. Human DNA topoisomerase inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7.

    Science.gov (United States)

    Tomczyk, M; Drozdowska, D; Bielawska, A; Bielawski, K; Gudej, J

    2008-05-01

    Two polyphenolics, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) and methyl brevifolincarboxylate (2) isolated from aerial parts of Potentilla argentea L. (Rosaceae) were evaluated for their cytotoxicities against human breast carcionoma cell line (MCF-7) and their DNA-binding ability. The DNA-binding ability of these compounds was studied by means of the human DNA topoisomerase I and II inhibition assay and ethidium displacement assay using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2. Compound 2 was much more active and showed a higher level of cytotoxic potency than compound 1, with IC50 values of 1.11 +/- 2 microM and 21.60 +/- 2 microM, respectively. In DNA topoisomerase I and II inhibition in vitro assays both investigated compounds 1 and 2 were more effective against topoisomerase II than I. The results of DNA binding studies reveal that methyl brevifolincarboxylate had a greater DNA binding affinity that tiliroside, which correlates with its greater potency as a topoisomerase I/II inhibitor.

  10. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  11. Anthocyanin Interactions with DNA: Intercalation, Topoisomerase I Inhibition and Oxidative Reactions.

    Science.gov (United States)

    Webb, Michael R; Min, Kyungmi; Ebeler, Susan E

    2008-09-23

    Anthocyanins and their aglycone anthocyanidins are pigmented flavonoids found in significant amounts in many commonly consumed foods. They exhibit a complex chemistry in aqueous solution, which makes it difficult to study their chemistry under physiological conditions. Here we used a gel electrophoresis assay employing supercoiled DNA plasmid to examine the ability of these compounds (1) to intercalate DNA, (2) to inhibit human topoisomerase I through both inhibition of plasmid relaxation activity (catalytic inhibition) and stabilization of the cleavable DNA-topoisomerase complex (poisoning), and (3) to inhibit or enhance oxidative single-strand DNA nicking. We found no evidence of DNA intercalation by anthocyan(id)ins in the physiological pH range for any of the compounds used in this study-cyanidin chloride, cyanidin 3-O-glucoside, cyanidin 3,5-O-diglucoside, malvidin 3-O-glucoside and luteolinidin chloride. The anthocyanins inhibited topoisomerase relaxation activity only at high concentrations (> 50 muM) and we could find no evidence of topoisomerase I cleavable complex stabilization by these compounds. However, we observed that all of the anthocyan(id)ins used in this study were capable of inducing significant oxidative DNA strand cleavage (nicking) in the presence of 1 mM DTT (dithiothreitol), while the free radical scavenger, DMSO, at concentrations typically used in similar studies, completely inhibited DNA nicking. Finally, we propose a mechanism to explain the anthocyan(id)in induced oxidative DNA cleavage observed under our experimental conditions. PMID:19924259

  12. Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant

    DEFF Research Database (Denmark)

    Arnò, Barbara; D'Annessa, Ilda; Tesauro, Cinzia;

    2013-01-01

    A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme, but it is chara...

  13. Structural and mechanistic insight into Holliday-junction dissolution by topoisomerase IIIα and RMI1

    DEFF Research Database (Denmark)

    Bocquet, Nicolas; Bizard, Anna H; Abdulrahman, Wassim;

    2014-01-01

    to TopIIIα influences it to behave as a hemicatenane dissolvase, rather than as an enzyme that relaxes DNA topology, is unknown. Here, we present the crystal structure of human TopIIIα complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIII assumes a toroidal type 1A topoisomerase...

  14. TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

    NARCIS (Netherlands)

    Gomez-Herreros, F.; Schuurs-Hoeijmakers, J.H.M.; McCormack, M.; Greally, M.T.; Rulten, S.; Romero-Granados, R.; Counihan, T.J.; Chaila, E.; Conroy, J.; Ennis, S.; Delanty, N.; Cortes-Ledesma, F.; Brouwer, A.P.M. de; Cavalleri, G.L.; El-Khamisy, S.F.; Vries, B.B. de; Caldecott, K.W.

    2014-01-01

    Topoisomerase II (TOP2) removes torsional stress from DNA and facilitates gene transcription by introducing transient DNA double-strand breaks (DSBs). Such DSBs are normally rejoined by TOP2 but on occasion can become abortive and remain unsealed. Here we identify homozygous mutations in the TDP2 ge

  15. Synthesis, DNA-damaging and cytotoxic properties of novel topoisomerase II-directed bisantrene analogues.

    Science.gov (United States)

    Zagotto, G; Oliva, A; Guano, F; Menta, E; Capranico, G; Palumbo, M

    1998-01-20

    New bisantrene analogues were synthesized, bearing one or two 4,5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1,4 or 9 of the anthracene ring system. A 10-azabioisostere was also prepared. The position of substituents in structurally isomeric drugs modulates topoisomerase II poisoning and specificity, along with cytotoxicity. PMID:9871638

  16. Apoptotic activity in Libyan breast cancer

    Directory of Open Access Journals (Sweden)

    Boder Jamela

    2012-06-01

    Full Text Available Abstract Background We evaluated the relationship of the apoptotic activity index (AI and the standardized mitotic-apoptotic ratio (SMI/AI with clinicopathological features and prognosis in Libyan female breast cancer (BC patients. We then compared our results with corresponding results in Finnish and Nigerian female BC patients. Methods Histological samples of breast carcinoma from 130 patients were retrospectively studied: an estimation of the apoptotic activity per square millimeter (expressed as apoptotic activity index (AI, and standardized mitotic-apoptotic ratio (SMI/AI was made, and the results compared with the clinicopathological features and the patient’s survival. Results There was a statistically significant correlation between the AI and most of the clinicopathological features; the strongest association was observed for clinical stage lymph node (LN status (P = 0.005. There were also correlations between AI and histological grade (P = 0.035, large tumor size (P = 0.011 and the clinical stage (P = 0.009. There were, however, prominent AI differences between Libyan, Nigerian and Finnish populations. The mean values of AI and SMI/AI in Libyan BC patients were 12.8 apoptotic figures per square millimeter and 2.8, respectively. The Libyan AI is slightly higher than in Nigeria, but much higher than in Finland. The differences between countries are seen throughout the samples as well as being present in certain subgroups. The survival analysis indicated that short survival time was associated with high apoptotic indices values and so can identify aggressive tumors and provide significant prognostic support. The cutoff (4 and 18 apoptosis/mm2 of AI might be applied as a quantitative criterion for Libyan BC to separate the patients into good, moderate and bad prognosis groups. Conclusions The results indicated that the differences in AI among the three countries may be due to the known variation in the distribution of

  17. Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

    Science.gov (United States)

    Kern, Gunther; Palmer, Tiffany; Ehmann, David E; Shapiro, Adam B; Andrews, Beth; Basarab, Gregory S; Doig, Peter; Fan, Jun; Gao, Ning; Mills, Scott D; Mueller, John; Sriram, Shubha; Thresher, Jason; Walkup, Grant K

    2015-08-21

    We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β. PMID:26149691

  18. Inhibition of Neisseria gonorrhoeae Type II Topoisomerases by the Novel Spiropyrimidinetrione AZD0914.

    Science.gov (United States)

    Kern, Gunther; Palmer, Tiffany; Ehmann, David E; Shapiro, Adam B; Andrews, Beth; Basarab, Gregory S; Doig, Peter; Fan, Jun; Gao, Ning; Mills, Scott D; Mueller, John; Sriram, Shubha; Thresher, Jason; Walkup, Grant K

    2015-08-21

    We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and β.

  19. Effect of topoisomerase II inhibitor on clonogen survival of FaDu tumor cells after irradiation in vitro

    International Nuclear Information System (INIS)

    The aim of this study is to examine the possible interaction between a topoisomerase II inhibitor (etoposide) and ionising radiation, since the combination of both therapeutic strategies is of great clinical interest. (orig.)

  20. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

    DEFF Research Database (Denmark)

    Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust;

    2013-01-01

    . The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor......Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I...... is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family...

  1. Ellagic acid and polyhydroxylated urolithins are potent catalytic inhibitors of human topoisomerase II: an in vitro study.

    Science.gov (United States)

    Furlanetto, Valentina; Zagotto, Giuseppe; Pasquale, Riccardo; Moro, Stefano; Gatto, Barbara

    2012-09-12

    Ellagic acid (EA), a natural polyphenol abundant in fruits and common in our diet, is under intense investigation for its chemopreventive activity resulting from multiple effects. EA inhibits topoisomerase II, but the effects on the human enzyme of urolithins, its monolactone metabolites, are not known. Therefore, the action of several synthetic urolithins toward topoisomerases II was evaluated, showing that polyhydroxylated urolithins, EA, and EA-related compounds are potent inhibitors of the α and β isoforms of human topoisomerase II at submicromolar concentrations. Competition tests demonstrate a dose-dependent relationship between ATP and the inhibition of the enzyme. Docking experiments show that the active compounds bind the ATP pocket of the human enzyme, thus supporting the hypothesis that EA and polyhydroxylated urolithins act as ATP-competitive inhibitors of human topoisomerase II. PMID:22924519

  2. Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

    Directory of Open Access Journals (Sweden)

    José A. Sánchez Alcázar

    2015-08-01

    Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

  3. Towards the Small and the Beautiful: A Small Dibromotyrosine Derivative from Pseudoceratina sp. Sponge Exhibits Potent Apoptotic Effect through Targeting IKK/NFκB Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Michael Y. Chiang

    2013-08-01

    Full Text Available A dibromotyrosine derivative, (1′R,5′S,6′S-2-(3′,5′-dibromo-1′,6′-dihydroxy-4′-oxocyclohex-2′-enyl acetonitrile (DT, was isolated from the sponge Pseudoceratina sp., and was found to exhibit a significant cytotoxic activity against leukemia K562 cells. Despite the large number of the isolated bromotyrosine derivatives, studies focusing on their biological mechanism of action are scarce. In the current study we designed a set of experiments to reveal the underlying mechanism of DT cytotoxic activity against K562 cells. First, the results of MTT cytotoxic and the annexin V-FITC/PI apoptotic assays, indicated that the DT cytotoxic activity is mediated through induction of apoptosis. This effect was also supported by caspases-3 and -9 activation as well as PARP cleavage. DT induced generation of reactive oxygen species (ROS and the disruption of mitochondrial membrane potential (MMP as indicated by flow cytometric assay. The involvement of ROS generation in the apoptotic activity of DT was further corroborated by the pretreatment of K562 cells with N-acetyl-cysteine (NAC, a ROS scavenger, which prevented apoptosis and the disruption of MMP induced by DT. Results of cell-free system assay suggested that DT can act as a topoisomerase II catalytic inhibitor, unlike the clinical anticancer drug, etoposide, which acts as a topoisomerase poison. Additionally, we found that DT treatment can block IKK/NFκB pathway and activate PI3K/Akt pathway. These findings suggest that the cytotoxic effect of DT is associated with mitochondrial dysfunction-dependent apoptosis which is mediated through oxidative stress. Therefore, DT represents an interesting reference point for the development of new cytotoxic agent targeting IKK/NFκB pathway.

  4. Towards the small and the beautiful: a small dibromotyrosine derivative from Pseudoceratina sp. sponge exhibits potent apoptotic effect through targeting IKK/NFκB signaling pathway.

    Science.gov (United States)

    Su, Jui-Hsin; Chen, Yu-Cheng; El-Shazly, Mohamed; Du, Ying-Chi; Su, Chiang-Wen; Tsao, Chia-Wei; Liu, Li-Lian; Chou, Yalan; Chang, Wen-Been; Su, Yin-Di; Chiang, Michael Y; Yeh, Yao-Tsung; Lu, Mei-Chin

    2013-09-01

    A dibromotyrosine derivative, (1'R,5'S,6'S)-2-(3',5'-dibromo-1',6'-dihydroxy-4'-oxocyclohex-2'-enyl) acetonitrile (DT), was isolated from the sponge Pseudoceratina sp., and was found to exhibit a significant cytotoxic activity against leukemia K562 cells. Despite the large number of the isolated bromotyrosine derivatives, studies focusing on their biological mechanism of action are scarce. In the current study we designed a set of experiments to reveal the underlying mechanism of DT cytotoxic activity against K562 cells. First, the results of MTT cytotoxic and the annexin V-FITC/PI apoptotic assays, indicated that the DT cytotoxic activity is mediated through induction of apoptosis. This effect was also supported by caspases-3 and -9 activation as well as PARP cleavage. DT induced generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) as indicated by flow cytometric assay. The involvement of ROS generation in the apoptotic activity of DT was further corroborated by the pretreatment of K562 cells with N-acetyl-cysteine (NAC), a ROS scavenger, which prevented apoptosis and the disruption of MMP induced by DT. Results of cell-free system assay suggested that DT can act as a topoisomerase II catalytic inhibitor, unlike the clinical anticancer drug, etoposide, which acts as a topoisomerase poison. Additionally, we found that DT treatment can block IKK/NFκB pathway and activate PI3K/Akt pathway. These findings suggest that the cytotoxic effect of DT is associated with mitochondrial dysfunction-dependent apoptosis which is mediated through oxidative stress. Therefore, DT represents an interesting reference point for the development of new cytotoxic agent targeting IKK/NFκB pathway. PMID:24065159

  5. A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes.

    Science.gov (United States)

    Kondapi, Anand K; Satyanarayana, Nathamu; Saikrishna, A D

    2006-06-15

    Human Topoisomerase II is present in two isoforms, 170KDa alpha and 180KDa beta. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II beta over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-1(93IN101) in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II alpha and beta isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication. PMID:16712776

  6. Antiproliferative and apoptotic effects of spanish honeys

    OpenAIRE

    Paloma Morales; Ana Isabel Haza

    2013-01-01

    Background: Current evidence supports that consumption of polyphenols has beneficial effects against numerous diseases mostly associated with their antioxidant activity. Honey is a good source of antioxidants since it contains a great variety of phenolic compounds. Objective: The main objective of this work was to investigate the antiproliferative and apoptotic effects of three crude commercial honeys of different floral origin (heather, rosemary and polyfloral honey) from Madrid Autonomic Co...

  7. Apoptotic regulation of epithelial cellular extrusion

    OpenAIRE

    De Andrade, Daniel,; Rosenblatt, Jody

    2011-01-01

    Cellular extrusion is a mechanism that removes dying cells from epithelial tissues to prevent compromising their barrier function. Extrusion occurs in all observed epithelia in vivo and can be modeled in vitro by inducing apoptosis in cultured epithelial monolayers. We established that actin and myosin form a ring that contracts in the surrounding cells that drives cellular extrusion. It is not clear, however, if all apoptotic pathways lead to extrusion and how apoptosis and extrusion are mol...

  8. Development of DNA topoisomerase-related therapeutics: a short perspective of new challenges.

    Science.gov (United States)

    Capranico, Giovanni; Zagotto, Giuseppe; Palumbo, Manlio

    2004-07-01

    Antitumor agents targeting DNA and DNA-associated processes are widely used in the treatment of human cancers and produce significant increases in the survival of patients. DNA topoisomerases remain the most significant target of these cytotoxic drugs and constitute a growing family of nuclear enzymes that regulate DNA topology during DNA replication and recombination, DNA transcription, chromosome condensation-decondensation and segregation. Major progress has been attained in recent years in the understanding of the structures of these enzymes and their main cellular functions, hopefully providing new opportunities for pharmacological interventions. New leads and derivatives of known structures have been reported recently, and here they will be discussed highlighting the challenges to find innovative and more effective drugs. Moreover, we will review novel and diverse approaches relevant to the development of new topoisomerase-related therapeutics. PMID:15281906

  9. Sophoridinol derivative 05D induces tumor cells apoptosis by topoisomerase1-mediated DNA breakage

    Directory of Open Access Journals (Sweden)

    Zhao W

    2016-05-01

    Full Text Available Wuli Zhao, Caixia Zhang, Chongwen Bi, Cheng Ye, Danqing Song, Xiujun Liu, Rongguang Shao Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People’s Republic of China Abstract: Sophoridine is a quinolizidine natural product of Sophora alopecuroides and has been applied for treatment of malignant trophoblastic tumors. Although characterized by low toxicity, the limited-spectrum antitumor activity hinders its further applications. 05D, a derivative of sophoridine, exhibits a better anticancer activity on diverse cancer cells, including solid tumors, and hematologic malignancy. It could inhibit topoisomerase 1 (top1 activity by stabilizing DNA–top1 complex and induce mitochondria-mediated apoptosis by promoting DNA single- and double-strand breakage mediated by top1. Also, 05D induced HCT116 cells arrest at G1 phase by inactivating CDK2/CDK4–Rb–E2F and cyclinD1–CDK4–p21 checkpoint signal pathways. 05D suppressed the ataxia telangiectasia mutated (ATM and ATM and Rad3-related (ATR activation and decreased 53BP level, which contributed to DNA damage repair, suggesting that the novel compound 05D might be helpful to improve the antitumor activity of DNA damaging agent by repressing ATM and ATR activation and 53BP level. In addition, the priorities in molecular traits and druggability, such as a simple structure and formulation for oral administration, further prove 05D to be a promising targeting topoisomerase agent. Keywords: topoisomerase inhibitor, topoisomerase 1, DNA breakage, sophoridinol, anticancer, apoptosis, cell cycle

  10. Topoisomerase II–DNA complexes trapped by ICRF-193 perturb chromatin structure

    OpenAIRE

    Germe, Thomas; Hyrien, Olivier

    2005-01-01

    DNA topoisomerase II (topo II) is involved in unlinking replicating DNA and organizing mitotic chromosomes. Topo II is the target of many antitumour drugs. Topo II inhibition results in extensive catenation of newly replicated DNA and may potentially perturb chromatin assembly. Here, we show that the topo II inhibitor ICRF-193 does not prevent the bulk of nucleosome deposition, but perturbs nucleosome spacing in Xenopus egg extracts. This is due to the trapping of topo II-closed clamps on the...

  11. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

    OpenAIRE

    Snapka, R M

    1986-01-01

    I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers ...

  12. Anthocyanin Interactions with DNA: Intercalation, Topoisomerase I Inhibition and Oxidative Reactions

    OpenAIRE

    Webb, Michael R.; Min, Kyungmi; Susan E. Ebeler

    2008-01-01

    Anthocyanins and their aglycone anthocyanidins are pigmented flavonoids found in significant amounts in many commonly consumed foods. They exhibit a complex chemistry in aqueous solution, which makes it difficult to study their chemistry under physiological conditions. Here we used a gel electrophoresis assay employing supercoiled DNA plasmid to examine the ability of these compounds (1) to intercalate DNA, (2) to inhibit human topoisomerase I through both inhibition of plasmid relaxation act...

  13. Position- and orientation-specific enhancement of topoisomerase I cleavage complexes by triplex DNA structures

    OpenAIRE

    Antony, Smitha; Arimondo, Paola B.; Sun, Jian-Sheng; Pommier, Yves

    2004-01-01

    Topoisomerase I (Top1) activities are sensitive to various endogenous base modifications, and anticancer drugs including the natural alkaloid camptothecin. Here, we show that triple helix-forming oligonucleotides (TFOs) can enhance Top1-mediated DNA cleavage by affecting either or both the nicking and the closing activities of Top1 depending on the position and the orientation of the triplex DNA structure relative to the Top1 site. TFO binding 1 bp downstream from the Top1 site enhances cleav...

  14. Topoisomerase IIIα is required for normal proliferation and telomere stability in alternative lengthening of telomeres

    OpenAIRE

    Temime-Smaali, Nassima; Guittat, Lionel; Wenner, Thomas; Bayart, Emilie; Douarre, Céline; Gomez, Dennis; Giraud-Panis, Marie-Josèphe; Londono-Vallejo, Arturo; Gilson, Eric; Amor-Guéret, Mounira; Riou, Jean-François

    2008-01-01

    Topoisomerase (Topo) IIIα associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIα colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIα immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immu...

  15. Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

    Science.gov (United States)

    Savage, Victoria J; Charrier, Cédric; Salisbury, Anne-Marie; Box, Helen; Chaffer-Malam, Nathan; Huxley, Anthony; Kirk, Ralph; Noonan, Gary M; Mohmed, Sarfraz; Craighead, Mark W; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

    2016-09-01

    There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections. PMID:27324777

  16. Influence of Cell Cycle and Oncogene Activity upon Topoisomerase IIα Expression and Drug Toxicity

    OpenAIRE

    Stacey, Dennis W.; Hitomi, Masahiro; Chen, Guan

    2000-01-01

    The cell cycle, oncogenic signaling, and topoisomerase (topo) IIα levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIα levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to i...

  17. Mutational analysis of a type II topoisomerase cleavage site: distinct requirements for enzyme and inhibitors.

    OpenAIRE

    Freudenreich, C H; Kreuzer, K. N.

    1993-01-01

    We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 type II topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cle...

  18. CS1 is a novel topoisomerase IIα inhibitor with favorable drug resistance profiles

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yan; Chen, Wang; Zhao, Baobing; Hao, Huilin; Li, Zhenyu; Lu, Chunhua; Shen, Yuemao, E-mail: yshen@sdu.edu.cn

    2014-10-24

    Highlights: • CS1 is a novel nonintercalating topoisomerase IIα poison. • CS1 shows potent in vitro and in vivo antitumor activity. • CS1 shows 6–10-fold less toxicity to normal cells compared with etoposide. • CS1 is not a substrate of P-glycoprotein and multidrug resistance irrelevant. - Abstract: DNA topoisomerase II (Topo II) is an essential nuclear enzyme and a validated target for anticancer agent screening. CS1, a novel 2-phenylnaphthalene, had potent cytotoxicity against nine tested tumor cell lines and showed 6–10-fold less toxicity against normal cell lines compared with etoposide. In addition, CS1 showed potential anti-multidrug resistance capabilities. kDNA decatenation, DNA relaxation and cleavage complex assays indicated that CS1 acted as a nonintercalating topoisomerase IIα (Topo IIα) inhibitor by stabilizing the DNA-Topo IIα cleavage complex. CS1 also induced DNA breaks in MDA-MB-231 cells evidenced by comet tails and the accumulation of γH2AX foci. The ability of CS1 in inducing DNA breaks mediated by Topo II resulted in G2/M phase arrest and apoptosis. Moreover, CS1 exhibited dramatic in vivo antitumor activity and lower toxicity compared with etoposide. This work supports the development of CS1 as a promising candidate for the treatment of cancer by targeting Topo IIα.

  19. Metal ion and inter-domain interactions as functional networks in E. coli topoisomerase I.

    Science.gov (United States)

    Sissi, Claudia; Cheng, Bokun; Lombardo, Valentina; Tse-Dinh, Yuk-Ching; Palumbo, Manlio

    2013-07-25

    Escherichia coli topoisomerase I (EcTopoI) is a type IA bacterial topoisomerase which is receiving large attention due to its potential application as novel target for antibacterial therapeutics. Nevertheless, a detailed knowledge of its mechanism of action at molecular level is to some extent lacking. This is partly due to the requirement of several factors (metal ions, nucleic acid) to the proper progress of the enzyme catalytic cycle. Additionally, each of them can differently affect the protein structure. Here we assess the role of the different components (DNA, metal ions, protein domains) in a dynamic environment as in solution by monitoring the catalytic as well as the structural properties of EcTopoI. Our results clearly indicated the interaction among these components as functionally relevant and underlined their mutual involvement. Some similarities with other enzymes of the same family emerged (for example DNA prevents divalent metal ions coordination at non selective binding sites). Interestingly, same interactions (C- and N-terminal domain interaction) appear to be peculiar of this bacterial topoisomerase which suggest they could be favorably exploited to the design of selective inhibitors for this class of enzyme.

  20. Mapping drug interactions at the covalent topoisomerase II-DNA complex by bisantrene/amsacrine congeners.

    Science.gov (United States)

    Capranico, G; Guano, F; Moro, S; Zagotto, G; Sissi, C; Gatto, B; Zunino, F; Menta, E; Palumbo, M

    1998-05-22

    To identify structural determinants for the sequence-specific recognition of covalent topoisomerase II-DNA complexes by anti-cancer drugs, we investigated a number of bisantrene congeners, including a 10-azabioisoster, bearing one or two 4, 5-dihydro-1H-imidazol-2-yl hydrazone side chains at positions 1, 4, or 9 of the anthracene ring system. The studied bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers were able to poison DNA topoisomerase II with an intermediate activity between those of bisantrene and m-AMSA. Moving the side chain from the central to a lateral ring (from C-9 to C-1/C-4) only slightly modified the drug DNA affinity, whereas it dramatically affected local base preferences of poison-stimulated DNA cleavage. In contrast, switching the planar aromatic systems of bisantrene and m-AMSA did not substantially alter the sequence specificity of drug action. A computer-assisted steric and electrostatic alignment analysis of the test compounds was in agreement with the experimental data, since a common pharmacophore was shared by bisantrene, m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of topoisomerase II poisons and in determining the poison pharmacophore. PMID:9582297

  1. Rubrofusarina, um policetídeo natural inibidor da topoisomerase II-α humana Rubrofusarin, a natural polyketide as new human topoisomerase II-α inhibitor

    Directory of Open Access Journals (Sweden)

    Alexsandro Branco

    2008-12-01

    Full Text Available Este trabalho descreve o efeito inibitório de rubrofusarina (5,6-diidroxi-8-metoxi-2-metilbenzo[g]cromen-4-ona, 1 sobre a enzima DNA topoisomerase II-α humana. Rubrofusarina mostrou total inibição da enzima topisomerase II-α na concentração de 120 µM. Este resultado é semelhante ao observado com etoposida, utilizada como controle positivo. Para a realização deste teste, rubrofusarina foi isolada de Senna macranthera var. nervosa (Voguel Irwin & Barnebyem e caracterizada por métodos espectroscópicos, incluindo RMN 2D, do produto natural bem como de seu derivado monoacetilado.This work describes the inhibitory effect of rubrofusarin (5,6-dihydroxy-8-methoxy-2-methylbenzo[g]cromen-4-one, 1 against human DNA topoisomerase II-α. The results for relaxation assays showed total inhibition of topisomerase II-α by rubrofusarin at 120 µM. This result is comparable to the one observed with etoposide as positive control. For this study, rubrofusarin was isolated from Senna macranthera var. nervosa (Voguel Irwin & Barnebyem and characterized by spectral data, including 2D NMR, as well as its acetylated derivative.

  2. Examination of the Impact of Copper(II) α-(N)-Heterocyclic Thiosemicarbazone Complexes on DNA Topoisomerase IIα.

    Science.gov (United States)

    Wilson, James T; Jiang, Xiaohua; McGill, Bradley C; Lisic, Edward C; Deweese, Joseph E

    2016-04-18

    Type II DNA topoisomerases resolve topological knots and tangles in DNA that result from routine cellular processes and are effective targets for anticancer therapeutics. To this end, thiosemicarbazones have been identified as having the ability to kill cancer cells from several cell lines. Literature evidence suggests that at least some thiosemicarbazones have an impact on topoisomerase II activity. However, the mechanism is not as clearly defined. Therefore, we set out to analyze the activity of four α-(N)-heterocyclic thiosemicarbazone compounds against topoisomerase IIα. The ligands, acetylpyridine-ethylthiosemicarbazone (APY-ETSC) and acetylpyrazine-methylthiosemicarbazone (APZ-MTSC), and their copper(II) [Cu(II)] complexes [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were examined for the ability to impact the catalytic cycle of human topoisomerase IIα. Both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] were more effective at inhibiting DNA relaxation compared with the ligands alone. Further, both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl] increased double-stranded DNA cleavage levels without inhibiting topoisomerase IIα-mediated DNA ligation. The Cu(II) complexes inactivate enzyme activity over time suggesting a critical interaction with the enzyme. Additionally, we found that the Cu(II)-thiosemicarbazone complexes do not significantly impact DNA cleavage by the catalytic core of the enzyme. This evidence is supported by the fact that both [Cu(APY-ETSC)Cl] and [Cu(APZ-MTSC)Cl], and to a lesser extent the ligands, inhibit topoisomerase IIα-mediated ATP hydrolysis. Based upon kinetic analysis, the Cu(II) complexes appear to be noncompetitive inhibitors of the ATPase domain of topoisomerase IIα. Taken together, our results provide evidence that Cu(II) complexes of α-(N)-heterocyclic thiosemicarbazones catalytically inhibit the enzyme through the ATPase domain but also promote double-stranded DNA cleavage by the enzyme. PMID:26982206

  3. Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

    Institute of Scientific and Technical Information of China (English)

    Sung Yi Hong; Myun Hee Lee; Kyung Sup Kim; Hyun Cheol Jung; Jae Kyung Roh; Woo Jin Hyung; Sung Hoon Noh; Seung Ho Choi

    2004-01-01

    AIM: rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer. However,a sustained and high protein delivery is required to achieve the desired therapeutic effects. We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model.METHODS: rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines. To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin, Western blotting and ELISA were performed. The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays.The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor, etoposide, were evaluated in a mouse liver tumor model.RESULTS: Topoisomerase inhibitors, including camptothecin and etoposide, were found to increase the endostatin expression level in vitro. The over-expressed endostatin,as a result of pretreatment with a topoisomerase inhibitor,was also biologically active. In animal experiments, the combined therapy of topoisomerase inhibitor, etoposide with the rAAV-endostatin vector had the best tumorsuppressive effect and tumor foci were barely observed in livers of the treated mice. Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice. Finally, the mice treated with rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models.CONCLUSION: rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

  4. Antiproliferative and apoptotic effects of spanish honeys

    Directory of Open Access Journals (Sweden)

    Paloma Morales

    2013-01-01

    Full Text Available Background: Current evidence supports that consumption of polyphenols has beneficial effects against numerous diseases mostly associated with their antioxidant activity. Honey is a good source of antioxidants since it contains a great variety of phenolic compounds. Objective: The main objective of this work was to investigate the antiproliferative and apoptotic effects of three crude commercial honeys of different floral origin (heather, rosemary and polyfloral honey from Madrid Autonomic Community (Spain as well as of an artificial honey in human peripheral blood promyelocytic leukemia cells (HL-60. Material and Methods: HL-60 cells were cultured in the presence of honeys at various concentrations for up to 72 hours and the percentage of cell viability was evaluated by MTT assay. Apoptotic cells were identified by chromatin condensation and flow cytometry analysis. ROS production was determined using 2΄,7΄-dichlorodihydrofluorescein diacetate (H 2 DCFDA. Results: The three types of crude commercial honey induced apoptosis in a concentration and time dependent-manner. In addition, honeys with the higher phenolic content, heather and polyfloral, were the most effective to induce apoptosis in HL-60 cells. However, honeys did not generate reactive oxygen species (ROS and N-acetyl-L-cysteine (NAC could not block honeys-induced apoptosis in HL-60 cells. Conclusion: These data support that honeys induced apoptosis in HL-60 cells through a ROS-independent cell death pathway.Moreover, our findings indicate that the antiproliferative and apoptotic effects of honey varied according to the floral origin and the phenolic content.

  5. Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster

    Indian Academy of Sciences (India)

    Madhu G Tapadia; Naveen K Gautam

    2011-08-01

    Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of -tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

  6. Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

    Science.gov (United States)

    Perng, Daw-Shyong; Tsai, Yu-Hsin; Cherng, Jonathan; Wang, Jeng-Shing; Chou, Kuo-Shen; Shih, Chia-Wen; Cherng, Jaw-Ming

    2016-01-01

    Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26792981

  7. Apoptotic cell clearance: basic biology and therapeutic potential.

    Science.gov (United States)

    Poon, Ivan K H; Lucas, Christopher D; Rossi, Adriano G; Ravichandran, Kodi S

    2014-03-01

    The prompt removal of apoptotic cells by phagocytes is important for maintaining tissue homeostasis. The molecular and cellular events that underpin apoptotic cell recognition and uptake, and the subsequent biological responses, are increasingly better defined. The detection and disposal of apoptotic cells generally promote an anti-inflammatory response at the tissue level, as well as immunological tolerance. Consequently, defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity. Conversely, under certain conditions, such as the killing of tumour cells by specific cell-death inducers, the recognition of apoptotic tumour cells can promote an immunogenic response and antitumour immunity. Here, we review the current understanding of the complex process of apoptotic cell clearance in physiology and pathology, and discuss how this knowledge could be harnessed for new therapeutic strategies.

  8. Isolation and partial characterisation of a mammalian cell mutant hypersensitive to topoisomerase II inhibitors and X-rays

    International Nuclear Information System (INIS)

    The authors have isolated, following one-step mutagenesis, a Chinese hamster ovary cell mutant hypersensitive to the intercalating agent, adriamycin. This agent exerts at least part of its cytotoxic action via inhibition of the nuclear enzyme, topoisomerase II. The mutant, designated ADR-3, showed hypersensitivity to all classes of topoisomerase II inhibitors, inlcuding actinomycin D, amsacrine (m-AMSA), etoposide (VP16) and mitoxantrone. ADR-3 cells also showed cross-sensitivity to ionizing radiation, but not no UV light. Topoisomerase II activity was elevated to a small but significant degree in ADR-3 cells, and this was reflected in a 1.5-fold higher level of topoisomerase II protein in ADR-3 than in CHO-K1 cells, as judged by Western blotting. ADR-3 cells were hypersensitive to cumene hydroperoxide but cross-resistant to hydrogen peroxide, suggesting possible abnormality in the detoxification of peroxides by glutathione peroxidase or catalase. Glutathione peroxidase activity against hydroperoxide was elevated to a small but significant extent in mutant cells. Catalase levels were not significantly different in ADR-3 and CHO-K1 cells. ADR-3 cells were recessive in hybrids with parental CHO-K1 cells with respect to sensitivity to topoisomerase II inhibitors and X-rays, and represent a different genetic complementation group from the previously reported adriamycin-sensitive mutant, ADR-1. (author). 34 refs.; 5 figs.; 3 tabs

  9. Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

    DEFF Research Database (Denmark)

    Li, Xiyang; Guo, Li; Deng, Ling;

    2011-01-01

    Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly t...

  10. Bcl-2 Inhibitors: Targeting Mitochondrial Apoptotic Pathways in Cancer Therapy

    OpenAIRE

    Kang, Min H.; Reynolds, C. Patrick

    2009-01-01

    Defects in apoptotic pathways can promote cancer cell survival and also confer resistance to antineoplastic drugs. One pathway being targeted for antineoplastic therapy is the anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1, and Bcl-B) that bind to and inactivate BH3-domain pro-apoptotic proteins. Signals transmitted by cellular damage (including antineoplastic drugs) or cytokine deprivation can initiate apoptosis via the intrinsic apoptotic ...

  11. Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening

    Institute of Scientific and Technical Information of China (English)

    张虹; 潘远江

    2004-01-01

    A method of screening assay is demonstrated. The approach is based on the affinity ofantitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concentration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detection (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.

  12. Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening

    Institute of Scientific and Technical Information of China (English)

    张虹; 潘远江

    2004-01-01

    A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concen-tration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detec-tion (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.

  13. Potent catenation of supercoiled and gapped DNA circles by topoisomerase I in the presence of a hydrophilic polymer.

    Science.gov (United States)

    Low, R L; Kaguni, J M; Kornberg, A

    1984-04-10

    An exceptionally potent DNA catenation activity, identified in an extract from Escherichia coli, has been purified and partially characterized. Catenation results from the sequential action of the following two polypeptides: beta, 34 kDa and identical to exonuclease III; and alpha, 101 kDa and identical to DNA topoisomerase I (omega protein). An additional requirement is that a small proportion of the circles be nicked in order to provide the substrate for exonuclease III to generate gaps, estimated to be about 100 nucleotides long. Following exonuclease III digestion, one molecule of topoisomerase I can interlock per minute at 30 degrees C about 20 supercoiled and gapped DNA circles into a massively catenated network. The reaction requires Mg2+ and a hydrophilic polymer (polyvinyl alcohol or polyethylene glycol) at about 7%, but neither ATP nor spermidine. The hydrophilic polymer appears to drive catenation by condensing the DNA; decatenation by topoisomerase I proceeds upon removal of the polymer. PMID:6323479

  14. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    G.P. Amarante-Mendes

    1999-09-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  15. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    Amarante-Mendes G.P.

    1999-01-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  16. Xer recombinase and genome integrity in Helicobacter pylori, a pathogen without topoisomerase IV.

    Directory of Open Access Journals (Sweden)

    Aleksandra W Debowski

    Full Text Available In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH and its cognate dif site (difH. Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.

  17. Interplay between type 1A topoisomerases and gyrase in chromosome segregation in Escherichia coli.

    Science.gov (United States)

    Usongo, Valentine; Tanguay, Cynthia; Nolent, Flora; Bessong, Jill Egbe; Drolet, Marc

    2013-04-01

    Escherichia coli possesses two type 1A topoisomerases, Topo I (topA) and Topo III (topB). Topo I relaxes excess negative supercoiling, and topA mutants can grow only in the presence of compensatory mechanisms, such as gyrase mutations. topB mutants grow as well as wild-type cells. In vitro, Topo III, but not Topo I, can efficiently decatenate DNA during replication. However, in vivo, a chromosome segregation defect is seen only when both type 1A topoisomerases are absent. Here we present experimental evidence for an interplay between gyrase and type 1A topoisomerases in chromosome segregation. We found that both the growth defect and the Par(-) phenotypes of a gyrB(Ts) mutant at nonpermissive temperatures were significantly corrected by deleting topA, but only when topB was present. Overproducing Topo IV, the major cellular decatenase, could not substitute for topB. We also show that overproducing Topo III at a very high level could suppress the Par(-) phenotype. We previously found that the growth and chromosome segregation defects of a triple topA rnhA gyrB(Ts) mutant in which gyrase supercoiling activity was strongly inhibited could be corrected by overproducing Topo III (V. Usongo, F. Nolent, P. Sanscartier, C. Tanguay, S. Broccoli, I. Baaklini, K. Drlica, and M. Drolet, Mol. Microbiol. 69:968-981, 2008). We show here that this overproduction could be bypassed by substituting the gyrB(Ts) allele for a gyrB(+) one or by growing cells in a minimal medium, conditions that reduced both topA- and rnhA-dependent unregulated replication. Altogether, our data point to a role for Topo III in chromosome segregation when gyrase is inefficient and suggest that Topo I plays an indirect role via supercoiling regulation. PMID:23396913

  18. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation

    OpenAIRE

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2008-01-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was express...

  19. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer

    OpenAIRE

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong; Kim, Wun-Jae

    2016-01-01

    Purpose Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Materials and Methods Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort ...

  20. Developing T lymphocytes are uniquely sensitive to a lack of topoisomerase III alpha

    DEFF Research Database (Denmark)

    Mönnich, Maren; Hess, Isabell; Wiest, Waltraud;

    2010-01-01

    , the heteromeric RTR complex consists of RMI1, RMI2, the Bloom's syndrome gene product (BLM), and topoisomerase 3A (TOP3A) proteins. Here, we describe the identification and characterization of two deleterious mutations in the zebrafish top3a gene that reveal an unexpected tissue-specific requirement of top3a...... function in developing thymocytes. Deficiency in top3a activates a p53-dependent check-point but does not affect VDJ recombination. Our results suggest that TOP3A could be a candidate gene involved in human primary immunodeficiency syndromes....

  1. Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator.

    Science.gov (United States)

    Choi, Byung-Min; Pae, Hyun-Ock; Jang, Seon Il; Kim, Young-Myeong; Chung, Hun-Taeg

    2002-01-31

    Nitric oxide (NO), synthesized from L-arginine by NO synthases, is a small, lipophilic, diffusible, highly reactive molecule with dichotomous regulatory roles in many biological events under physiological and pathological conditions. NO can promote apoptosis (pro-apoptosis) in some cells, whereas it inhibits apoptosis (anti-apoptosis) in other cells. This complexity is a consequence of the rate of NO production and the interaction with biological molecules such as metal ion, thiol, protein tyrosine, and reactive oxygen species. Long-lasting overproduction of NO acts as a pro-apoptotic modulator, activating caspase family proteases through the release of mitochondrial cytochrome c into cytosol, up-regulation of the p53 expression, and alterations in the expression of apoptosis-associated proteins, including the Bcl-2 family. However, low or physiological concentrations of NO prevent cells from apoptosis that is induced by the trophic factor withdrawal, Fas, TNFalpha/ActD, and LPS. The anti-apoptotic mechanism is understood on the basis of gene transcription of protective proteins. These include: heat shock protein, hemeoxygenase, or cyclooxygenase-2 and direct inhibition of the apoptotic executive effectors caspase family protease by S-nitrosylation of the cysteine thiol group in their catalytic site in a cell specific way. Our current understanding of the mechanisms by which NO exerts both pro- and anti-apototic action is discussed in this review article. PMID:16248976

  2. Combination phenylbutyrate/gemcitabine therapy effectively inhibits in vitro and in vivo growth of NSCLC by intrinsic apoptotic pathways

    Directory of Open Access Journals (Sweden)

    Schniewind Bodo

    2006-11-01

    Full Text Available Abstract Background Standard chemotherapy protocols in NSCLC are of limited clinical benefit. Histone deacetylase (HDAC inhibitors represent a new strategy in human cancer therapy. In this study the combination of the HDAC inhibitor phenylbutyrate (PB and the nucleoside analogue gemcitabine (GEM was evaluated and the mechanisms underlying increased cell death were analyzed. Methods Dose escalation studies evaluating the cytotoxicity of PB (0.01–100 mM, GEM (0.01–100 μg/ml and a combination of the two were performed on two NSCLC cell lines (BEN and KNS62. Apoptotic cell death was quantified. The involvement of caspase-dependent cell death and MAP-kinase activation was analyzed. Additionally, mitochondrial damage was determined. In an orthotopic animal model the combined effect of PB and GEM on therapy was analyzed. Results Applied as a single drug both GEM and PB revealed limited potential to induce apoptosis in KNS62 and Ben cells. Combination therapy was 50–80% (p = 0.012 more effective than either agent alone. On the caspase level, combination therapy significantly increased cleavage of the pro-forms compared to single chemotherapy. The broad spectrum caspase-inhibitor zVAD was able to inhibit caspase cleavage completely, but reduced the frequency of apoptotic cells only by 30%. Combination therapy significantly increased changes in MTP and the release of cyto-c, AIF and Smac/Diabolo into the cytoplasm. Furthermore, the inhibitors of apoptosis c-IAP1 and c-IAP2 were downregulated and it was shown that in combination therapy JNK activation contributed significantly to induction of apoptosis. The size of the primary tumors growing orthotopically in SCID mice treated for 4 weeks with GEM and PB was significantly reduced (2.2–2.7 fold compared to GEM therapy alone. The Ki-67 (KNS62: p = 0.015; Ben: p = 0.093 and topoisomerase IIα (KNS62: p = 0.008; Ben: p = 0.064 proliferation indices were clearly reduced in tumors treated by combination

  3. Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface

    International Nuclear Information System (INIS)

    The pentapeptide repeat protein AlbG, provides self-resistance to the nonribosomally encoded hybrid polyketide-peptide termed albicidin. Analysis of the AlbG three-dimensional structure and the sequences of other pentapeptide repeat proteins that confer resistance to topiosomerase poisons suggests they have a similar dimer interface which may be critical to their interaction with topoisomerases. The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Å resolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral β-helix composed of approximately eight semi-regular coils. The regularity of the β-helix is blemished by a large loop/deviation in the β-helix between coils 4 and 5. The C-terminus of the β-helix is capped by a dimerization module, yielding a dimer with a 110 Å semi-collinear β-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric

  4. Identification, characteristic and phylogenetic analysis of type Ⅱ DNA topoisomerase gene in Giardia lamblia

    Institute of Scientific and Technical Information of China (English)

    De HE; Jian Fan WEN; Wan Qun CHEN; Si Qi LU; De Dong XIN

    2005-01-01

    The genes encoding type Ⅱ DNA topoisomerases were investigated in Giardia lamblia genome, and a type ⅡA gene,GlTop 2 was identified. It is a single copy gene with a 4476 bp long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a ~100 aa longer central domain; a ~200 aa shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate" G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type ⅡA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

  5. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    Science.gov (United States)

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1) at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1) occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1) at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1) in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1) nucleosomes. PMID:23516381

  6. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    Directory of Open Access Journals (Sweden)

    Ulrika Norman-Axelsson

    Full Text Available Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1 nucleosomes.

  7. Irreversible and reversible topoisomerase II DNA cleavage stimulated by clerocidin: sequence specificity and structural drug determinants.

    Science.gov (United States)

    Binaschi, M; Zagotto, G; Palumbo, M; Zunino, F; Farinosi, R; Capranico, G

    1997-05-01

    In contrast to other topoisomerase II poisons, the microbial terpenoid clerocidin was shown to stimulate irreversible topoisomerase II-mediated DNA cleavage. To establish the structural determinants for drug activity, in this study we have investigated intensity patterns and sequence specificity of clerocidin-stimulated DNA cleavage using 5'-end 32P-labeled DNA fragments. At a majority of the sites, clerocidin-stimulated cleavage did not revert upon NaCl addition; nevertheless, at some sites, cleavage completely reverted. Statistical analyses showed that drug-preferred bases were different in the two cases: guanine and cytosine were highly preferred at position -1 at irreversible and reversible sites, respectively. These results demonstrated that cleavage irreversibility was site selective and required a guanine at the 3' end of the cut. Further experiments revealed that some irreversible sites showed an abnormal electrophoretic mobility in sequencing gels with respect to cleaved bands generated by 4-(9-acridinylamino)methanesulfon-m-anisidide, suggesting a chemical alteration of the DNA strand. Interestingly, the ability to stimulate irreversible cleavage progressively decreased over time when clerocidin was stored in ethanol. Under these conditions, nuclear magnetic resonance measurements demonstrated that the drug underwent structural modifications that involved the C-12-C-15 side chain. Thus, the results indicate that a specific moiety of clerocidin may react with the DNA (guanine at -1) in the ternary complex, resulting in cleavage irreversibility and in altered DNA mobility in sequencing gels. PMID:9135013

  8. RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

    Science.gov (United States)

    Baranello, Laura; Wojtowicz, Damian; Cui, Kairong; Devaiah, Ballachanda N; Chung, Hye-Jung; Chan-Salis, Ka Yim; Guha, Rajarshi; Wilson, Kelli; Zhang, Xiaohu; Zhang, Hongliang; Piotrowski, Jason; Thomas, Craig J; Singer, Dinah S; Pugh, B Franklin; Pommier, Yves; Przytycka, Teresa M; Kouzine, Fedor; Lewis, Brian A; Zhao, Keji; Levens, David

    2016-04-01

    We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

  9. Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation.

    Science.gov (United States)

    Yang, Jenny; Annamalai, Thirunavukkarasu; Cheng, Bokun; Banda, Srikanth; Tyagi, Rakhi; Tse-Dinh, Yuk-Ching

    2015-10-01

    Topoisomerase functions are required in all organisms for many vital cellular processes, including transcription elongation. The C terminus domains (CTD) of Escherichia coli topoisomerase I interact directly with RNA polymerase to remove transcription-driven negative supercoiling behind the RNA polymerase complex. This interaction prevents inhibition of transcription elongation from hypernegative supercoiling and R-loop accumulation. The physiological function of bacterial topoisomerase I in transcription is especially important for a rapid network response to an antibiotic challenge. In this study, Escherichia coli with a topA66 single nucleotide deletion mutation, which results in a frameshift in the TopA CTD, was shown to exhibit increased sensitivity to trimethoprim and quinolone antimicrobials. The topoisomerase I-RNA polymerase interaction and the SOS response to the antimicrobial agents were found to be significantly reduced by this topA66 mutation. Consequently, the mutation frequency measured by rifampin selection following SOS induction was diminished in the topA66 mutant. The increased antibiotic sensitivity for the topA66 mutant can be reversed by the expression of recombinant E. coli topoisomerase I but not by the expression of recombinant Mycobacterium tuberculosis topoisomerase I that has a nonhomologous CTD even though the recombinant M. tuberculosis topoisomerase I can restore most of the plasmid DNA linking number deficiency caused by the topA66 mutation. Direct interactions of E. coli topoisomerase I as part of transcription complexes are likely to be required for the rapid network response to an antibiotic challenge. Inhibitors of bacterial topoisomerase I functions and interactions may sensitize pathogens to antibiotic treatment and limit the mutagenic response.

  10. c-erbB2 and topoisomerase IIα protein expression independently predict poor survival in primary human breast cancer: a retrospective study

    International Nuclear Information System (INIS)

    c-erbB2 (also known as HER-2/neu) and topoisomerase IIα are frequently overexpressed in breast cancer. The aim of the study was to analyze retrospectively whether the expression of c-erbB2 and topoisomerase IIα protein influences the long-term outcome of patients with primary breast cancer. In this study c-erbB2 and topoisomerase IIα protein were evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tissue from 225 samples of primary breast cancer, obtained between 1986 and 1998. The prognostic value of these markers was analyzed. Of 225 primary breast tumor samples, 78 (34.7%) showed overexpression of either c-erbB2 (9.8%) or topoisomerase IIα protein (24.9%), whereas in 21 tumors (9.3%) both proteins were found to be overexpressed. Patients lacking both c-erbB2 and topoisomerase IIα overexpression had the best long-term survival. Overexpression of either c-erbB2 or topoisomerase IIα was associated with shortened survival, whereas patients overexpressing both c-erbB2 and topoisomerase IIα showed the worst disease outcome (P < 0.0001). Treatment with anthracyclines was not capable of reversing the negative prognostic impact of topoisomerase IIα or c-erbB2 overexpression. The results of this exploratory study suggest that protein expression of c-erbB2 and topoisomerase IIα in primary breast cancer tissues are independent prognostic factors and are not exclusively predictive factors for anthracycline response in patients with primary breast cancer

  11. Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

    Science.gov (United States)

    Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

    2016-05-01

    A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

  12. Topoisomerase 1(TOP1) gene copy number in stage III colorectal cancer patients and its relation to prognosis

    DEFF Research Database (Denmark)

    Rømer, Maria Unni Koefoed; Nygård, Sune Boris; Christensen, Ib Jarle;

    2013-01-01

    A Topoisomerase 1 (Top1) poison is frequently included in the treatment regimens for metastatic colorectal cancer (mCRC). However, no predictive biomarkers for Top1 poisons are available. We here report a study on the TOP1 gene copy number in CRC patients and its association with patient prognosis...

  13. Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport.

    Science.gov (United States)

    Laponogov, Ivan; Veselkov, Dennis A; Crevel, Isabelle M-T; Pan, Xiao-Su; Fisher, L Mark; Sanderson, Mark R

    2013-11-01

    Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first 'open clamp' structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important β-pinwheel DNA-binding domain. The protein N gate is present in an 'arms-wide-open' state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

  14. Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors.

    Science.gov (United States)

    Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M Thirumala; Mazumdar, H K; Munshi, Parthapratim; Sen, Subhabrata

    2016-01-01

    A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency. PMID:27221589

  15. In vitro study of immunosuppressive effect of apoptotic cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wen-jin; ZHENG Shu-sen

    2005-01-01

    Recent studies revealed that apoptotic cells are actively involved in immunosuppression and anti-inflammation. After being phagocytosed by macrophages, apoptotic cells can actively regulate cytokines secretion from lipopolysaccharide (LPS)-stimulated macrophages, in which the secretion of immunosuppressive cytokines such as interleukin-10 (IL-10) is increased while the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα), interleukin-1beta (IL-1β) and leukin-8 (IL-8) are suppressed. In this paper, we first present evidence that phagocytosed apoptotic cells regulate cytokine secretion of LPS-stimulated macrophages, but also inhibit the activation of T lymphocytes stimulated by ConA. These data suggest that apoptotic cells can alter the biological behavior of macrophages which gain immunosuppressive property.

  16. Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents.

    Science.gov (United States)

    Walker, John; Saravia, Nancy G

    2004-10-01

    We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives. PMID:15562618

  17. Apoptotic Genes are Differentially Expressed in Aged Gingival Tissue

    OpenAIRE

    González, O. A.; Stromberg, A.J.; Huggins, P. M.; Gonzalez-Martinez, J.; Novak, M.J.; Ebersole, J. L.

    2011-01-01

    Cellular and molecular changes of the periodontium associated with a higher prevalence of oral diseases (e.g., chronic periodontitis) in aged populations have received little attention. Since impaired apoptosis during aging appears to be related to chronic inflammatory disorders, we hypothesized that the expression of genes associated with apoptotic processes are altered in aged healthy and periodontitis-affected gingival tissue. Ontology analysis of 88 genes related to apoptotic pathways was...

  18. Biological evaluation and molecular modelling study of thiosemicarbazide derivatives as bacterial type IIA topoisomerases inhibitors.

    Science.gov (United States)

    Paneth, Agata; Stączek, Paweł; Plech, Tomasz; Strzelczyk, Aleksandra; Dzitko, Katarzyna; Wujec, Monika; Kuśmierz, Edyta; Kosikowska, Urszula; Grzegorczyk, Agnieszka; Paneth, Piotr

    2016-01-01

    In the present article, we describe the inhibitory potency of nine thiosemicarbazide derivatives against bacterial type IIA topoisomerases, their antibacterial profile and molecular modelling evaluation. We found that one of the tested compounds, compound 7, significantly inhibits activity of Staphylococcus aureus DNA gyrase with an IC(50) below 15 μM. Besides, this compound displays antibacterial activity on reference Staphylococuss spp. and Enterococcus faecalis strains as well as clinical S. aureus isolates at non-cytotoxic concentrations in mammalian cells with MIC values ranging from 16 to 32 μg/mL thereby indicating, in some cases, equipotent or even more effective action than standard drugs such as vancomycin, ampicillin and nitrofurantoin. The computational studies showed that both molecular geometry and the electron density distribution have a great impact on antibacterial activity of thiosemicarbazide derivatives. PMID:25792505

  19. PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

    Science.gov (United States)

    Nielsen, Christian F; Huttner, Diana; Bizard, Anna H; Hirano, Seiki; Li, Tian-Neng; Palmai-Pallag, Timea; Bjerregaard, Victoria A; Liu, Ying; Nigg, Erich A; Wang, Lily Hui-Ching; Hickson, Ian D

    2015-01-01

    PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

  20. Topoisomerase-1 gene copy aberrations are frequent in patients with breast cancer

    DEFF Research Database (Denmark)

    Kümler, Iben; Balslev, Eva; Poulsen, Tim S.;

    2015-01-01

    Topoisomerase-1 (Top1) targeting drugs have shown promising efficacy in patients with metastatic breast cancer (BC). However, these drugs are rather toxic calling for development and validation of predictive biomarkers to increase the therapeutic index. As these drugs are targeting the Top1 protein......, and since no validated anti-Top1 antibodies for immunohistochemistry have been reported, we raised the hypothesis that TOP1 gene amplifications may serve as a proxy for the Top1 protein and thereby a biomarker of response to treatment with Top1 inhibitors in BC. The aim was to determine the prevalence...... of TOP1 gene copy gain in BC. The prevalence of TOP1 gene copy gain was investigated by fluorescence in situ hybridization with a TOP1/CEN-20 probemix in normal breast tissue (N=100) and in tissue from patients with metastatic BC in a discovery (N=100) and a validation cohort (N=205). As amplification...

  1. Salvicine, a novel topoisomerase II inhibitor, exerts its potent anticancer activity by ROS generation.

    Science.gov (United States)

    Meng, Ling-hua; Ding, Jian

    2007-09-01

    Salvicine is a novel diterpenoid quinone compound obtained by structural modification of a natural product lead isolated from a Chinese herb with potent growth inhibitory activity against a wide spectrum of human tumor cells in vitro and in mice bearing human tumor xenografts. Salvicine has also been found to have a profound cytotoxic effect on multidrug-resisitant (MDR) cells. Moreover, Salvicine significantly reduced the lung metastatic foci of MDA-MB-435 orthotopic xenograft. Recent studies demonstrated that salvicine is a novel non-intercalative topoisomerase II (Topo II) poison by binding to the ATPase domain, promoting DNA-Topo II binding and inhibiting Topo II-mediated DNA relegation and ATP hydrolysis. Further studies have indicated that salcivine-elicited ROS plays a central role in salvicine-induced cellular response including Topo II inhibition, DNA damage, circumventing MDR and tumor cell adhesion inhibition. PMID:17723179

  2. DNA Topoisomerase-I Inhibition due to Exposure to X-Rays

    International Nuclear Information System (INIS)

    In events such as radiological terrorism, accidents involving radioactive materials and occupational exposures, there is a great need to identify exposures to relatively low radiation levels. In many situations, the evaluation of radiation doses is not possible using physical dosimeters as they are not worn, and it is desirable to achieve this based on sensitive biomarkers (1, 2, 3). DNA Topoisomerase-I (Topo-I) is an essential nuclear enzyme that is responsible for the topological state of the DNA. The enzyme is involved in a variety of DNA transactions, including replication, transcription, recombination and DNA repair (4,5). The aim of the present work was to investigate the influence of X-ray radiation on the catalytic activity of this enzyme, and to evaluate its applicability as a biological dosimeter

  3. Phaeophytins from Thyrsacanthus ramosissimus Moric. with inhibitory activity on human DNA topoisomerase II-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Cabral, Analucia Guedes Silveira; Tenorio-Souza, Fabio Henrique; Moura, Marcelo Dantas; Mota, Sabrina Gondim Ribeiro; Silva Lins, Antonio Claudio da; Dias, Celidarque da Silva; Barbosa-Filho, Jose Maria [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Ciencias Frmaceuticas; Giulietti, Ana Maria [Universidade Estadual de Feira de Santana, Feira de Santana, BA (Brazil). Dept. de Ciencias Biologicas; Silva, Tania Maria Sarmento da [Universidade Federal Rural de Pernambuco, Recife, PE (Brazil). Dept. de Ciencias Moleculares; Santos, Creusioni Figueredo dos, E-mail: jbarbosa@ltf.ufpb.br [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil). Dept. de Biologia Molecular

    2012-07-01

    Our study reports the extraction and isolation of a new phaeophytin derivative 15{sup 1}-hydroxy-(15{sup 1}-S)-porphyrinolactone, designated anamariaine (1) herein, isolated from the chloroform fraction of aerial parts of Thyrsacanthus ramosissimus Moric. along with the known 15{sup 1}-ethoxy-(15{sup 1}-S)-porphyrinolactone (2). These compounds were identified by usual spectroscopic methods. Both compounds were subjected to in vitro (inhibitory activity) tests by means of supercoiled DNA relaxation techniques and were shown to display inhibitory activity against human DNA topoisomerase II-{alpha} at 50 {mu}M. Interconversion of these two pigments under the mild conditions of the isolation techniques should be highly unlikely but cannot be entirely ruled out. (author)

  4. Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation.

    Directory of Open Access Journals (Sweden)

    Katherine L Furniss

    Full Text Available By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.

  5. Phylogenetic positions of several amitochondriate protozoa-Evidence from phylogenetic analysis of DNA topoisomerase II

    Institute of Scientific and Technical Information of China (English)

    HE; De; DONG; Jiuhong; WEN; Jianfan; XIN; Dedong; LU; Siqi

    2005-01-01

    Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented

  6. QSAR and docking studies on xanthone derivatives for anticancer activity targeting DNA topoisomerase IIα

    Directory of Open Access Journals (Sweden)

    Alam S

    2014-01-01

    Full Text Available Sarfaraz Alam, Feroz KhanMetabolic and Structural Biology Department, Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research, Lucknow, Uttar Pradesh, IndiaAbstract: Due to the high mortality rate in India, the identification of novel molecules is important in the development of novel and potent anticancer drugs. Xanthones are natural constituents of plants in the families Bonnetiaceae and Clusiaceae, and comprise oxygenated heterocycles with a variety of biological activities along with an anticancer effect. To explore the anticancer compounds from xanthone derivatives, a quantitative structure activity relationship (QSAR model was developed by the multiple linear regression method. The structure–activity relationship represented by the QSAR model yielded a high activity–descriptors relationship accuracy (84% referred by regression coefficient (r2=0.84 and a high activity prediction accuracy (82%. Five molecular descriptors – dielectric energy, group count (hydroxyl, LogP (the logarithm of the partition coefficient between n-octanol and water, shape index basic (order 3, and the solvent-accessible surface area – were significantly correlated with anticancer activity. Using this QSAR model, a set of virtually designed xanthone derivatives was screened out. A molecular docking study was also carried out to predict the molecular interaction between proposed compounds and deoxyribonucleic acid (DNA topoisomerase IIα. The pharmacokinetics parameters, such as absorption, distribution, metabolism, excretion, and toxicity, were also calculated, and later an appraisal of synthetic accessibility of organic compounds was carried out. The strategy used in this study may provide understanding in designing novel DNA topoisomerase IIα inhibitors, as well as for other cancer targets.Keywords: drug likeness, ADMET, regression model, HeLa cell line

  7. cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

    International Nuclear Information System (INIS)

    cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

  8. Surface code—biophysical signals for apoptotic cell clearance

    Science.gov (United States)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, Luis E.

    2013-12-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

  9. Surface code—biophysical signals for apoptotic cell clearance

    International Nuclear Information System (INIS)

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes. (paper)

  10. Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    William L Riles; Jason Erickson; Sanjay Nayyar; Mary Jo Atten; Bashar M Attar; Oksana Holian

    2006-01-01

    AIM: To investigate the intracellular apoptotic signals engaged by resveratrol in three gastric adenocarcinoma cancer cell lines, two of which (AGS and SNU-1) express p53 and one (KATO-Ⅲ) with deleted p53.METHODS: Nuclear fragmentation was used to quantitate apoptotic cells; caspase activity was determined by photometric detection of cleaved substrates; formation of oxidized cytochrome C was used to measure cytochrome C activity, and Western blot analysis was used to determine protein expression.RESULTS: Gastric cancer cells, irrespective of their p53 status, responded to resveratrol with fragmentation of DNA and cleavage of nuclear lamins A and B and PARP, Resveratrol, however, has no effect on mitochondria-associated apoptotic proteins Bcl-2, Bclxl, Bax, Bid or Smac/Diablo, and did not promote subcellular redistribution of cytochrome C, indicating that resveratrol-induced apoptosis of gastric carcinoma cells does not require breakdown of mitochondrial membrane integrity. Resveratrol up-regulated p53 protein in SNU-1 and AGS cells but there was a difference in response of intracellular apoptotic signals between these cell lines.SNU-1 cells responded to resveratrol treatment with down-regulation of survivin, whereas in AGS and KATO-Ⅲ cells resveratrol stimulated caspase 3 and cytochrome C oxidase activities.CONCLUSION: These findings indicate that even within a specific cancer the intracellular apoptotic signals engaged by resveratrol are cell type dependent and suggest that such differences may be related to differentiation or lack of differentiation of these cells.

  11. Discovery of 7-Methyl-10-Hydroxyhomocamptothecins with 1,2,3-Triazole Moiety as Potent Topoisomerase I Inhibitors.

    Science.gov (United States)

    Xu, Xiguo; Wu, Yuelin; Liu, Wenfeng; Sheng, Chuanquan; Yao, Jianzhong; Dong, Guoqiang; Fang, Kun; Li, Jin; Yu, Zhiliang; Min, Xiao; Zhang, Huojun; Miao, Zhenyuan; Zhang, Wannian

    2016-09-01

    Homocamptothecin is emerging as an important topoisomerase I inhibitor originating in natural product camptothecin. We report the modifications and SAR of homocamptothecin on position C10 to develop potent topoisomerase I inhibitors for anticancer drug discovery. Based on click chemistry, twenty-one 1,2,3-triazole-substituted homocamptothecin derivatives were readily synthesized in two steps. For A549, cycloalkyl- and alkyl-substituted compounds 6j, 6l, and 6o revealed highly antiproliferative inhibitory activities with IC50 value of 30, 30, and 50 nm, respectively. In addition, cyclopropyl 6j exhibited greater Topo I inhibitory activity than 20(S)-Camptothecin, which indicated suitability for further drug development. PMID:27062430

  12. ERK1/2 Signaling Plays an Important Role in Topoisomerase II Poison-Induced G2/M Checkpoint Activation

    OpenAIRE

    Kolb, Ryan H.; Greer, Patrick M.; Cao, Phu T.; Cowan, Kenneth H.; Ying Yan

    2012-01-01

    Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-...

  13. Depletion of RNase HI activity in Escherichia coli lacking DNA topoisomerase I leads to defects in DNA supercoiling and segregation.

    Science.gov (United States)

    Usongo, Valentine; Nolent, Flora; Sanscartier, Patrick; Tanguay, Cynthia; Broccoli, Sonia; Baaklini, Imad; Drlica, Karl; Drolet, Marc

    2008-08-01

    Gyrase-mediated hypernegative supercoiling is one manifestation of R-loop formation, a phenomenon that is normally suppressed by topoisomerase I (topA) in Escherichia coli. Overproduction of RNase HI (rnhA), an enzyme that removes the RNA moiety of R-loops, prevents hypernegative supercoiling and allows growth of topA null mutants. We previously showed that topA and rnhA null mutations are incompatible. We now report that such mutants were viable when RNase HI or topoisomerase III was expressed from a plasmid-borne gene. Surprisingly, DNA of topA null mutants became relaxed rather than hypernegatively supercoiled following depletion of RNase HI activity. This result failed to correlate with the cellular concentration of gyrase or topoisomerase IV (the other relaxing enzyme in the cell) or with transcription-induced supercoiling. Rather, intracellular DNA relaxation in the absence of RNase HI was related to inhibition of gyrase activity both in vivo and in extracts. Cells lacking topA and rnhA also exhibited properties consistent with segregation defects. Overproduction of topoisomerase III, an enzyme that can carry out DNA decatenation, corrected the segregation defects without restoring supercoiling activity. Collectively these data reveal (i) the existence of a cellular response to loss of RNase HI that counters the supercoiling activity of gyrase, and (ii) supercoiling-independent segregation defects due to loss of RNase HI from topA null mutants. Thus RNase HI plays a more central role in DNA topology than previously thought. PMID:18554330

  14. DNA TOPOISOMERASE I INHIBITORS AMELIORATE SEIZURE-LIKE BEHAVIORS AND PARALYSIS IN A DROSOPHILA MODEL OF EPILEPSY

    OpenAIRE

    Song, Juan; Parker, Louise; Hormozi, Linda; Tanouye, Mark A

    2008-01-01

    The Drosophila DNA topoisomerase type I mutant allele, top1JS is an effective general seizure-suppressor mutation, reverting seizure-sensitive phenotypes of several mutant strains in a genetic model of epilepsy. Seizure-suppression is caused by reduced transcription of the top1 gene (Song et al., 2007). Here, we examine the possibility that pharmaceutical inhibition of Top1 enzymatic activity may also be effective at reducing seizure phenotypes. We investigate the effect of vertebrate Top1 in...

  15. Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

    Directory of Open Access Journals (Sweden)

    Yu-Shih Lin

    Full Text Available Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α. The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

  16. QSAR Modeling on Benzo[c]phenanthridine Analogues as Topoisomerase I Inhibitors and Anti-cancer Agents

    Directory of Open Access Journals (Sweden)

    Thi-Ngoc-Phuong Huynh

    2012-05-01

    Full Text Available Benzo[c]phenanthridine (BCP derivatives were identified as topoisomerase I (TOP-I targeting agents with pronounced antitumor activity. In this study, hologram-QSAR, 2D-QSAR and 3D-QSAR models were developed for BCPs on topoisomerase I inbibitory activity and cytotoxicity against seven tumor cell lines including RPMI8402, CPT-K5, P388, CPT45, KB3-1, KBV-1and KBH5.0. The hologram, 2D, and 3D-QSAR models were obtained with the square of correlation coefficient R2 = 0.58 − 0.77, the square of the crossvalidation coefficient q2 = 0.41 − 0.60 as well as the external set’s square of predictive correlation coefficient r2 = 0.51 − 0.80. Moreover, the assessment method based on reliability test with confidence level of 95% was used to validate the predictive power of QSAR models and to prevent over-fitting phenomenon of classical QSAR models. Our QSAR model could be applied to design new analogues of BCPs with higher antitumor and topoisomerase I inhibitory activity.

  17. Topoisomerase II alpha and p27; alternative markers to decide on the proliferation capacity of astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Evrim ÖZTÜRK

    2008-05-01

    Full Text Available Proliferation capacity is an important parameter which enables us to predict the prognosis of tumours. Many immunohistochemical studies were conducted to search the relation of proliferative capacity with different clinical and histological parameters. Ki67 is a well known immunohistochemical marker of proliferation and some standard values have been established for Ki67 indexes of astrocytic tumours. For this purpose, considering the roles of proteins in cell cycle, some immunohistochemical markers other than Ki67 can be suggested. In this study, expressions of topoisomerase II alpha, a nuclear protein in mitotically active cells and p27, a cylin-dependent kinase inhibitor, were correlated with the grade and Ki67 indexes of 67 astrocytomas. Topoisomerase expressions demonstrated an increase with increasing grade. It also followed a parallel curve with Ki67. On the other hand, p27 had an inverse correlation with the tumor grade. The cut-off value for topoisomerase was calculated to vary 3.5% between low and high grade tumours. No cut-off value could be obtained for p27.

  18. Upregulation of Phagocytic Clearance of Apoptotic Cells by Autoimmune Regulator

    Institute of Scientific and Technical Information of China (English)

    石亮; 胡丽华; 李一荣

    2010-01-01

    To investigate the effect of autoimmune regulator(AIRE) on phagocytic clearance of apoptotic cells,a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells.After incubation with transfected 16HBE cells,engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase(MPO) staining.The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting.The results showed that the phagocytosis perce...

  19. Crystallization and X-ray diffraction studies of DNA-free and DNA-bound forms of EcoO109I DNA methyltransferase

    International Nuclear Information System (INIS)

    The crystallization and diffraction studies of EcoO109I DNA methyltransferase are described. EcoO109I DNA methyltransferase (M.EcoO109I) is a type II modification enzyme from the EcoO109I restriction-modification system identified in Escherichia coli strain H709c. M.EcoO109I recognizes double-stranded RGGNCCY (where R = A or G, Y = T or C and N is any base) and transfers a methyl group to the C5 of the inner cytosines from S-adenosylmethionine. To reveal the mechanism of substrate recognition by M.EcoO109I, DNA-free and DNA-bound forms of M.EcoO109I were successfully crystallized. Crystals of the DNA-free and DNA-bound forms belonged to space groups P42212, with unit-cell parameters a = b = 120.5, c = 79.8 Å, and P21, with unit-cell parameters a = 55.8, b = 77.4, c = 117.4 Å, β = 93.5°, respectively

  20. Irradiation with ultraviolet light and gamma-rays increases the level of DNA topoisomerase II in nuclei of normal and xeroderma pigmentosum fibroblasts.

    Science.gov (United States)

    Thielmann, H W; Popanda, O

    1998-02-01

    DNA topoisomerase II was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with 254-nm UV light and -rays from a 137Cs source. DNA topoisomerase II was localized immunohistochemically as bright fluorescent dots in the karyoplasm. Investigated fibroblasts originated from normal human donors and a xeroderma pigmentosum patient (XP12BE). All cell lines showed a time and dose-dependent increase in DNA topoisomerase II abundance after irradiation. The increase may reflect enhanced accessibility of the enzyme, enhanced gene expression or enhanced stabilization of mRNA or protein molecules. The effect was detectable as early as 1 h after irradiation at doses 3 J/m2 or 3 Gy. It passed through a maximum and decreased within 18 h (UV light) or 6 h ( -rays). Except for the duration of the response, no principal differences were seen between the effects caused by UV light and those elicited by -rays. The increase in enzyme levels might be part of the well-known DNA damage responses which operate in cell-protective or DNA-reparative pathways or may reflect initiation of apoptosis. DNA topoisomerase I was detected with a commercially available polyclonal antibody raised against human DNA topoisomerase I. In unirradiated cells, DNA topoisomerase I was found to be mainly concentrated in nucleoli. Irradiation with -rays changed the staining pattern in that it caused a multitude of DNA topoisomerase I-rich centers to occur which may reflect sites of transcription of radiation-inducible genes.

  1. Identification of pathogenesis pathway in basal-like breast cancer based on mutant p53 protein and topoisomerase-IIα expression

    Directory of Open Access Journals (Sweden)

    Yayi Dwina

    2015-01-01

    Full Text Available Background: Basal-like breast cancer is difficult to treat with standard regimen therapy, because it doesn’t express hormone receptors or epidermal growth factor receptors. Identification of oncogenesis pathway is expected to find molecules which can be used as target for therapy. One candidate molecule is topoisomerase-IIα whose expression is regulated by p53. This study aimed to compare the expression of mutant p53 proteins and topoisomerase IIα in basal-like and non basal-like breast cancer, and to determine the association between mutant p53 proteins and topoisomerase IIα in basal-like group.Methods: The samples were 40 formalin fixed paraffin embedded tissues from verified triple negative breast cancer tissue. The samples were divided into 2 groups, basal-like and non basal-like breast cancer, based on cytokeratin - 5 (CK-5 expression. Mutant p53 proteins and topoisomerase IIα were stained using immunohistochemistry method, scored and compared. Statistical test used SPSS software version 16 for descriptive statistics, kappa test, normality test, comparison of two mean, and categorical comparison.Results: Median (min-max of mutant p53 protein expression in basal-like group was 21 (0-100, the non basal-like group was 2 (0-80, p = 0.061. Min-max of topoisomerase IIα in basal-like group was 263 (15-492, non basal-like group was 262 (0-481, p = 0.409. There was an association between mutant p53 positivity with breast cancer subtype (p = 0.027 and between mutant p53-topoisomerase IIα coexpression with breast cancer subtype (p = 0.018.Conclusion: Co-expression of mutant p53 with topoisomerase IIα has the role in one of the pathway of basal-like breast cancer pathogenesis.

  2. Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

    Directory of Open Access Journals (Sweden)

    Maria Cristina P. Araújo

    1998-09-01

    Full Text Available Combined radiation and antineoplastic drug treatment have important applications in cancer therapy. In the present work, an evaluation was made of two known topoisomerase II inhibitors, doxorubicin (DXR and mitoxantrone (MXN, with g-radiation. The effects of DXR or MXN on g-radiation-induced chromosome aberrations in Chinese hamster ovary (CHO cells were analyzed. Two concentrations of each drug, 0.5 and 1.0 µg/ml DXR, and 0.02 and 0.04 µg/ml MXN, were applied in combination with two doses of g-radiation (20 and 40 cGy. A significant potentiating effect on chromosomal aberrations was observed in CHO cells exposed to 1.0 µg/ml DXR plus 40 cGy. In the other tests, the combination of g-radiation with DXR or MXN gave approximately additive effects. Reduced mitotic indices reflected higher toxicity of the drugs when combined with radiation.A associação de radiação ionizante com drogas antineoplásicas tem importante aplicação na terapia do câncer. No presente trabalho, foram avaliados os efeitos de dois inibidores de topoisomerase II, doxorubicina (DXR e mitoxantrona (MXN, sobre as aberrações cromossômicas induzidas pelas radiações-g em células do ovário de hamster chinês (CHO. Foram usadas as concentrações 0,5 e 1,0 mg/ml de DXR e 0,02 e 0,04 mg/ml de MXN, combinadas com duas doses de radiações gama (20 e 40 cGy. Um significativo efeito potenciador das aberrações cromossômicas foi observado em células CHO tratadas com 1,0 mg/ml de DXR e expostas a 40 cGy de radiação. Nos outros testes, a combinação da radiação-g com a DXR ou MXN apresentou um efeito próximo ao aditivo. A redução dos índices mitóticos refletiu a alta citotoxicidade das drogas quando combinadas às radiações-g.

  3. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  4. The apoptotic thanatotranscriptome associated with the liver of cadavers.

    Science.gov (United States)

    Javan, Gulnaz T; Can, Ismail; Finley, Sheree J; Soni, Shivani

    2015-12-01

    Gene expression investigations are well-established components of ante mortem studies with broad applications ranging from elucidating basic mechanisms responsible for normal physiological processes to discovering therapeutic targets in pathophysiological conditions. However, gene expression studies and their application in the medico-legal field are still in their infancy. Therefore, the present study focuses on RNA using PCR array in the analysis of gene expression associated with tissues taken from actual criminal cases. RNA was extracted from the liver tissues of bodies with PMIs between 6 and 48 h. The results demonstrated that mRNA was stable up to 48 h postmortem. Further, as cell death is an indispensable and necessary part of the biological life cycle, apoptotic gene expression profiles were investigated. The gene expression related to the programmed cell death found in body tissues after death is defined as the apoptotic thanatotranscriptome (thanatos-, Greek for death). On comparison of control and decaying tissues, the results show that with time, pro-apoptotic genes such as caspases are up-regulated and the expression of genes responsible for anti-apoptosis such as BCL2 and BAG3 were down-regulated. Thus, this current work gives a unique perspective of the apoptotic thanatotranscriptome that is affected after death. Up to the present time, gene expression in bodies from criminal cases has not been reported in literature using PCR array techniques. Thus, this thanatotranscriptome study provides insight into postmortem gene activity with potential applications in medico-legal investigations. PMID:26318598

  5. Genotoxic profile of inhibitors of topoisomerases I (camptothecin) and II (etoposide) in a mitotic recombination and sex-chromosome loss somatic eye assay of Drosophila melanogaster.

    Science.gov (United States)

    Sortibrán, América Nitxin Castañeda; Téllez, María Guadalupe Ordaz; Rodríguez-Arnaiz, Rosario

    2006-04-30

    Genotoxic carcinogens which interact with DNA may produce double-strand breaks as normal intermediates of homologous mitotic recombination, and may give rise to structural chromosome aberrations and inter-chromosomal deletion-recombination. The genotoxic profile of two inhibitors of DNA topoisomerases were evaluated using an in vivo somatic w/w+ eye assay of Drosophila melanogaster for the detection of loss of heterozygosity (LOH) by homologous mitotic recombination, intra-chromosomal recombination and structural chromosomal aberrations. We studied camptothecin (CPT) as a topoisomerase-I-interactive agent and etoposide (ETOP) as a topoisomerase II inhibitor. These drugs act by stabilizing a ternary complex consisting of topoisomerases covalently linked to DNA at single-strand or at double-strand breaks, thereby preventing the relegation step of the breakage/rejoining reaction mediated by the enzyme. The genotoxic profiles were determined from the appearance of eye tissue in adult flies, in which LOH and expression of the reporter gene white produced light clones. The results demonstrated that both compounds were significantly genotoxic, with CPT being more effective than ETOP. Inter-chromosomal mitotic recombination was the major mechanism responsible for the induction of light spots by both compounds in XX females. Loss of the ring X chromosome (rX), was significantly enhanced by CPT, and this topoisomerase blocker also produced intra-chromosomal recombination (XY males). PMID:16529987

  6. Mechanism of action of ferrocene derivatives on the catalytic activity of topoisomerase IIalpha and beta--distinct mode of action of two derivatives.

    Science.gov (United States)

    Sai Krishna, A D; Panda, Gayatri; Kondapi, Anand K

    2005-06-15

    Topoisomerase II is found to be present in two isoforms alpha and beta, and both the isoforms are regulated in cancerous tissue. Development of isoform-specific topoisomerase II poisons has been of great interest for cancer-specific drug targeting. In the present investigation using quantitative structure-activity analysis of ferrocene derivatives, we show that two derivatives of ferrocene, azalactone ferrocene and thiomorpholide amido methyl ferrocene, can preferentially inhibit topoisomerase IIbeta activity. Thiomorpholide amido methyl ferrocene shows higher inhibition of catalytic activity (IC(50) = 50 microM) against topoisomerase IIbeta compared to azalactone ferrocene (IC(50) = 100 microM). The analysis of protein DNA intermediates formed in the presence of these two compounds suggests that azalactone ferrocene readily induces formation of cleavable complex in a dose-dependent manner, in comparison with thiomorpholide amido methyl ferrocene. Both the compounds show significant inhibition of DNA-dependent ATPase activity of enzyme. These results suggest that azalactone ferrocene inhibits DNA passage activity of enzyme leading to the formation of cleavable complex, while thiomorpholide amido methyl ferrocene competes with ATP binding resulting in the inhibition of catalytic activity of enzyme. In summary, thiomorpholide amido methyl ferrocene and azalactone ferrocene show distinctly different mechanisms in inhibition of catalytic activity of topoisomerase IIbeta. PMID:15907782

  7. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    Science.gov (United States)

    El Sayyed, Hafez; Le Chat, Ludovic; Lebailly, Elise; Vickridge, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco; Espéli, Olivier

    2016-05-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  8. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    Science.gov (United States)

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-09-30

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

  9. Resveratrol Modulates the Topoisomerase Inhibitory Potential of Doxorubicin in Human Colon Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Anika Schroeter

    2014-12-01

    Full Text Available Resveratrol (RSV is currently being widely discussed as potentially useful for anticancer therapy in combination with classical chemotherapeutics, e.g., the topoisomerase II (TOP II poison doxorubicin (DOX. However, there is still a lack of knowledge of possible interference at the target enzyme, especially since RSV itself has recently been described to act as a TOP poison. We therefore sought to address the question whether RSV affects DOX-induced genotoxic and cytotoxic effects with special emphasis on TOP II in HT-29 colon carcinoma cells. RSV was found to counteract DOX-induced formation of DNA-TOP-intermediates at ≥100 µM for TOP IIα and at 250 µM for TOP IIβ. As a consequence, RSV modulated the DNA-strand breaking potential of DOX by mediating protective effects with an apparent maximum at 100 µM. At higher concentration ranges (≥200 µM RSV diminished the intracellular concentrations of DOX. Nevertheless, the presence of RSV slightly enhanced the cytotoxic effects of DOX after 1.5 h and 24 h of incubation. Taken together, at least in cell culture RSV was found to affect the TOP-poisoning potential of DOX and to modulate its cytotoxic effectiveness. Thus, further studies are needed to clarify the impact of RSV on the therapeutic effectiveness of DOX under in vivo conditions.

  10. Charcterization of Type Ⅱ Topoisomerase Gene Mutations in Clinical Isolates of Mycoplasma Hominis Resistant to Fluoroquinolones

    Institute of Scientific and Technical Information of China (English)

    吴移谋; 张文波; 姚艳冰

    2002-01-01

    Objective: To analyze type Ⅱ topoisomerase genes inclinical isolates of fluoroquinolone-resistant Mycoplasmahominis. Methods: Eight isolates of M.hominis cross resistant to 6fluoroquinolones were selected from 103 clinical strains ofM.hominis using a broth microdilution method. Type IItopoisomerase genes were amplified by PCR and directlysequenced. Nucleotide sequences were compared to sequencesfrom a susceptible strain (M.hominis PG2I). Results: MICs of resistant Mh isolates were 4- to 512-fold higher than MICs from the susceptible reference strain.Sequence comparison revealed a C to T change at 113nt ingyrA QRDR led to the substitution of Ser83 by Leucine and noamino acid change in gyrB. A change of G to T at 134nt inparC QRDR led to the substitution of Ser80 by Isoleucine anda G to A change at 70nt in parE QRDR led to the substitutionof Aspartic acid by Asparagine. Conclusion: These results suggest that a C to T change atll3nt in gyrA, a G to T change at 134nt in parC and a G to Achange at 70nt in patrE are associated with fluoroquinoloneresistance of M.hominis.

  11. Bioassay-Guided Isolation of Two Flavonoids from Derris scandens with Topoisomerase II Poison Activity.

    Science.gov (United States)

    Sangmalee, Suphattra; Laorpaksa, Areerat; Sritularak, Boonchoo; Sukrong, Suchada

    2016-01-01

    Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4'-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment. PMID:26754253

  12. Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer

    Science.gov (United States)

    Tsai, Hsiang-Ping; An, Herng-Wei; Lee, Chi-Ming; Wu, Jen-Chine; Chen, Chien-Shu; Huang, Shih-Hao; Hwang, Jaulang; Cheng, Kur-Ta; Leiw, Phui-Ly; Chen, Chi-Long; Lin, Chun-Mao

    2015-01-01

    DNA topoisomerase I (TOP1) levels of several human neoplasms are higher than those of normal tissues. TOP1 inhibitors are widely used in treating conventional therapy-resistant ovarian cancers. However, patients may develop resistance to TOP1 inhibitors, hampering chemotherapy success. In this study, we examined the mechanisms associated with the development of camptothecin (CPT) resistance in ovarian cancers and identified evodiamine (EVO), a natural product with TOP1 inhibiting activity that overcomes the resistance. The correlations among TOP1 levels, cancer staging, and overall survival (OS) were analyzed. The effect of EVO on CPT-resistant ovarian cancer was evaluated in vitro and in vivo. TOP1 was associated with poor prognosis in ovarian cancers (p = 0.024). EVO induced apoptosis that was detected using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The tumor size decreased significantly in the EVO treatment group compared with the control group (p < 0.01) in a xenograft mouse model. Effects of drugs targeting TOP1 for prognosis and therapy in CPT-resistant ovarian cancer are anticipated. EVO with TOP1 can be developed as an antiproliferative agent for overcoming CPT resistance in ovarian cancers. PMID:26207989

  13. Targeting of Topoisomerase I for Prognoses and Therapeutics of Camptothecin-Resistant Ovarian Cancer.

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Lee

    Full Text Available DNA topoisomerase I (TOP1 levels of several human neoplasms are higher than those of normal tissues. TOP1 inhibitors are widely used in treating conventional therapy-resistant ovarian cancers. However, patients may develop resistance to TOP1 inhibitors, hampering chemotherapy success. In this study, we examined the mechanisms associated with the development of camptothecin (CPT resistance in ovarian cancers and identified evodiamine (EVO, a natural product with TOP1 inhibiting activity that overcomes the resistance. The correlations among TOP1 levels, cancer staging, and overall survival (OS were analyzed. The effect of EVO on CPT-resistant ovarian cancer was evaluated in vitro and in vivo. TOP1 was associated with poor prognosis in ovarian cancers (p = 0.024. EVO induced apoptosis that was detected using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay. The tumor size decreased significantly in the EVO treatment group compared with the control group (p < 0.01 in a xenograft mouse model. Effects of drugs targeting TOP1 for prognosis and therapy in CPT-resistant ovarian cancer are anticipated. EVO with TOP1 can be developed as an antiproliferative agent for overcoming CPT resistance in ovarian cancers.

  14. Topoisomerase-Based Preparation and AFM Imaging of Multi-Interlocked Circular DNA.

    Science.gov (United States)

    Li, Tevin; Zhang, Hao; Hu, Lianzhe; Shao, Fangwei

    2016-03-16

    Multi-interlocked circular DNA structures have been in high demand for fabricating complicated functional DNA architectures and nanodevices such as molecular switches, shuttles, and motors. Even though various innovative methods have been developed in the past, creation of multi-interlocked circular DNA structures with defined numbers of DNA molecules and linking patterns is still a challenging task nowadays. Here, we propose a top-down decatenation of kinetoplast DNA as a new approach for creating multi-interlocked circular DNA structures. Through optimizing the amount and reaction time of topoisomerase II, we synthesized completely mutually interlocked tricircular, tetra-circular, and oligo-circular DNA structures, which have not yet been acquirable through any other existing synthetic means. The catenation structures of multiple circular DNA were further verified through atomic force microscopic analysis of the backbone overlapping patterns and the circumference. It accordingly is our expectation that the top-down enzymatic approaches could offer a highly interlocked network with defined numbers of circular DNA with simple protocols, and could consequently be beneficial to the design and fabrication of sophisticated functional molecules and nanodevices in the areas of supramolecular chemistry, DNA nanotechnology, and material science. PMID:26745453

  15. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms

    Science.gov (United States)

    Mabb, Angela M.; Simon, Jeremy M.; King, Ian F.; Lee, Hyeong-Min; An, Lin-Kun; Philpot, Benjamin D.; Zylka, Mark J.

    2016-01-01

    Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression. PMID:27231886

  16. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    Science.gov (United States)

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  17. Catalytic inhibitors of DNA topoisomerase II suppress the androgen receptor signaling and prostate cancer progression.

    Science.gov (United States)

    Li, Haolong; Xie, Ning; Gleave, Martin E; Dong, Xuesen

    2015-08-21

    Although the new generation of androgen receptor (AR) antagonists like enzalutamide (ENZ) prolong survival of metastatic castration-resistant prostate cancer (CRPC), AR-driven tumors eventually recur indicating that additional therapies are required to fully block AR function. Since DNA topoisomerase II (Topo II) was demonstrated to be essential for AR to initiate gene transcription, this study tested whether catalytic inhibitors of Topo II can block AR signaling and suppress ENZ-resistant CRPC growth. Using multiple prostate cancer cell lines, we showed that catalytic Topo II inhibitors, ICRF187 and ICRF193 inhibited transcription activities of the wild-type AR, mutant ARs (F876L and W741C) and the AR-V7 splice variant. ICRF187 and ICRF193 decreased AR recruitment to target promoters and reduced AR nuclear localization. Both ICRF187 and ICRF193 also inhibited cell proliferation and delayed cell cycling at the G2/M phase. ICRF187 inhibited tumor growth of castration-resistant LNCaP and 22RV1 xenografts as well as ENZ-resistant MR49F xenografts. We conclude that catalytic Topo II inhibitors can block AR signaling and inhibit tumor growth of CRPC xenografts, identifying a potential co-targeting approach using these inhibitors in combination with AR pathway inhibitors in CRPC.

  18. Topoisomerase II inhibition suppresses the proliferation of telomerase-negative cancers.

    Science.gov (United States)

    Hsieh, Meng-Hsun; Tsai, Cheng-Hui; Lin, Chuan-Chuan; Li, Tsai-Kun; Hung, Ting-Wei; Chang, Li-Te; Hsin, Ling-Wei; Teng, Shu-Chun

    2015-05-01

    Telomere maintenance is required for chromosome stability, and telomeres are typically elongated by telomerase following DNA replication. In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. Previous studies have indicated that yeast Sgs1 and Top3 may work together to remove highly negative supercoils that are generated from recombination. However, the mechanism by which cells eradicate highly positive supercoils during recombination remains unclear. In the present study, we demonstrate that TOP2 is involved in telomere-telomere recombination. Disturbance of telomeric structure by RIF1 or RIF2 deletion alleviates the requirement for TOP2 in telomere-telomere recombination. In human telomerase-negative alternative lengthening of telomere (ALT) cells, TOP2α or TOP2β knockdown decreases ALT-associated PML bodies, increases telomere dysfunction-induced foci and triggers telomere shortening. Similar results were observed when ALT cells were treated with ICRF-193, a TOP2 inhibitor. Importantly, ICRF-193 treatment blocks ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation in mice. Taken together, these findings imply that TOP2 is involved in the ALT pathway, perhaps by resolving the highly positive supercoil structure at the front of the helicase. Inhibition of topoisomerase II may be a promising therapeutic approach that can be used to prevent cell proliferation in ALT-type cancer cells. PMID:25430478

  19. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

    Science.gov (United States)

    Farr, Christine J; Antoniou-Kourounioti, Melissa; Mimmack, Michael L; Volkov, Arsen; Porter, Andrew C G

    2014-04-01

    As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.

  20. Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2

    Science.gov (United States)

    Schellenberg, Matthew J.; Perera, Lalith; Strom, Christina N.; Waters, Crystal A.; Monian, Brinda; Appel, C. Denise; Vilas, Caroline K.; Williams, Jason G.; Ramsden, Dale A.; Williams, R. Scott

    2016-01-01

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. PMID:27060144

  1. Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

    Science.gov (United States)

    Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N; Waters, Crystal A; Monian, Brinda; Appel, C Denise; Vilas, Caroline K; Williams, Jason G; Ramsden, Dale A; Williams, R Scott

    2016-05-01

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. PMID:27060144

  2. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Angela M Mabb

    Full Text Available Topoisomerase 1 (TOP1 inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc's that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc's, and a TOP1 mutation (T718A that stabilizes TOP1cc's. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression.

  3. Synthesis, DNA Binding and Topoisomerase I Inhibition Activity of Thiazacridine and Imidazacridine Derivatives

    Directory of Open Access Journals (Sweden)

    Elizabeth Almeida Lafayette

    2013-12-01

    Full Text Available Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 104 to 6.01 × 104 M−1. UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  4. Sequence-specific interactions of drugs interfering with the topoisomerase-DNA cleavage complex.

    Science.gov (United States)

    Palumbo, Manlio; Gatto, Barbara; Moro, Stefano; Sissi, Claudia; Zagotto, Giuseppe

    2002-07-18

    DNA-processing enzymes, such as the topoisomerases (tops), represent major targets for potent anticancer (and antibacterial) agents. The drugs kill cells by poisoning the enzymes' catalytic cycle. Understanding the molecular details of top poisoning is a fundamental requisite for the rational development of novel, more effective antineoplastic drugs. In this connection, sequence-specific recognition of the top-DNA complex is a key step to preferentially direct the action of the drugs onto selected genomic sequences. In fact, the (reversible) interference of drugs with the top-DNA complex exhibits well-defined preferences for DNA bases in the proximity of the cleavage site, each drug showing peculiarities connected to its structural features. A second level of selectivity can be observed when chemically reactive groups are present in the structure of the top-directed drug. In this case, the enzyme recognizes or generates a unique site for covalent drug-DNA binding. This will further subtly modulate the drug's efficiency in stimulating DNA damage at selected sites. Finally, drugs can discriminate not only among different types of tops, but also among different isoenzymes, providing an additional level of specific selection. Once the molecular basis for DNA sequence-dependent recognition has been established, the above-mentioned modes to generate selectivity in drug poisoning can be rationally exploited, alone or in combination, to develop tailor-made drugs targeted at defined loci in cancer cells. PMID:12084456

  5. DNA topoisomerase II structures and anthracycline activity: insights into ternary complex formation.

    Science.gov (United States)

    Dal Ben, D; Palumbo, M; Zagotto, G; Capranico, G; Moro, S

    2007-01-01

    DNA Topoisomerase II (Top2) is an essential nuclear enzyme that regulates the topological state of the DNA, and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have revised the published crystal structures of eukaryotic and prokaryotic Top2s and relevant biochemical investigations of enzyme activity and anthracycline action. In particular, we have considered Top2 mutations conferring resistance to anthracyclines and related agents. Following a previous study (Moro et al, Biochemistry, 2004; 43: 7503-13), we have then re-built a molecular model of the entire enzyme in complex with DNA after the cleavage reaction, and used it to define the receptor site of anthracyclines. The results suggest a model wherein the drug specifically contacts the cleaved DNA as well as amino acid residues of the enzyme CAP-like domain. The findings can explain several established structure-activity relationships of antitumour anthracyclines, and provide a framework for further developments of effective Top2 poison. PMID:17897022

  6. Apoptotic bone marrow CD34+ cells in cirrhotic patients

    Institute of Scientific and Technical Information of China (English)

    Shuang-Suo Dang; Wen-Jun Wang; Ning Gao; Shun-Da Wang; Mei Li; La-Yang Liu; Ming-Zhun Sun; Tao Dong

    2011-01-01

    AIM: To access the frequency and level of apoptotic CD34+ cells isolated from the marrow fluid of patients with post-hepatitis cirrhosis.METHODS: The frequency of bone marrow CD34+ cells and apoptotic bone marrow CD34+ cells in 31 in-patients with post-hepatitis cirrhosis (cirrhosis group), and 15 out-patients without liver or blood disorders (control group) was calculated by flow cytometry. Pa-rameters were collected to evaluate liver functions of patients in cirrhosis group.RESULTS: The percentage of normal bone marrow CD34+ cells was 6.30% ± 2.48% and 1.87% ± 0.53% (t = 3.906, P < 0.01) while that of apoptotic marrow CD34+ cells was 15.00% ± 15.81% and 5.73% ± 1.57% (t = 2.367, P < 0.05) in cirrhosis and control groups, re-spectively. The percentage of apoptotic marrow CD34+ cells was 6.25% ± 3.30% and 20.92 ± 18.5% (t = 2.409, P < 0.05) in Child-Pugh A and Child-Pugh B + C cirrhotic patients, respectively. The percentage of late apoptotic marrow CD34+ cells was positively correlated with the total bilirubin and aspartate aminotransferase serum levels in patients with cirrhosis.CONCLUSION: The status of CD34+ marrow cells in cirrhotic patients may suggest that the ability of he-matopoietic progenitor cells to transform into mature blood cells is impaired.

  7. QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACID LEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

    NARCIS (Netherlands)

    WITHOFF, S; SMIT, EF; MEERSMA, GJ; van den Berg, Anke; TIMMERBOSSCHA, H; KOK, K; POSTMUS, PE; MULDER, NH; DEVRIES, EGE; BUYS, CHCM

    1994-01-01

    BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase I

  8. Apoptotic machinery diversity in Multiple Myeloma molecular subtypes.

    Directory of Open Access Journals (Sweden)

    Patricia eGomez-Bougie

    2013-12-01

    Full Text Available Multiple myeloma (MM is a plasma cell malignancy that is heterogeneous in its clinical presentation and prognosis. Monoclonal gammopathy of undetermined significance (MGUS consistently preceded development of MM. The presence of primary IgH translocations and the universal over-expression of cyclin D genes led to a molecular classification of MM patients into different disease subtypes. Since Bcl-2 family proteins determine cell fate, we analyzed a publicly available Affymetrix gene expression of 44 MGUS and 414 newly diagnosed MM patients to investigate (1 the global change of Bcl-2 family members in MM versus MGUS (2 whether the four major subtypes defined as hyperdiploid, CCND1, MAF and MMSET, display specific apoptotic machineries.We showed that among the main anti-apoptotic members (Bcl-2, Bcl-xL and Mcl-1, Mcl-1 up-regulation discriminated MM from MGUS, in agreement with the prominent role of Mcl-1 in plasma cell differentiation. Surprisingly, the expression of multi-domain pro-apoptotic Bak and Bax were increased during the progression of MGUS to MM. The combined profile of Bcl-2, Bcl-xL and Mcl-1 was sufficient to distinguish MM molecular groups. While specific pro-apoptotic members expression was observed for each MM subtypes, CCDN1 subgroup, was identified as a particular entity characterized by a low expression of both BH3-only (Puma, Bik and Bad and multi-domain pro-apoptotic members (Bax and Bak. Our analysis supports the notion that MM heterogeneity is extended to the differential expression of the Bcl-2 family content in each MM subgroup. The influence of Bcl-2 family profile in the survival of the different patient groups will be further discussed to establish the potential consequences for therapeutic interventions. Finally, the use of distinct pro-survival members in the different steps of immune responses to antigen rise also the question of whether the different Bcl-2 anti-apoptotic profile could reflect a different origin of

  9. Apoptotic cell death and its relationship to gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Ferda Bir; Nese Calli-Demirkan; A Cevik Tufan; Metin Akbulut; N Lale Satiroglu-Tufan

    2007-01-01

    AIM: To investigate the apoptotic process of cells within the intestinal metaplasia areas co-localizing with chronic gastritis and gastric carcinomas and to analyze the involvement of proteins regulating apoptosis in the process of intestinal metaplasia related gastric carcinogenesis.METHODS: Forty-two gastric carcinoma and seventeen chronic gastritis cases were included in this study. All cases were examined for the existence of intestinal metaplasia. Ten cases randomly selected from each group were processed for TUNEL assay. TUNEL positive cells within the intestinal metaplasia areas, colocalizing either to gastric carcinoma or chronic gastritis,were counted and converted to apoptotic indices.In addition, p53, bcl-2 and bax expression patterns within these tissues were analyzed on the basis of immunohistochemistry.RESULTS: Twenty-eight of the cases were intestinal and 14 of the cases were diffuse type adenocarcinomas.64% (27/42) of the gastric carcinoma cases had intestinal metaplasia. Intestinal metaplasia co-localized more with intestinal type carcinomas compared with diffuse type carcinomas [75% (21/28) vs 42% (6/14),respectively; P≤0.05]. The mean apoptotic index in tumor cells was 0.70±0.08. The mean apoptotic index in intestinal metaplasias co-localizing to tumors was significantly higher than that of intestinal metaplasias co-localizing to chronic gastritis (0.70±0.03 vs 0.09±0.01, respectively; P≤0.05). P53 positivity was not observed in areas of intestinal metaplasia adjacent to tumors or chronic gastritis. Intestinal metaplasia areas adjacent to tumors showed lower cytoplasmic bcl-2 positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [55.5% (15/27) vs 70.5%(12/17), respectively]. On the other hand, intestinal metaplasia areas adjacent to tumors showed significantly higher cytoplasmic bax positivity compared to intestinal metaplasia areas adjacent to chronic gastritis [44.4%(12/27) vs 11.7% (2/17), respectively; P≤0

  10. Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.

    Science.gov (United States)

    Schneider, E; Horton, J K; Yang, C H; Nakagawa, M; Cowan, K H

    1994-01-01

    A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity. PMID:7903202

  11. Apoptotic Cells Are Cleared by Directional Migration and elmo1-Dependent Macrophage Engulfment

    NARCIS (Netherlands)

    van Ham, Tjakko J.; Kokel, David; Peterson, Randall T.

    2012-01-01

    Apoptotic cell death is essential for development and tissue homeostasis [1, 2]. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity [3, 4]. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptot

  12. DNA-binding preferences of bisantrene analogues: relevance to the sequence specificity of drug-mediated topoisomerase II poisoning.

    Science.gov (United States)

    Sissi, C; Bolgan, L; Moro, S; Zagotto, G; Bailly, C; Menta, E; Capranico, G; Palumbo, M

    1998-12-01

    To elucidate structure-activity relationships for drugs that are able to poison or inhibit topoisomerase II, we investigated the thermodynamics and stereochemistry of the DNA binding of a number of anthracene derivatives bearing one or two 4, 5-dihydro-1H-imidazol-2-yl-hydrazone side chains (characteristic of bisantrene) at different positions of the planar aromatic system. An aza-bioisostere, which can be considered a bisantrene-amsacrine hybrid, was also tested. The affinity for nucleic acids in different sequence contexts was evaluated by spectroscopic techniques, using various experimental conditions. DNA-melting and DNase I footprinting experiments were also performed. The location and number of the otherwise identical side chains dramatically affected the affinity of the test compounds for the nucleic acid. In addition, the new compounds exhibited different DNA sequence preferences, depending on the locations of the dihydroimidazolyl-hydrazone groups, which indicates a major role for the side-chain position in generating specific contacts with the nucleic acid. Molecular modeling studies of the intercalative binding of the 1- or 9-substituted isomers to DNA fully supported the experimental data, because a substantially more favorable recognition of A-T steps, compared with G-C steps, was found for the 9-substituted derivative, whereas a much closer energy balance was found for the 1-substituted isomer. These results compare well with the alteration of base specificity found for the topoisomerase II-mediated DNA cleavage stimulated by the isomeric drugs. Therefore, DNA-binding specificity appears to represent an important determinant for the recognition of the topoisomerase-DNA cleavable complex by the drug, at least for poisons belonging to the amsacrine-bisantrene family. PMID:9855632

  13. Identification of pathogenesis pathway in basal-like breast cancer based on mutant p53 protein and topoisomerase-IIα expression

    OpenAIRE

    Yayi Dwina; Ria Kodariah; Endang S.R. Hardjolukito

    2015-01-01

    Background: Basal-like breast cancer is difficult to treat with standard regimen therapy, because it doesn’t express hormone receptors or epidermal growth factor receptors. Identification of oncogenesis pathway is expected to find molecules which can be used as target for therapy. One candidate molecule is topoisomerase-IIα whose expression is regulated by p53. This study aimed to compare the expression of mutant p53 proteins and topoisomerase IIα in basal-like and non basal-like breast cance...

  14. Correlation between topoisomerase I and tyrosyl-DNA phosphodiesterase 1 activities in non-small cell lung cancer tissue

    DEFF Research Database (Denmark)

    Jakobsen, Ann-Katrine; Lauridsen, Kristina Lystlund; Samuel, Evelyn Benuja;

    2015-01-01

    camptothecin family (CPT). TDP1 has in cell line based assays been shown to counteract the effect of CPT. We have quantified the enzymatic activities of TOP1 and TDP1 in paired (tumor and adjacent non-tumor) samples from non-small cell lung cancer (NSCLC) patients and show that in NSCLC TOP1 and TDP1......Topoisomerase I (TOP1) regulates DNA topology during replication and transcription whereas tyrosyl-DNA phosphodiesterase 1 (TDP1) is involved in the repair of several types of DNA damages, including damages from defective TOP1 catalysis. TOP1 is the target of chemotherapeutic drugs of the...

  15. Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers

    DEFF Research Database (Denmark)

    Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup;

    2015-01-01

    . MATERIAL AND METHODS: Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18......INTRODUCTION: Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair...

  16. Apoptotic clearance in rabbits with experimental pulmonary emphysema

    OpenAIRE

    Žunić-Božinovski Snežana; Žunić Svetlana; Mladenović-Đorđević Aleksandra; Ruždijić Sabera; Kanazir Selma

    2011-01-01

    In order to better understand pathogenesis of pulmonary emphysema, the model of experimentally induced pulmonary emphysema in Chinchilla rabbits was used for the estimation of apoptotic clearance of pulmonary tissue. Bronchoalveolar lavage was performed in three groups of animals: experimental group-E on hypercholesterolemic diet (4% edible oil solution of crystalline cholesterol), control group-C1 on standard diet for that animal species and animals on oil...

  17. Catalytic inhibitors of topoisomerase II differently modulate the toxicity of anthracyclines in cardiac and cancer cells.

    Directory of Open Access Journals (Sweden)

    Anna Vavrova

    Full Text Available Anthracyclines (such as doxorubicin or daunorubicin are among the most effective anticancer drugs, but their usefulness is hampered by the risk of irreversible cardiotoxicity. Dexrazoxane (ICRF-187 is the only clinically approved cardioprotective agent against anthracycline cardiotoxicity. Its activity has traditionally been attributed to the iron-chelating effects of its metabolite with subsequent protection from oxidative stress. However, dexrazoxane is also a catalytic inhibitor of topoisomerase II (TOP2. Therefore, we examined whether dexrazoxane and two other TOP2 catalytic inhibitors, namely sobuzoxane (MST-16 and merbarone, protect cardiomyocytes from anthracycline toxicity and assessed their effects on anthracycline antineoplastic efficacy. Dexrazoxane and two other TOP2 inhibitors protected isolated neonatal rat cardiomyocytes against toxicity induced by both doxorubicin and daunorubicin. However, none of the TOP2 inhibitors significantly protected cardiomyocytes in a model of hydrogen peroxide-induced oxidative injury. In contrast, the catalytic inhibitors did not compromise the antiproliferative effects of the anthracyclines in the HL-60 leukemic cell line; instead, synergistic interactions were mostly observed. Additionally, anthracycline-induced caspase activation was differentially modulated by the TOP2 inhibitors in cardiac and cancer cells. Whereas dexrazoxane was upon hydrolysis able to significantly chelate intracellular labile iron ions, no such effect was noted for either sobuzoxane or merbarone. In conclusion, our data indicate that dexrazoxane may protect cardiomyocytes via its catalytic TOP2 inhibitory activity rather than iron-chelation activity. The differential expression and/or regulation of TOP2 isoforms in cardiac and cancer cells by catalytic inhibitors may be responsible for the selective modulation of anthracycline action observed.

  18. Cytotoxic and DNA-topoisomerase effects of lapachol amine derivatives and interactions with DNA

    Directory of Open Access Journals (Sweden)

    A. Esteves-Souza

    2007-10-01

    Full Text Available The cytotoxic activity of amino (3a-e, aza-1-antraquinone (4a-e lapachol derivatives against Ehrlich carcinoma and human K562 leukemia cells was investigated. Cell viability was determined using MTT assay, after 48 (Ehrlich or 96 h (K562 of culture, and vincristine (for K562 leukemia and quercetin (for Ehrlich carcinoma were used as positive controls. The results showed dose-dependent growth-inhibiting activities and that the amino derivatives were active against the assayed cells, whereas the 4a-e derivatives were not. The allylamine derivative 3a was the most active against Ehrlich carcinoma, with IC50 = 16.94 ± 1.25 µM, and against K562 leukemia, with IC50 = 14.11 ± 1.39 µM. The analogous lawsone derivative, 5a, was also active against Ehrlich carcinoma (IC50 = 23.89 ± 2.3 µM, although the 5d and 5e derivatives showed lower activity. The interaction between 3a-d and calf thymus DNA was investigated by fluorimetric titration and the results showed a hyperchromic effect indicating binding to DNA as presented of ethidium bromide, used as positive control. The inhibitory action on DNA-topoisomerase II-a was also evaluated by a relaxation assay of supercoiled DNA plasmid, and the etoposide (200 µM was used as positive control. Significant inhibitory activities were observed for 3a-d at 200 µM and a partial inhibitory action was observed for lapachol and methoxylapachol.

  19. Combined MET inhibition and topoisomerase I inhibition block cell growth of small cell lung cancer.

    Science.gov (United States)

    Rolle, Cleo E; Kanteti, Rajani; Surati, Mosmi; Nandi, Suvobroto; Dhanasingh, Immanuel; Yala, Soheil; Tretiakova, Maria; Arif, Qudsia; Hembrough, Todd; Brand, Toni M; Wheeler, Deric L; Husain, Aliya N; Vokes, Everett E; Bharti, Ajit; Salgia, Ravi

    2014-03-01

    Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)-induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC. PMID:24327519

  20. Novel insights into the apoptosis mechanism of DNA topoisomerase I inhibitor isoliquiritigenin on HCC tumor cell

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ze-xin; Li, Jian; Li, Yan; You, Kun; Xu, Hongwei; Wang, Jianguo, E-mail: wangjianguoxx@163.com

    2015-08-21

    The inhibitory effect of DNA topoisomerase (Top I) by isoliquiritigenin(ISO) were investigated and their interaction mechanism was evaluated using methods including UV–vis absorption, fluorescence, coupled with molecular simulation, and using the MTT method of inhibition rate of HCC tumor cell SNU475 proliferation assay, finally, the interaction of ISO with calf thymus DNA was investigated by melting measurements and molecular docking studies. It was found that isoliquiritigenin reversibly inhibited DNA Top I in a competitive manner with the concentrations of ISO resulting in 50% activity lost (IC{sub 50}) were estimated to be 0.178 ± 0.12 mM. Isoliquiritigenin exhibited a strong ability to quench the intrinsic fluorescence of Top I through a static quenching procedure. The positive values of enthalpy change and entropy change suggested that the binding of isoliquiritigenin to Top I was driven mainly by hydrophobic interactions. The molecular docking results revealed isoliquiritigenin actually interacted with the primary amino acid residues on the active site of Top I, and the detection results of fluorescence staining and the inhibitory effect on the growth of HCC SUN475 showed that isoliquiritigenin induced the apoptosis cells increased gradually. The interaction of ISO with DNA can cause the denaturation temperature to be increased, which indicated that the stabilization of the DNA helix was increased in the presence of ISO, which indicated that the results provide strong evidence for intercalative binding of ISO with DNA. - Highlights: • ISO reversibly inhibits TOP I activity in an A dose dependent manner. • Hydrophobic interactions play a major role in ISO–TOP I interaction. • ISO has a high affinity close to the active site pocket of TOP I. • The binding of ISO to DNA induces the stability of the structure of DNA.

  1. Inhibitory effects of docosyl p-coumarate on DNA topoisomerase activity and human cancer cell growth.

    Science.gov (United States)

    Mizushina, Yoshiyuki; Nishimura, Katsumi; Takenaka, Yukiko; Takeuchi, Toshifumi; Sugawara, Fumio; Yoshida, Hiromi; Tanahashi, Takao

    2010-10-01

    We previously found six compounds of alkyl p-coumarates from a composite plant Artemisia annua L., and chemically synthesized these compounds (cis-isomer of C20, C22 and C24, and trans-isomer of C20, C22 and C24 of p-coumarates are compounds 1-6, respectively). This report describes the inhibitory activities of these alkyl p-coumarates against DNA polymerase (pol), DNA topoisomerase (topo), and human cancer cell growth. Among the compounds tested, compounds 1 and 4 weakly inhibited repair-related pol beta activity, but no compound influenced the activity of replicative pol alpha. Compounds 4-6 and compounds 2 and 5 were potent inhibitors of human topos I and II, respectively. Compounds 2, 4, 5 and 6 also suppressed the growth of human colon carcinoma cell line, HCT116, with or without p53, suggesting that cell growth inhibition had the same tendency as the inhibition of topos rather than pols. Compound 5 (docosyl p-coumarate), which was the strongest inhibitor of topo II and cancer cell growth in the compounds tested, halted HCT116 p53(+/+) cells in G2/M phases, and induced apoptosis, although this compound did not affect the cell cycle of HCT116 p53(-/-) cells. These results suggest that the effect of p53-dependent cell cycle arrest may be effective for topo inhibition by com-pound 5. From these findings, the action mode of alkyl p-coumarates as an anti-cancer agent is discussed. PMID:20811721

  2. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro

    Indian Academy of Sciences (India)

    Swathi Kota; Hari S Misra

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase lb of D. radiodurans (DraTopolB) could change the electrophoretic mobility of fast migrating intramolecular rec-G4 DNA into the slow migrating species. DraTopolB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular rec-G4 DNA structures stabilized by K+. On the contrary, when DraTopolB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopolB. This implies that DraTopolB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopolB activity on intramolecular G4 DNA structures. These results suggested that DraTopolB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  3. Novel insights into the apoptosis mechanism of DNA topoisomerase I inhibitor isoliquiritigenin on HCC tumor cell

    International Nuclear Information System (INIS)

    The inhibitory effect of DNA topoisomerase (Top I) by isoliquiritigenin(ISO) were investigated and their interaction mechanism was evaluated using methods including UV–vis absorption, fluorescence, coupled with molecular simulation, and using the MTT method of inhibition rate of HCC tumor cell SNU475 proliferation assay, finally, the interaction of ISO with calf thymus DNA was investigated by melting measurements and molecular docking studies. It was found that isoliquiritigenin reversibly inhibited DNA Top I in a competitive manner with the concentrations of ISO resulting in 50% activity lost (IC50) were estimated to be 0.178 ± 0.12 mM. Isoliquiritigenin exhibited a strong ability to quench the intrinsic fluorescence of Top I through a static quenching procedure. The positive values of enthalpy change and entropy change suggested that the binding of isoliquiritigenin to Top I was driven mainly by hydrophobic interactions. The molecular docking results revealed isoliquiritigenin actually interacted with the primary amino acid residues on the active site of Top I, and the detection results of fluorescence staining and the inhibitory effect on the growth of HCC SUN475 showed that isoliquiritigenin induced the apoptosis cells increased gradually. The interaction of ISO with DNA can cause the denaturation temperature to be increased, which indicated that the stabilization of the DNA helix was increased in the presence of ISO, which indicated that the results provide strong evidence for intercalative binding of ISO with DNA. - Highlights: • ISO reversibly inhibits TOP I activity in an A dose dependent manner. • Hydrophobic interactions play a major role in ISO–TOP I interaction. • ISO has a high affinity close to the active site pocket of TOP I. • The binding of ISO to DNA induces the stability of the structure of DNA

  4. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer

    Science.gov (United States)

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong

    2016-01-01

    Purpose Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Materials and Methods Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort 2 (83 BC patients, 54 nonmalignant hematuric patients, and 61 normal controls) provided urine samples. Real-time quantitative polymerase chain reaction was used to measure expression of TopoIIA mRNA in tissues and TopoIIA cell-free DNA in urine samples. Results The results showed that expression of TopoIIA mRNA in BC tissues was significantly higher than that in noncancer control tissues (p<0.001). The expression of urinary TopoIIA cell-free DNA in BC patients was also significantly higher than that in noncancer patient controls and hematuria patients (p < 0.001 and p < 0.001, respectively). High expression of urinary TopoIIA cell-free DNA was also detected in muscle invasive bladder cancer (MIBC) when compared with nonmuscle invasive bladder cancer (NMIBC) (p=0.002). Receiver operating characteristics (ROC) curve analysis was performed to examine the sensitivity/specificity of urinary TopoIIA cell-free DNA for diagnosing BC, NMIBC, and MIBC. The areas under the ROC curve for BC, NMIBC, and MIBC were 0.741, 0.701, and 0.838, respectively. Conclusions In summary, the results of this study provide evidence that cell-free TopoIIA DNA may be a potential biomarker for BC. PMID:26981592

  5. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

    Science.gov (United States)

    Kota, Swathi; Misra, Hari S

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  6. Extracellular Matrix Proteins Modulate Antimigratory and Apoptotic Effects of Doxorubicin

    Directory of Open Access Journals (Sweden)

    Georges Said

    2012-01-01

    Full Text Available Anticancer drug resistance is a multifactorial process that includes acquired and de novo drug resistances. Acquired resistance develops during treatment, while de novo resistance is the primary way for tumor cells to escape chemotherapy. Tumor microenvironment has been recently shown to be one of the important factors contributing to de novo resistance and called environment-mediated drug resistance (EMDR. Two forms of EMDR have been described: soluble factor-mediated drug resistance (SFM-DR and cell adhesion-mediated drug resistance (CAM-DR. Anthracyclines, among the most potent chemotherapeutic agents, are widely used in clinics against hematopoietic and solid tumors. Their main mechanism of action relies on the inhibition of topoisomerase I and/or II and the induction of apoptosis. Beyond this well-known antitumor activity, it has been recently demonstrated that anthracyclines may display potent anti-invasive effects when used at subtoxic concentrations. In this paper, we will describe two particular modes of EMDR by which microenvironment may influence tumor-cell response to one of these anthracyclines, doxorubicin. The first one considers the influence of type I collagen on the antimigratory effect of doxorubicin (CAM-DR. The second considers the protection of tumor cells by thrombospondin-I against doxorubicin-induced apoptosis (SFM-DR.

  7. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

    Science.gov (United States)

    Kim, H R; Luo, Y; Li, G; Kessel, D

    1999-07-15

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

  8. Cell shape and organelle modification in apoptotic U937 cells

    Directory of Open Access Journals (Sweden)

    MR Montinari

    2009-12-01

    Full Text Available U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis were simultaneously analyzed.

  9. The inflammatory role of phagocyte apoptotic pathways in rheumatic diseases.

    Science.gov (United States)

    Cuda, Carla M; Pope, Richard M; Perlman, Harris

    2016-08-23

    Rheumatoid arthritis affects nearly 1% of the world's population and is a debilitating autoimmune condition that can result in joint destruction. During the past decade, inflammatory functions have been described for signalling molecules classically involved in apoptotic and non-apoptotic death pathways, including, but not limited to, Toll-like receptor signalling, inflammasome activation, cytokine production, macrophage polarization and antigen citrullination. In light of these remarkable advances in the understanding of inflammatory mechanisms of the death machinery, this Review provides a snapshot of the available evidence implicating death pathways, especially within the phagocyte populations of the innate immune system, in the perpetuation of rheumatoid arthritis and other rheumatic diseases. Elevated levels of signalling mediators of both extrinsic and intrinsic apoptosis, as well as the autophagy, are observed in the joints of patients with rheumatoid arthritis. Furthermore, risk polymorphisms are present in signalling molecules of the extrinsic apoptotic and autophagy death pathways. Although research into the mechanisms underlying these pathways has made considerable progress, this Review highlights areas where further investigation is particularly needed. This exploration is critical, as new discoveries in this field could lead to the development of novel therapies for rheumatoid arthritis and other rheumatic diseases. PMID:27549026

  10. PDT-treated apoptotic cells induce macrophage synthesis NO

    Science.gov (United States)

    Song, S.; Xing, D.; Zhou, F. F.; Chen, W. R.

    2009-11-01

    Nitric oxide (NO) is a biologically active molecule which has multi-functional in different species. As a second messenger and neurotransmitter, NO is not only an important regulatory factor between cells' information transmission, but also an important messenger in cell-mediated immunity and cytotoxicity. On the other side, NO is involving in some diseases' pathological process. In pathological conditions, the macrophages are activated to produce a large quantity of nitric oxide synthase (iNOS), which can use L-arginine to produce an excessive amount of NO, thereby killing bacteria, viruses, parasites, fungi, tumor cells, as well as in other series of the immune process. In this paper, photofrin-based photodynamic therapy (PDT) was used to treat EMT6 mammary tumors in vitro to induce apoptotic cells, and then co-incubation both apoptotic cells and macrophages, which could activate macrophage to induce a series of cytotoxic factors, especially NO. This, in turn, utilizes macrophages to activate a cytotoxic response towards neighboring tumor cells. These results provided a new idea for us to further study the immunological mechanism involved in damaging effects of PDT, also revealed the important function of the immune effect of apoptotic cells in PDT.

  11. Effect of PDT-treated apoptotic cells on macrophages

    Science.gov (United States)

    Song, Sheng; Xing, Da; Zhou, Fei-fan; Chen, Wei R.

    2009-02-01

    Recently, the long-term immunological effects of photodynamic therapy have attracted much attention. PDT induced immune response was mainly initiated through necrotic cells and apoptotic cells, as well as immune cells such as macrophages. Nitric oxide (NO) as an important regulatory factor in signal transfer between cells has been wildly studied for generation, development, and metastasis of tumors. NO synthase is a key enzyme in nitric oxide synthesis. However, inducible nitric oxide synthase (iNOS) is usually activated under pathological conditions, such as stress and cancer, which can produce high levels of nitric oxide and contribute to tumor cytotoxicity. In addition, increased NO production by iNOS has been associated with the host immune response and cell apoptosis, which play an important role in many carcinogenesis and anti-carcinoma mechanisms. This study focuses on the NO production in macrophages, induced by mouse breast carcinoma apoptotic cells treated by PDT in vitro, and on the effects of immune response induced by apoptotic cells in tumor cells growth.

  12. Targeting topoisomerase IIa in endometrial adenocarcinoma: a combined chromogenic in situ hybridization and immunohistochemistry study based on tissue microarrays.

    Science.gov (United States)

    Tsiambas, E; Alexopoulou, D; Lambropoulou, S; Gerontopoulos, K; Karakitsos, P; Karameris, A

    2006-01-01

    Topoisomerase IIa is a nucleic enzyme that affects the topological structure of DNA and also is a target for chemotherapy (ie, anthracyclines). In this study, we coevaluated its protein expression with chromosome 17 and gene status. Using tissue microarrays, 40 cases of sporadic, primary endometrial adenocarcinomas, 5 cases of atypical hyperplasia, and 5 cases of benign hyperplasia were obtained and reembedded into two paraffin blocks with a core diameter of 1 mm. Immunohistochemistry combined with chromogenic in situ hybridization was performed in 2 and 5 microm sections, respectively. Finally using a semiautomated Image Analysis System, we evaluated the levels of Nuclear labeling index of topoisomerase IIa expression. Statistical analysis was performed by SPSS version 11.0 software. The results indicate that chromosome 17 instability (aneuploidy in 7/40 cases) and Topo IIa gene deregulation (amplification in 3/40 and deletion in 1/40 cases) are significant genetic events correlated with biologic behavior in endometrial adenocarcinoma. Because protein overexpression was observed in a significant proportion of the tumors (18/40), detection of the specific gene deregulation mechanism is a crucial process for application of targeted chemotherapies, which are characterized by different levels of cardiotoxicity and other serious effects.

  13. Flavonoids from Annona dioica leaves and their effects in Ehrlich carcinoma cells, DNA-topoisomerase I and II

    International Nuclear Information System (INIS)

    Chemical investigation of methanol extract leaves from Annona dioica (Annonaceae) resulted in the identification of flavonoids kaempferol (1), 3-O-[3'',6''-di-O-p-hydroxycinnamoyl]-β- galactopyranosyl-kaempferol (2), 6''-O-p-hydroxycinnamoyl-β-galactopyranosyl-kaempferol (3) and 3-O-β-galactopyranosyl-kaempferol (4). The structures were unequivocally characterized by 1H and 13C NMR spectroscopic analyses using 1D and 2D experiments. The cytotoxic effects of the flavonoids and flavonoid fraction (FF) were evaluated by MTT (3-(4,5-dimethylthiazole-2- yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma cells. The results indicated that 1, 2, 3 and FF exhibit significant antiproliferative action when compared to quercetin. The inhibitory action on DNA-topoisomerase I and II of all the flavonoids was evaluated by relaxation assays on pBR322 plasmid DNA. The results indicated the inhibitory and non-selective effects of the flavonoids on DNA-topoisomerase I and II. (author)

  14. Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target.

    Directory of Open Access Journals (Sweden)

    Ivan Laponogov

    Full Text Available The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo IV subunit A (ParC from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA, but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.

  15. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, M; Kveiborg, Marie; Kassem, M;

    2000-01-01

    In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitative...... reverse transcriptase-polymerase chain reaction (RT-PCR). There is a progressive and significant reduction of the CBFA1 steady-state mRNA level down to 50% during cellular aging of human osteoblasts. In comparison to the normal cells, human osteosarcoma cell lines SaOS-2 and KHOS/NP, and the SV40......-transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40...

  16. Flavonoids from Annona dioica leaves and their effects in Ehrlich carcinoma cells, DNA-topoisomerase I and II

    Energy Technology Data Exchange (ETDEWEB)

    Vega, Maria R.G.; Esteves-Souza, Andressa; Echevarria, Aurea [Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropedica, RJ (Brazil). Dept. de Quimica]. E-mail: echevarr@ufrrj.br; Vieira, Ivo J.C.; Mathias, Leda; Braz-Filho, Raimundo [Universidade Estadual do Norte Fluminense (UENF), Campos dos Goytacases, RJ (Brazil). Lab. de Ciencias Quimicas

    2007-07-01

    Chemical investigation of methanol extract leaves from Annona dioica (Annonaceae) resulted in the identification of flavonoids kaempferol (1), 3-O-[3'',6''-di-O-p-hydroxycinnamoyl]-{beta}- galactopyranosyl-kaempferol (2), 6''-O-p-hydroxycinnamoyl-{beta}-galactopyranosyl-kaempferol (3) and 3-O-{beta}-galactopyranosyl-kaempferol (4). The structures were unequivocally characterized by {sup 1}H and {sup 13}C NMR spectroscopic analyses using 1D and 2D experiments. The cytotoxic effects of the flavonoids and flavonoid fraction (FF) were evaluated by MTT (3-(4,5-dimethylthiazole-2- yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma cells. The results indicated that 1, 2, 3 and FF exhibit significant antiproliferative action when compared to quercetin. The inhibitory action on DNA-topoisomerase I and II of all the flavonoids was evaluated by relaxation assays on pBR322 plasmid DNA. The results indicated the inhibitory and non-selective effects of the flavonoids on DNA-topoisomerase I and II. (author)

  17. Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2

    Energy Technology Data Exchange (ETDEWEB)

    Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay; Robertson, Patrick D; Ramsden, Dale A; Williams, R Scott [NIH; (Georgetown); (UNC)

    2012-10-28

    The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.

  18. The cytotoxicity of benzaldehyde nitrogen mustard-2-pyridine carboxylic acid hydrazone being involved in topoisomerase IIα inhibition.

    Science.gov (United States)

    Fu, Yun; Zhou, Sufeng; Liu, Youxun; Yang, Yingli; Sun, Xingzhi; Li, Changzheng

    2014-01-01

    The antitumor property of iron chelators and aromatic nitrogen mustard derivatives has been well documented. Combination of the two pharmacophores in one molecule in drug designation is worth to be explored. We reported previously the syntheses and preliminary cytotoxicity evaluation of benzaldehyde nitrogen mustard pyridine carboxyl acid hydrazones (BNMPH) as extended study, more tumor cell lines (IC50 for HepG2: 26.1 ± 3.5 μM, HCT-116: 57.5 ± 5.3 μM, K562: 48.2 ± 4.0 μM, and PC-12: 19.4 ± 2.2 μM) were used to investigate its cytotoxicity and potential mechanism. In vitro experimental data showed that the BNMPH chelating Fe(2+) caused a large number of ROS formations which led to DNA cleavage, and this was further supported by comet assay, implying that ROS might be involved in the cytotoxicity of BNMPH. The ROS induced changes of apoptosis related genes, but the TFR1 and NDRG1 metastatic genes were not obviously regulated, prompting that BNMPH might not be able to deprive Fe(2+) of ribonucleotide reductase. The BNMPH induced S phase arrest was different from that of iron chelators (G1) and alkylating agents (G2). BNMPH also exhibited its inhibition of human topoisomerase IIα. Those revealed that the cytotoxic mechanism of the BNMPH could stem from both the topoisomerase II inhibition, ROS generation and DNA alkylation.

  19. Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

    DEFF Research Database (Denmark)

    Wang, Zhenxing; D'Annessa, Ilda; Tesauro, Cinzia;

    2015-01-01

    Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein...

  20. Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort

    DEFF Research Database (Denmark)

    Smith, David Hersi; Christensen, Ib Jarle; Jensen, Niels Frank;

    2013-01-01

    Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III C...

  1. Responding to the challenge of untreatable gonorrhea: ETX0914, a first-in-class agent with a distinct mechanism-of-action against bacterial Type II topoisomerases.

    Science.gov (United States)

    Basarab, Gregory S; Kern, Gunther H; McNulty, John; Mueller, John P; Lawrence, Kenneth; Vishwanathan, Karthick; Alm, Richard A; Barvian, Kevin; Doig, Peter; Galullo, Vincent; Gardner, Humphrey; Gowravaram, Madhusudhan; Huband, Michael; Kimzey, Amy; Morningstar, Marshall; Kutschke, Amy; Lahiri, Sushmita D; Perros, Manos; Singh, Renu; Schuck, Virna J A; Tommasi, Ruben; Walkup, Grant; Newman, Joseph V

    2015-01-01

    With the diminishing effectiveness of current antibacterial therapies, it is critically important to discover agents that operate by a mechanism that circumvents existing resistance. ETX0914, the first of a new class of antibacterial agent targeted for the treatment of gonorrhea, operates by a novel mode-of-inhibition against bacterial type II topoisomerases. Incorporating an oxazolidinone on the scaffold mitigated toxicological issues often seen with topoisomerase inhibitors. Organisms resistant to other topoisomerase inhibitors were not cross-resistant with ETX0914 nor were spontaneous resistant mutants to ETX0914 cross-resistant with other topoisomerase inhibitor classes, including the widely used fluoroquinolone class. Preclinical evaluation of ETX0914 pharmacokinetics and pharmacodynamics showed distribution into vascular tissues and efficacy in a murine Staphylococcus aureus infection model that served as a surrogate for predicting efficacious exposures for the treatment of Neisseria gonorrhoeae infections. A wide safety margin to the efficacious exposure in toxicological evaluations supported progression to Phase 1. Dosing ETX0914 in human volunteers showed sufficient exposure and minimal adverse effects to expect a highly efficacious anti-gonorrhea therapy. PMID:26168713

  2. Lack of cross-resistance to fostriecin in a human small-cell lung carcinoma cell line showing topoisomerase II-related drug resistance

    NARCIS (Netherlands)

    de Jong, Steven; Zijlstra, J G; Mulder, Nanno; de Vries, Liesbeth

    1991-01-01

    Cells exhibiting decreased topoisomerase II (Topo II) activity are resistant to several drugs that require Topo II as an intermediate. These drugs are cytotoxic due to the formation of a cleavable complex between the drug, Topo II and DNA. Fostriecin belongs to a new class of drugs that inhibit Topo

  3. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    International Nuclear Information System (INIS)

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P21, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P21212, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism

  4. The impact of the human DNA topoisomerase II C-terminal domain on activity.

    Directory of Open Access Journals (Sweden)

    Emma L Meczes

    Full Text Available BACKGROUND: Type II DNA topoisomerases (topos are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. METHODOLOGY/PRINCIPLE FINDINGS: We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity. CONCLUSIONS/SIGNIFICANCE: In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro

  5. Roles of type 1A topoisomerases in genome maintenance in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Valentine Usongo

    2014-08-01

    Full Text Available In eukaryotes, type 1A topoisomerases (topos act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA and III (topB enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i by preventing over-replication originating from oriC (topo I alone and R

  6. Roles of type 1A topoisomerases in genome maintenance in Escherichia coli.

    Science.gov (United States)

    Usongo, Valentine; Drolet, Marc

    2014-08-01

    In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii

  7. The broken MLL gene is frequently located outside the inherent chromosome territory in human lymphoid cells treated with DNA topoisomerase II poison etoposide.

    Directory of Open Access Journals (Sweden)

    Sergey I Glukhov

    Full Text Available The mixed lineage leukaemia (MLL gene is frequently rearranged in secondary leukaemias, in which it could fuse to a variety of different partners. Breakage in the MLL gene preferentially occurs within a ~8 kb region that possesses a strong DNA topoisomerase II cleavage site. It has been proposed that DNA topoisomerase II-mediated DNA cleavage within this and other regions triggers translocations that occur due to incorrect joining of broken DNA ends. To further clarify a possible mechanism for MLL rearrangements, we analysed the frequency of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison commonly used as an anticancer drug, and positioning of the broken 3'-end of the MLL gene in respect to inherent chromosomal territories. It was demonstrated that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL genes in ~17% of nuclei. Using confocal microscopy and 3D modelling, we demonstrated that in cells treated with etoposide and cultivated for 1 h under normal conditions, ~9% of the broken MLL alleles were present outside the chromosome 11 territory, whereas in both control cells and cells inspected immediately after etoposide treatment, virtually all MLL alleles were present within the chromosomal territory. The data are discussed in the framework of the "breakage first" model of juxtaposing translocation partners. We propose that in the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of stalled DNA topoisomerase II complexes and non-homologous end joining, DNA ends acquire additional mobility, which allows the meeting and incorrect joining of translocation partners.

  8. DNA Topoisomerase IIα contributes to the early steps of adipogenesis in 3T3-L1 cells.

    Science.gov (United States)

    Jacobsen, Rhîan G; Mazloumi Gavgani, Fatemeh; Mellgren, Gunnar; Lewis, Aurélia E

    2016-10-01

    DNA topoisomerases (Topo) are multifunctional enzymes resolving DNA topological problems such as those arising during DNA replication, transcription and mitosis. Mammalian cells express 2 class II isoforms, Topoisomerases IIα (Topo IIα) and IIβ (Topo IIβ), which have similar enzymatic properties but are differently expressed, in dividing and pluripotent cells, and in post-mitotic and differentiated cells respectively. Pre-adipocytes re-enter the cell cycle prior to committing to their differentiation and we hypothesised that Topo II could contribute to these processes. We show that Topo IIα expression in 3T3-L1 cells is induced within 16h after the initiation of the differentiation programme, peaks at 24h and rapidly declines thereafter. In contrast Topo IIβ was present both in pre-adipocytes and throughout differentiation. Inhibition of PI3K with LY294002, known to prevent adipocyte differentiation, consistently reduced the expression of Topo IIα, whereas a clear effect on Topo IIβ was not apparent. In addition, inhibition of mTOR with rapamycin also reduced the protein levels of Topo IIα. Using specific class IA PI3K catalytic subunit inhibitors, we show that p110α inhibition with A66 has the greatest reduction of Topo IIα expression and of differentiation, as measured by triglyceride storage. The timing of Topo IIα expression coincides with the mitotic clonal expansion (MCE) phase of differentiation and inhibition of Topo II with ICRF-187 during this stage decreased PPARγ1 and 2 protein levels and triglyceride storage, whereas inhibition later on has little impact. Moreover, the addition of ICRF-187 had no effect on the incorporation of EdU during S-phase at day 1 but lowered the relative cell numbers on day 2. ICRF-187 also induced an increase in the centri/pericentromeric heterochromatin localisation of Topo IIα, indicating a role for Topo IIα at these locations during MCE. In summary, we present evidence that Topo IIα plays an important role

  9. Interaction of late apoptotic and necrotic cells with vitronectin.

    Directory of Open Access Journals (Sweden)

    Ondrej Stepanek

    Full Text Available BACKGROUND: Vitronectin is an abundant plasma glycoprotein identified also as a part of extracellular matrix. Vitronectin is substantially enriched at sites of injured, fibrosing, inflamed, and tumor tissues where it is believed to be involved in wound healing and tissue remodeling. Little is known about the mechanism of vitronectin localization into the damaged tissues. METHODOLOGY/PRINCIPAL FINDINGS: 2E12 antibody has been described to bind a subset of late apoptotic cells. Using immunoisolation followed by mass spectrometry, we identified the antigen recognized by 2E12 antibody as vitronectin. Based on flow cytometry, we described that vitronectin binds to the late apoptotic and necrotic cells in cell cultures in vitro as well as in murine thymus and spleen in vivo. Confocal microscopy revealed that vitronectin binds to an intracellular cytoplasmic structure after the membrane rupture. CONCLUSIONS/SIGNIFICANCE: We propose that vitronectin could serve as a marker of membrane disruption in necrosis and apoptosis for flow cytometry analysis. Moreover, we suggest that vitronectin binding to dead cells may represent one of the mechanisms of vitronectin incorporation into the injured tissues.

  10. Heat-induced fibrillation of BclXL apoptotic repressor.

    Science.gov (United States)

    Bhat, Vikas; Olenick, Max B; Schuchardt, Brett J; Mikles, David C; Deegan, Brian J; McDonald, Caleb B; Seldeen, Kenneth L; Kurouski, Dmitry; Faridi, Mohd Hafeez; Shareef, Mohammed M; Gupta, Vineet; Lednev, Igor K; Farooq, Amjad

    2013-09-01

    The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease. PMID:23714425

  11. Development of novel cyclic peptides as pro-apoptotic agents.

    Science.gov (United States)

    Brindisi, Margherita; Maramai, Samuele; Brogi, Simone; Fanigliulo, Emanuela; Butini, Stefania; Guarino, Egeria; Casagni, Alice; Lamponi, Stefania; Bonechi, Claudia; Nathwani, Seema M; Finetti, Federica; Ragonese, Francesco; Arcidiacono, Paola; Campiglia, Pietro; Valenti, Salvatore; Novellino, Ettore; Spaccapelo, Roberta; Morbidelli, Lucia; Zisterer, Daniela M; Williams, Clive D; Donati, Alessandro; Baldari, Cosima; Campiani, Giuseppe; Ulivieri, Cristina; Gemma, Sandra

    2016-07-19

    Our recent finding that paclitaxel behaves as a peptidomimetic of the endogenous protein Nur77 inspired the design of two peptides (PEP1 and PEP2) reproducing the effects of paclitaxel on Bcl-2 and tubulin, proving the peptidomimetic nature of paclitaxel. Starting from these peptide-hits, we herein describe the synthesis and the biological investigation of linear and cyclic peptides structurally related to PEP2. While linear peptides (2a,b, 3a,b, 4, 6a-f) were found inactive in cell-based assays, biological analysis revealed a pro-apoptotic effect for most of the cyclic peptides (5a-g). Cellular permeability of 5a (and also of 2a,b) on HL60 cells was assessed through confocal microscopy analysis. Further cellular studies on a panel of leukemic cell lines (HL60, Jurkat, MEC, EBVB) and solid tumor cell lines (breast cancer MCF-7 cells, human melanoma A375 and 501Mel cells, and murine melanoma B16F1 cells) confirmed the pro-apoptotic effect of the cyclic peptides. Cell cycle analysis revealed that treatment with 5a, 5c, 5d or 5f resulted in an increase in the number of cells in the sub-G0/G1 peak. Direct interaction with tubulin (turbidimetric assay) and with microtubules (immunostaining experiments) was assessed in vitro for the most promising compounds. PMID:27150036

  12. Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient

    Science.gov (United States)

    Sun, E.; Gao, Y.; Chen, J.; Roberts, A. I.; Wang, X.; Chen, Z.; Shi, Y.

    2004-01-01

    Cell death through apoptosis plays a critical role in regulating cellular homeostasis. Whether the disposal of apoptotic cells through phagocytosis can actively induce immune tolerance in vivo, however, remains controversial. Here, we report in a rat model that without using immunosuppressants, transfusion of apoptotic splenocytes from the donor strain prior to transplant dramatically prolonged survival of heart allografts. Histological analysis verified that rejection signs were significantly ameliorated. Splenocytes from rats transfused with donor apoptotic cells showed a dramatically decreased response to donor lymphocyte stimulation. Most importantly, blockade of phagocytosis in vivo, either with gadolinium chloride to disrupt phagocyte function or with annexin V to block binding of exposed phosphotidylserine to its receptor on phagocytes, abolished the beneficial effect of transfused apoptotic cells on heart allograft survival. Our results demonstrate that donor apoptotic cells promote specific allograft acceptance and that phagocytosis of apoptotic cells in vivo plays a crucial role in maintaining immune tolerance.

  13. Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

    OpenAIRE

    Fotios Drakopanagiotakis; Areti Xifteri; Evaggelos Tsiambas; Andreas Karameris; Konstantina Tsakanika; Napoleon Karagiannidis; Demetrios Mermigkis; Vlasis Polychronopoulos; Demosthenes Bouros

    2012-01-01

    Introduction. Increased apoptosis of epithelial cells and decreased apoptosis of myofibroblasts are involved in the pathogenesis of IPF. The apoptotic profile of alveolar macrophages (AMs) in IPF is unclear. Aim. To investigate whether AMs of patients with IPF exhibit a different apoptotic profile compared to normal subjects. Methods. We analyzed, by immunohistochemistry, the expression of the apoptotic markers fas, fas ligand , bcl-2, and bax in AM obtained from bronchoalveolar lavage fluid ...

  14. HMM Search for Apoptotic Domains (MOLECULAR BIOLOGY AND INFORMATION-Biological Information Science)

    OpenAIRE

    Hattori, Masahiro; Kanehisa, Minoru

    2000-01-01

    For the purpose of analyzing apoptotic molecular interactions, we have developed a knowledge base, which consists of apoptotic molecular interactions, together with the WWW interface for it. This database and the user interface enabled us to find out entries containing various information about cell death. This information tells us that the apoptotic molecular interactions are likely to be controlled under a series of specific conserved domains. Thus, the viewpoint of domain seems to be more ...

  15. Age-related activation of mitochondrial caspase-independent apoptotic signaling in rat gastrocnemius muscle

    OpenAIRE

    Marzetti, Emanuele; Wohlgemuth, Stephanie Eva; Lees, Hazel Anne; Chung, Hae-young; Giovannini, Silvia; Leeuwenburgh, Christiaan

    2008-01-01

    Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in...

  16. Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

    OpenAIRE

    Fricker, M; O'Prey, J.; Tolkovsky, A M; Ryan, K M

    2010-01-01

    Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first ...

  17. A Support Vector Machine Classification Model for Benzo[c]phenathridine Analogues with Topoisomerase-I Inhibitory Activity

    Directory of Open Access Journals (Sweden)

    Thanh-Dao Tran

    2012-04-01

    Full Text Available Benzo[c]phenanthridine (BCP derivatives were identified as topoisomerase I (TOP-I targeting agents with pronounced antitumor activity. In this study, a support vector machine model was performed on a series of 73 analogues to classify BCP derivatives according to TOP-I inhibitory activity. The best SVM model with total accuracy of 93% for training set was achieved using a set of 7 descriptors identified from a large set via a random forest algorithm. Overall accuracy of up to 87% and a Matthews coefficient correlation (MCC of 0.71 were obtained after this SVM classifier was validated internally by a test set of 15 compounds. For two external test sets, 89% and 80% BCP compounds, respectively, were correctly predicted. The results indicated that our SVM model could be used as the filter for designing new BCP compounds with higher TOP-I inhibitory activity.

  18. Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Pedersen, Mette Ø; Poulsen, Tim S; Gang, Anne O;

    2015-01-01

    Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number...... changes at relapse, with possible implications for relapse therapy efficacy. TOP gene alterations in lymphoma are poorly investigated. In this study, TOP1 and TOP2A gene alterations were investigated in patients with de novo diffuse large B-cell lymphoma (DLBCL) (n = 33) and relapsed DLBCL treated...... with chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13...

  19. Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso (NWU); (SKI)

    2010-10-22

    Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

  20. Discovery of membrane active benzimidazole quinolones-based topoisomerase inhibitors as potential DNA-binding antimicrobial agents.

    Science.gov (United States)

    Zhang, Ling; Addla, Dinesh; Ponmani, Jeyakkumar; Wang, Ao; Xie, Dan; Wang, Ya-Nan; Zhang, Shao-Lin; Geng, Rong-Xia; Cai, Gui-Xin; Li, Shuo; Zhou, Cheng-He

    2016-03-23

    A series of novel benzimidazole quinolones as potential antimicrobial agents were designed and synthesized. Most of the prepared compounds exhibited good or even stronger antimicrobial activities in comparison with reference drugs. The most potent compound 15m was membrane active and did not trigger the development of resistance in bacteria. It not only inhibited the formation of biofilms but also disrupted the established Staphylococcus aureus and Escherichia coli biofilms. It was able to inhibit the relaxation activity of E. coli topoisomerase IV at 10 μM concentration. Moreover, this compound also showed low toxicity against mammalian cells. Molecular modeling and experimental investigation of compound 15m with DNA suggested that this compound could effectively bind with DNA to form a steady 15m-DNA complex which might further block DNA replication to exert the powerful bioactivities.

  1. Inhibition of topoisomerase IIα activity in CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

    International Nuclear Information System (INIS)

    The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector and antimutagenic agent when it is administered 30 min prior to radiation exposure to Chinese hamster ovary Kl cells at a concentration of 4 mM. Under these exposure conditions, topoisomerase (topo) I and II activities and associated protein contents were measured in the K1 cell line using the DNA relaxation assay, the P4 unknotting assay, and immunoblotting, respectively. WR-1065 was ineffective in modifying topo I activity, but it did reduce topo IIa activity by an average of 50 percent. The magnitude of topo IIa protein content, however, was not affected by these exposure conditions. Cell cycle effects were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for a period of up to 6 h resulted in a buildup of cells in the G2 compartment. However, in contrast to topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotector agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of radiation protection attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by 2-[(aminopropyl)amino]ethanethiol on type II topoisomerase, which is involved in DNA synthesis

  2. Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie L Boerner

    Full Text Available Patients with metastatic triple-negative breast cancer (TNBC have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose polymerase (PARP, a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

  3. Topoisomerase I inhibitors, shikonin and topotecan, inhibit growth and induce apoptosis of glioma cells and glioma stem cells.

    Directory of Open Access Journals (Sweden)

    Feng-Lei Zhang

    Full Text Available Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

  4. PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy

    Directory of Open Access Journals (Sweden)

    Byeon HJ

    2015-01-01

    Full Text Available Hyeong Jun Byeon,1 Insoo Kim,1 Ji Su Choi,1 Eun Seong Lee,2 Beom Soo Shin,3 Yu Seok Youn11Department of Pharmaceutical Sciences, School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea; 2Division of Biotechnology, The Catholic University of Korea, Bucheon-si, Republic of Korea; 3Department of Pharmacy, College of Pharmacy, Catholic University of Daegu, Gyeongsan-si, Republic of KoreaAbstract: The aim of the current study was to investigate the antitumor potential of poly(D,L-lactic-co-glycolic acid microspheres (PLGA MSs containing polyethylene glycol (PEG-conjugated (PEGylated tumor necrosis factor–related apoptosis-inducing ligand (PEG-TRAIL. PEG-TRAIL PLGA MSs were prepared by using a water-in-oil-in-water double-emulsion method, and the apoptotic activities of supernatants released from the PLGA MSs at days 1, 3, and 7 were examined. The antitumor effect caused by PEG-TRAIL PLGA MSs was evaluated in pancreatic Mia Paca-2 cell-xenografted mice. PEG-TRAIL PLGA MS was found to be spherical and 14.4±1.06 µm in size, and its encapsulation efficiency was significantly greater than that of TRAIL MS (85.7%±4.1% vs 43.3%±10.9%, respectively. The PLGA MS gradually released PEG-TRAIL for 14 days, and the released PEG-TRAIL was shown to have clear apoptotic activity in Mia Paca-2 cells, whereas TRAIL released after 1 day had a negligible activity. Finally, PEG-TRAIL PLGA MS displayed remarkably greater antitumor efficacy than blank or TRAIL PLGA MS in Mia Paca-2 cell-xenografted mice in terms of tumor volume and weight, apparently due to increased stability and well-retained apoptotic activity of PEG-TRAIL in PLGA MS. We believe that this PLGA MS system, combined with PEG-TRAIL, should be considered a promising candidate for treating pancreatic cancer.Keywords: Poly(D,L-lactic-co-glycolic acid, controlled release, PEGylation, TRAIL, pancreatic cancer

  5. In vitro apoptotic cell death during erythroid differentiation.

    Science.gov (United States)

    Zamai, L; Burattini, S; Luchetti, F; Canonico, B; Ferri, P; Melloni, E; Gonelli, A; Guidotti, L; Papa, S; Falcieri, E

    2004-03-01

    Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro. PMID:15004520

  6. PARP Inhibition Restores Extrinsic Apoptotic Sensitivity in Glioblastoma

    Science.gov (United States)

    Karpel-Massler, Georg; Pareja, Fresia; Aimé, Pascaline; Shu, Chang; Chau, Lily; Westhoff, Mike-Andrew; Halatsch, Marc-Eric; Crary, John F.; Canoll, Peter; Siegelin, Markus D.

    2014-01-01

    Background Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM). We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281) or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. Methods The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. Results The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with up-regulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP) homology protein (CHOP). Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. Conclusions PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM. PMID:25531448

  7. PARP inhibition restores extrinsic apoptotic sensitivity in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Georg Karpel-Massler

    Full Text Available BACKGROUND: Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM. We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281 or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. METHODS: The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. RESULTS: The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with up-regulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP homology protein (CHOP. Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. CONCLUSIONS: PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM.

  8. Docking studies, synthesis, characterization of some novel oxazine substituted 9-anilinoacridine derivatives and evaluation for their antioxidant and anticancer activities as topoisomerase II inhibitors.

    Science.gov (United States)

    Kalirajan, R; Kulshrestha, Vivek; Sankar, S; Jubie, S

    2012-10-01

    A series of 9-anilinoacridines substituted with oxazine derivatives were synthesized to evaluate their antioxidant and anticancer activity against Daltons Lymphoma Ascites (DLA) cell growth by in vitro method. It was revealed that these conjugates exhibited significant antioxidant and anticancer activity (inhibition of DLA cell proliferation). Among these agents, compounds 5a, 5h, 5i, 5j were the most cytotoxic with CTC(50) value of 140-250 μg/mL. The docking studies of the synthesized compounds were performed towards the key Topoisomerase II (1QZR) by using Schrodinger Maestro 9.2 version. The oxazine substituted 9-anilinoacridine derivatives 5a, 5h, 5i, 5j have significant anticancer activity as topoisomerase II inhibitors. PMID:22982526

  9. A murine experimental anthracycline extravasation model: pathology and study of the involvement of topoisomerase II alpha and iron in the mechanism of tissue damage

    DEFF Research Database (Denmark)

    2010-01-01

    The bisdioxopiperazine topoisomerase II catalytic inhibitor dexrazoxane has successfully been introduced into the clinic as an antidote to accidental anthracycline extravasation based on our preclinical mouse studies. The histology of this mouse extravasation model was investigated and found...... with dense dermal connective tissue. The extension of this fibrosis was quantified, and dexrazoxane intervention resulted in a statistically significant decrease in fibrosis extension, as also observed in the clinic. Several mechanisms have been proposed in anthracycline extravasation cytotoxicity, and we...... of topoisomerase II alpha and thereby prevents access of anthracycline to the enzyme and thus cytotoxicity, and also acts as a strong iron chelator following opening of its two bisdioxopiperazine rings. Using the model of extravasation in a dexrazoxane-resistant transgenic mouse with a heterozygous mutation...

  10. Assessment of the topoisomerase I gene copy number as a predictive biomarker of objective response to irinotecan in metastatic colorectal cancer

    DEFF Research Database (Denmark)

    Nygård, Sune Boris; Christensen, Ib Jarle; Nielsen, Signe Lykke;

    2014-01-01

    Abstract Objective. DNA topoisomerase I is a putative biomarker of irinotecan efficacy with clinical associations previously demonstrated at the protein level. The purpose of the present study was to perform the first clinical investigation of the association between the DNA topoisomerase I gene...... (TOP1) copy number and objective response following irinotecan treatment in patients with metastatic colorectal cancer. Materials and methods. Formalin-fixed, paraffin-embedded tumor samples from 78 patients, who received irinotecan monotherapy in second line, were included. TOP1 was assessed....... Despite limitations of the study the positive associations between TOP1 and objective response suggest that further analysis in larger tumor material, preferably in a randomized setting, is highly warranted....

  11. Pro-apoptotic gene regulation in the Caribbean fruit fly, Anastrepha suspensa

    Science.gov (United States)

    Transcriptional activation of pro-apoptotic genes in response to cytotoxic stimuli is a conserved feature of the cell death pathway proposed for metazoans. However, understanding the extent of this conservation in insects, as well as other organisms, has been limited by the lack of known pro-apoptot...

  12. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

    Directory of Open Access Journals (Sweden)

    Hafner Martin

    2004-08-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. Results Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. Conclusion Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance.

  13. Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations

    OpenAIRE

    Jensen, Niels Frank; Agama, Keli; Roy, Amit; Smith, David Hersi; Pfister, Thomas D.; Rømer, Maria Unni; Zhang, Hong-Liang; Doroshow, James H.; Knudsen, Birgitta R.; Stenvang, Jan; Brünner, Nils; Pommier, Yves

    2016-01-01

    Background DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resis...

  14. Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

    OpenAIRE

    Caroline Ballot; Alain Martoriati; Manel Jendoubi; Sébastien Buche; Pierre Formstecher; Laurent Mortier; Jérome Kluza; Philippe Marchetti

    2014-01-01

    Lamellarin D (LamD) is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2) and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytosta...

  15. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides

    OpenAIRE

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B.

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents ‘poison’ the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called tre...

  16. The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

    OpenAIRE

    Nassima Temime-Smaali; Lionel Guittat; Assitan Sidibe; Kazuo Shin-ya; Chantal Trentesaux; Jean-François Riou

    2009-01-01

    In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIalpha (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was s...

  17. Genetic resistance determinants, in vitro time-kill curve analysis and pharmacodynamic functions for the novel topoisomerase II inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae

    OpenAIRE

    Sunniva eFoerster; Daniel eGolparian; Susanne eJacobsson; Lucy eHathaway; Nicola eLow; William eShafer; Christian eAlthaus; Magnus eUnemo

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the gro...

  18. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

    OpenAIRE

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J; Low, Nicola; Shafer, William M.; Althaus, Christian L.; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the gro...

  19. Identification of cyclohexanone derivatives that act as catalytic inhibitors of topoisomerase I: effects on tamoxifen-resistant MCF-7 cancer cells.

    Science.gov (United States)

    Leung, Euphemia; Rewcastle, Gordon W; Joseph, Wayne R; Rosengren, Rhonda J; Larsen, Lesley; Baguley, Bruce C

    2012-12-01

    Breast cancer is commonly treated with anti-estrogens or aromatase inhibitors, but resistant disease eventually develops and new therapies for such resistance are of great interest. We have previously isolated several tamoxifen-resistant variant sub-lines of the MCF-7 breast cancer cell line and provided evidence that they arose from expansion of pre-existing minor populations. We have searched for therapeutic agents that exhibit selective growth inhibition of the resistant lines and here investigate 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91). We found that two of the tamoxifen-resistant sub-lines (TamR3 and TamC3) unexpectedly showed increased sensitivity to RL90 and RL91. We utilized growth inhibition assays, flow cytometry and immunoblotting to establish a mechanistic basis for their action. Treated sensitive cells showed S-phase selective DNA damage, as detected by histone H2AX phosphorylation. Cellular responses were similar to those induced by the topoisomerase I poison camptothecin. Although IC(50) values of camptothecin, RL90, RL91 were correlated, studies with purified mammalian topoisomerase I suggested that RL90 and RL91 differed from camptothecin by acting as catalytic topoisomerase I inhibitors. These drugs provide a platform for the further development of DNA damaging drugs that have selective effects on tamoxifen resistant breast cancer cells. The results also raise the question of whether clinical topoisomerase I poisons such as irinotecan and topotecan might be active in the treatment of some types of tamoxifen-resistant cancer.

  20. Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants

    OpenAIRE

    Sirikantaramas, Supaart; Yamazaki, Mami; Saito, Kazuki

    2008-01-01

    Plants produce a variety of toxic compounds, which are often used as anticancer drugs. The self-resistance mechanism to these toxic metabolites in the producing plants, however, remains unclear. The plant-derived anticancer alkaloid camptothecin (CPT) induces cell death by targeting DNA topoisomerase I (Top1), the enzyme that catalyzes changes in DNA topology. We found that CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila, and Ophiorrhiza liukiuensis, have Top1s with ...

  1. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H. [School of Chemistry, University of Edinburgh, The King' s Buildings, Edinburgh, EH9 3JJ (United Kingdom); Dryden, David T.F., E-mail: david.dryden@ed.ac.uk [School of Chemistry, University of Edinburgh, The King' s Buildings, Edinburgh, EH9 3JJ (United Kingdom)

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  2. Role of Topoisomerases in Pediatric High Grade Osteosarcomas: TOP2A Gene Is One of the Unique Molecular Biomarkers of Chemoresponse

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Aurelia; Lasthaus, Christelle; Guerin, Eric [Laboratoire de Biochimie et Biologie Moléculaire, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France); EA4438, Groupe Marqueurs Moléculaires de Progression Tumorale et de Sensibilisation aux Drogues Anti-Cancéreuses, University of Strasbourg, 3 Avenue Molière, Strasbourg 67000 (France); Marcellin, Luc [Laboratoire d’Anatomie Pathologique, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France); Pencreach, Erwan; Gaub, Marie-Pierre [Laboratoire de Biochimie et Biologie Moléculaire, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France); EA4438, Groupe Marqueurs Moléculaires de Progression Tumorale et de Sensibilisation aux Drogues Anti-Cancéreuses, University of Strasbourg, 3 Avenue Molière, Strasbourg 67000 (France); Guenot, Dominique [Laboratoire de Biochimie et Biologie Moléculaire, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France); Entz-Werle, Natacha, E-mail: Natacha.entz-werle@chru-strasbourg.fr [Laboratoire de Biochimie et Biologie Moléculaire, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France); Service de Pédiatrie Onco-Hématologie, CHRU Hautepierre, Avenue Molière, Strasbourg Cedex 67098 (France)

    2013-06-04

    Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness.

  3. Role of Topoisomerases in Pediatric High Grade Osteosarcomas: TOP2A Gene Is One of the Unique Molecular Biomarkers of Chemoresponse

    International Nuclear Information System (INIS)

    Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness

  4. The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster.

    Science.gov (United States)

    Rahden-Staron, Iwonna

    2002-07-25

    In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10-20% inhibition of activity in the range of 10-100microM). In contrast, both chemicals decreased the activity of topoisomerase II already at 1microM concentration (50 and 20% inhibition of activity by captafol and captan, respectively).Genotoxicity was tested in vivo by administrating both compounds by acute (3h) and chronic feeding (48h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10-100mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides.

  5. Discovery of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine as a non-intercalative DNA-binding topoisomerase II-specific catalytic inhibitor.

    Science.gov (United States)

    Jun, Kyu-Yeon; Kwon, Hanbyeol; Park, So-Eun; Lee, Eunyoung; Karki, Radha; Thapa, Pritam; Lee, Jun-Ho; Lee, Eung-Seok; Kwon, Youngjoo

    2014-06-10

    We describe our rationale for designing specific catalytic inhibitors of topoisomerase II (topo II) over topoisomerase I (topo I). Based on 3D-QSAR studies of previously published dihydroxylated 2,4-diphenyl-6-aryl pyridine derivatives, 9 novel dihydroxylated 2,4-diphenyl-6-thiophen-2-yl pyridine compounds were designed, synthesized, and their biological activities were evaluated. These compounds have 2-thienyl ring substituted on the R(3) group on the pyridine ring and they all showed excellent specificity toward topo II compared to topo I. In vitro experiments were performed for compound 13 to determine the mechanism of action for this series of compounds. Compound 13 inhibited topoisomerase II specifically by non-intercalative binding to DNA and did not stabilize enzyme-cleavable DNA complex. Compound 13 efficiently inhibited cell viability, cell migration, and induced G1 arrest. Also from 3D-QSAR studies, the results were compared with other previously published dihydroxylated 2,4-diphenyl-6-aryl pyridine derivatives to explain the structure-activity relationships.

  6. Role of Topoisomerases in Pediatric High Grade Osteosarcomas: TOP2A Gene Is One of the Unique Molecular Biomarkers of Chemoresponse

    Directory of Open Access Journals (Sweden)

    Natacha Entz-Werle

    2013-06-01

    Full Text Available Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness.

  7. Thyroid hormone regulation of apoptotic tissue remodeling during anuran metamorphosis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Anuran metamorphosis involves systematic transformations of individual organs in a thyroid hormone (TH)-dependent manner. Morphological and cellular studies have shown that the removal of larval or gans/tissues such the tail and the tadpole intestinal epithelium is through programmed cell death or apop tosis. Recent molecular investigations suggest that TH regulates metamorphosis by regulating target gene expression through thyroid hormone receptors (TRs), which are DNA-binding transcription factors. Cloning and characterization of TH response genes show that diverse groups of early response genes are induced by TH. The products of these TH response genes are believed to directly or indirectly affect the expression and/or functions of cell death genes, which are conserved at both sequence and function levels in different animal species. A major challenge for future research lies at determining the signaling pathways leading to the activation of apoptotic processes and whether different death genes are involved in the regulation of apoptosis in different tissues/organs to effect tissue-specific transformations.

  8. Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Noelia Estévez-Calvar

    Full Text Available Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.

  9. Genotoxic and apoptotic effects of Goeckerman therapy for psoriasis

    Energy Technology Data Exchange (ETDEWEB)

    Borska, L.; Andrys, C.; Krejsek, J.; Hamakova, K.; Kremlacek, J.; Palicka, V.; Ranna, D.; Fiala, Z. [Charles University Prague, Prague (Czech Republic). Faculty of Medicine

    2010-03-15

    Goeckerman therapy (GT) for psoriasis is based on cutaneous application of crude coal tar (polycyclic aromatic hydrocarbons (PAH)) and exposure to ultraviolet radiation (UVR). PAH and UVR are mutagenic, carcinogenic and immunotoxic agents that promote apoptosis. We evaluated dermal absorption of PAH as well as the genotoxic and apoptotic effects of GT in 20 patients with psoriasis, by determining numbers of chromosomal abnormalities in peripheral lymphocytes, and levels of 1-hydroxypyrene (1-OHP), p53 protein and soluble FasL (sFasL) in urine and/or blood, before and after GT. Psoriasis Area and Severity Index (PASI) score was used to evaluate clinical efficacy of GT. Compared with pre-treatment levels, there was a significant increase in urine 1-OHP, indicating a high degree of dermal absorption of PAH (P <0.01). We also found a significant increase in the number of chromosomal abnormalities in peripheral blood lymphocytes (P <0.001), suggesting that GT is genotoxic; significantly increased p53 protein in plasma (P <0.05), an indicator of cell response to DNA damage; and significantly increased sFasL in serum (P <0.01), an indicator of apoptosis. The PASI score was significantly decreased after GT (P <0.001), confirming clinical benefit of this treatment. Our results demonstrate high dermal absorption of PAH during GT and provide evidence that GT promotes genotoxicity and apoptosis.

  10. Non-apoptotic cell death associated with perturbations of macropinocytosis

    Directory of Open Access Journals (Sweden)

    William A. Maltese

    2015-02-01

    Full Text Available Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed ‘methuosis’, from the Greek methuo (to drink to intoxication. It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

  11. Bak apoptotic function is not directly regulated by phosphorylation.

    Science.gov (United States)

    Tran, V H; Bartolo, R; Westphal, D; Alsop, A; Dewson, G; Kluck, R M

    2013-01-01

    During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation. PMID:23303126

  12. Multimodal Interaction with BCL-2 Family Proteins Underlies the Pro-Apoptotic Activity of PUMA BH3

    OpenAIRE

    Edwards, Amanda L.; Gavathiotis, Evripidis; LaBelle, James L.; Braun, Craig R.; Opoku-Nsiah, Kwadwo A.; Bird, Gregory H.; Walensky, Loren D.

    2013-01-01

    PUMA is a pro-apoptotic BCL-2 family member that drives the apoptotic response to a diversity of p53-dependent and independent cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent pro-apoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally-stabilized PUMA BH3 helices that, in addition to broadly targeting anti-apoptotic proteins, directly bind to BAX. NMR, photocrosslinking, and biochemical analy...

  13. The replication of human immunodeficiency virus type 1 in macrophages is enhanced after phagocytosis of apoptotic cells

    OpenAIRE

    Lima, Rosangela G; Van Weyenbergh, Johan; Saraiva, Elvira M. B.; Barral-Netto, Manoel; Galvão-Castro, Bernardo; Bou-Habib, Dumith Chequer

    2002-01-01

    Clearance of apoptotic cells increases macrophage secretion of antiinflammatory mediators and might modulate viral replication in human immunodeficiency virus (HIV) type 1-infected macrophages. To study this, primary macrophages were infected with HIV-1 and exposed to apoptotic cells. It was found that phagocytosis of apoptotic cells potently enhanced HIV-1 growth. The peptide Arg-Gly-Asp-Ser, which binds to integrin receptors, inhibited the uptake of apoptotic cells and the subsequent enhanc...

  14. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.

    Science.gov (United States)

    Srivastava, M; Scherr, H; Lackey, M; Xu, D; Chen, Z; Lu, J; Bergmann, A

    2007-01-01

    In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.

  15. Quinoline-2-carboxaldehyde thiosemicarbazones and their Cu(II) and Ni(II) complexes as topoisomerase IIa inhibitors.

    Science.gov (United States)

    Bisceglie, Franco; Musiari, Anastasia; Pinelli, Silvana; Alinovi, Rossella; Menozzi, Ilaria; Polverini, Eugenia; Tarasconi, Pieralberto; Tavone, Matteo; Pelosi, Giorgio

    2015-11-01

    A series of quinoline-2-carboxaldehyde thiosemicarbazones and their copper(II) and nickel(II) complexes were synthesized and characterized. In all complexes the ligands are in the E configuration with respect to the imino bond and behave as terdentate. The copper(II) complexes form square planar derivatives with one molecule of terdentate ligand and chloride ion. A further non-coordinated chloride ion compensates the overall charge. Nickel(II) ions form instead octahedral complexes with two ligands for each metal ion, independently from the stoichiometric metal:ligand ratio used in the synthesis. Ligands and complexes were tested for their antiproliferative properties on histiocytic lymphoma cell line U937. Copper(II) derivatives are systematically more active than the ligands and the nickel complexes. All copper derivatives result in inhibiting topoisomerase IIa in vitro. Computational methods were used to propose a model to explain the different extent of inhibition presented by these compounds. The positive charge of the dissociated form of the copper complexes may play a key role in their action. PMID:26335598

  16. The role of the Zn(II binding domain in the mechanism of E. coli DNA topoisomerase I

    Directory of Open Access Journals (Sweden)

    Tse-Dinh Yuk-Ching

    2002-05-01

    Full Text Available Abstract Background Escherichia coli DNA topoisomerase I binds three Zn(II with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain. The 67 kDa N-terminal domain (Top67 has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism. Results Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain. Conclusions We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.

  17. Hypoxia-Targeted Drug Q6 Induces G2-M Arrest and Apoptosis via Poisoning Topoisomerase II under Hypoxia.

    Directory of Open Access Journals (Sweden)

    Linlin Chang

    Full Text Available In spite of the tremendous efforts dedicated to developing hypoxia-activated prodrugs, no agents yet have been approved for clinical therapy. In the present study, the hypoxic selective anti-cancer activity as well as the cellular target of a novel tirapazamine (TPZ analogue, 7-methyl-3-(3-chlorophenyl-quinoxaline-2-carbonitrile 1,4-dioxide (Q6 were investigated. Q6 implemented anti-cancer effects via poisoning topoisomerase II (topo II under hypoxia. Modified trapped in agarose DNA immunostaining (TARDIS assay showed more topo II-DNA cleavage complexes trapped by Q6 than TPZ at even lower concentration. In addition, by introducing ataxia-telangiectasia-mutated (ATM kinase inhibitors caffeine and KU-60019, we displayed that Q6-triggered apoptosis was attributed, at least partially, to DNA double-strand breaks generated by the topo II-targeting effect. Collectively, Q6 stood out for its better hypoxia-selectivity and topo II-poisoning than the parental compound TPZ. All these data shed light on the research of Q6 as a promising hypoxia-activated prodrug candidate for human hepatocellular carcinoma therapy.

  18. Synthesis of novel naphthoquinone-spermidine conjugates and their effects on DNA-topoisomerases I and II-α

    International Nuclear Information System (INIS)

    Novel derivatives of lapachol 2, nor-lapachol 3 and lawsone 4 have been synthesized by nucleophilic displacement of the methoxynaphthoquinones 2a, 3a and 4a with the polyamine (PA) N1-Boc-N5-Bn-spermidine 1a. The respective products 2b-4b were obtained in good yields and characterized by spectroscopic and analytical methods. The inhibitory action of these naphthoquinone-PA conjugates on DNA-topoisomerases (topo) I and II-α was evaluated by relaxation assay of supercoiled DNA plasmid. All compounds (1a 2b, 3b and 4b) presented significant inhibition of topo II-α catalytic activity at the 2 μM dose. Considering that only PA 1a did not inhibit the enzyme catalytic activity at the 0.2 μM dose, the appended naphthoquinone moiety acts as a 'value added' fragment. Compounds 1a 2b, 3b and 4b did not inhibit the enzyme DNA-topo I at the 200 μM dose. (author)

  19. Synthesis and biological evaluation of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives as potential topoisomerase I inhibitors.

    Science.gov (United States)

    Ge, Raoling; Zhao, Qian; Xie, Zhouling; Lu, Lu; Guo, Qinglong; Li, Zhiyu; Zhao, Li

    2016-10-21

    The design and synthesis of a new series of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives, built on the structure of 1-ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4(1H)-quinolone, are described. These compounds provide new scaffold for the discovery of Topoisomerase I (Top I) inhibitors and target based assay showed that they can obviously inhibited Top I at 100 μM. The in vitro anti-proliferative activity of these new compounds was evaluated against A549, Hela, BGC-823, and HepG2 cell lines. Compounds 18a-g showed potent inhibitory activity against the growth of those cancer cell lines. The most positive compounds 18f and 18g demonstrated as potent as camptothecin in Top I inhibition assay and MTT assay. Compounds 18f and 18g led to an obvious increase in the percentage of S phase of the cells in 24 h. The in vivo data showed that 18f and 18g inhibited tumor growth with the inhibitory rate of 29.25% and 42.75% at 20 mg/kg, respectively. The data suggested the therapeutic potential for further development. PMID:27416553

  20. Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases α and β

    International Nuclear Information System (INIS)

    Inhibitors of (a) DNA topoisomerases (novobiocin and nalidixic acid) and of (b) eukaryotic DNA polymerases α (cytosine arabinoside) and β (dideoxythymidine) blocked different steps of DNA repair, demonstrated by the effects of the inhibitors on the relaxation of supercoiled DNA nucleoids following treatment of human cell cultures with ultraviolet light (1-3 J/m2) or MNNG (5 or 20 μM) and the subsequent restoration of the supercoiled nucleoids during repair incubation. Inhibition of repair by novobiocin was partially reversible; upon its removal from the culture medium, the nucleoid DNA of repairing cells became relaxed. The DNA polymerase inhibitors allowed the initial relaxation of DNA after treatment of the cells with ultraviolet or MNNG but delayed the regeneration of rapidly-sedimenting (supercoiled) nucleoid DNA for 2-4 h. Dideoxythymidine (1 mM) was more effective than cytosine arabinoside (1 μM) in producing this delay, but neither inhibitor by itself blocked repair permanently. Incubation of ultraviolet-irradiated cells with 1 μM cytosine arabinoside plus 1 mM dideoxythymidine blocked the completion of repair for 24 h, whereas incubation with 10 μM cytosine arabinoside or 5 mM dideoxythymidine produced only temporary repair delays of 2-4 h. Thus, it is likely that the two DNA polymerase inhibitors act upon separate targets and that both targets are involved in repair. (Auth.)

  1. Synthesis of novel naphthoquinone-spermidine conjugates and their effects on DNA-topoisomerases I and II-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Andrea S.; Lima, Edson L.S.; Pinto, Angelo C.; Esteves-Souza, Andressa; Torrese, Jose C. [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Quimica; Echevarria, Aurea [Universidade Federal Rural do Rio de Janeiro, RJ (Brazil). Dept. de Quimica; Camara, Celso A. [Paraiba Univ., Joao Pessoa, PB (Brazil). Lab. de Tecnologia Farmaceutica; Vargas, Maria D. [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Inst. de Quimica]. E-mail: mdvargas@vm.uff.br

    2006-05-15

    Novel derivatives of lapachol 2, nor-lapachol 3 and lawsone 4 have been synthesized by nucleophilic displacement of the methoxynaphthoquinones 2a, 3a and 4a with the polyamine (PA) N{sup 1}-Boc-N{sup 5}-Bn-spermidine 1a. The respective products 2b-4b were obtained in good yields and characterized by spectroscopic and analytical methods. The inhibitory action of these naphthoquinone-PA conjugates on DNA-topoisomerases (topo) I and II-{alpha} was evaluated by relaxation assay of supercoiled DNA plasmid. All compounds (1a 2b, 3b and 4b) presented significant inhibition of topo II-{alpha} catalytic activity at the 2 {mu}M dose. Considering that only PA 1a did not inhibit the enzyme catalytic activity at the 0.2 {mu}M dose, the appended naphthoquinone moiety acts as a 'value added' fragment. Compounds 1a 2b, 3b and 4b did not inhibit the enzyme DNA-topo I at the 200 {mu}M dose. (author)

  2. Gene delivery of the elastase inhibitor elafin protects macrophages from neutrophil elastase-mediated impairment of apoptotic cell recognition.

    Science.gov (United States)

    Henriksen, Peter A; Devitt, Andrew; Kotelevtsev, Yuri; Sallenave, Jean-Michel

    2004-09-10

    The resolution of inflammation is dependent on recognition and phagocytic removal of apoptotic cells by macrophages. Receptors for apoptotic cells are sensitive to degradation by human neutrophil elastase (HNE). We show in the present study that HNE cleaves macrophage cell surface CD14 and in so doing, reduces phagocytic recognition of apoptotic lymphocytic cells (Mutu 1). Using an improved method of adenovirus-mediated transfection of macrophages with the HNE inhibitor elafin, we demonstrate that elafin overexpression prevents CD14 cleavage and restores apoptotic cell recognition by macrophages. This approach of genetic modification of macrophages could be used to restore apoptotic cell recognition in inflammatory conditions. PMID:15358543

  3. Phototherapy-treated apoptotic tumor cells induce pro-inflammatory cytokines production in macrophage

    Science.gov (United States)

    Lu, Cuixia; Wei, Yanchun; Xing, Da

    2014-09-01

    Our previous studies have demonstrated that as a mitochondria-targeting cancer phototherapy, high fluence low-power laser irradiation (HF-LPLI) induces mitochondrial superoxide anion burst, resulting in oxidative damage to tumor cells. In this study, we further explored the immunological effects of HF-LPLI-induced apoptotic tumor cells. When macrophages were co-incubated with apoptotic cells induced by HF-LPLI, we observed the increased levels of TNF-α secretion and NO production in macrophages. Further experiments showed that NF-κB was activated in macrophages after co-incubation with HF-LPLI-induced apoptotic cells, and inhibition of NF-κB activity by pyrrolidinedithiocarbamic acid (PDTC) reduced the elevated levels of TNF-α secretion and NO production. These data indicate that HF-LPLI-induced apoptotic tumor cells induce the secretion of pro-inflammatory cytokines in macrophages, which may be helpful for better understanding the biological effects of cancer phototherapy.

  4. Adiponectin modulates inflammatory reactions via calreticulin receptor–dependent clearance of early apoptotic bodies

    OpenAIRE

    Takemura, Yukihiro; Ouchi, Noriyuki; Shibata, Rei; Aprahamian, Tamar; Kirber, Michael T.; Summer, Ross S; Kihara, Shinji; Walsh, Kenneth

    2007-01-01

    Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone tr...

  5. Decreased Apoptotic Rate of Alveolar Macrophages of Patients with Idiopathic Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Fotios Drakopanagiotakis

    2012-01-01

    and control group. No difference was found between the respiratory function parameters of the two treatment groups after six months. A positive correlation was found between the number of bcl-2 positive stained macrophages and DLCO after treatment. Conclusions. The decreased apoptotic rate of AM of patients with IPF is not associated with decreased expression of apoptosis mediators involved in the external or internal apoptotic pathway.

  6. The role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.

    Science.gov (United States)

    Grabiec, Aleksander M; Hussell, Tracy

    2016-07-01

    Acute and chronic inflammatory responses in the lung are associated with the accumulation of large quantities of immune and structural cells undergoing apoptosis, which need to be engulfed by phagocytes in a process called 'efferocytosis'. Apoptotic cell recognition and removal from the lung is mediated predominantly by airway macrophages, though immature dendritic cells and non-professional phagocytes, such as epithelial cells and mesenchymal cells, can also display this function. Efficient clearance of apoptotic cells from the airways is essential for successful resolution of inflammation and the return to lung homeostasis. Disruption of this process leads to secondary necrosis of accumulating apoptotic cells, release of necrotic cell debris and subsequent uncontrolled inflammatory activation of the innate immune system by the released 'damage associated molecular patterns' (DAMPS). To control the duration of the immune response and prevent autoimmune reactions, anti-inflammatory signalling cascades are initiated in the phagocyte upon apoptotic cell uptake, mediated by a range of receptors that recognise specific phospholipids or proteins externalised on, or secreted by, the apoptotic cell. However, prolonged activation of apoptotic cell recognition receptors, such as the family of receptor tyrosine kinases Tyro3, Axl and MerTK (TAM), may delay or prevent inflammatory responses to subsequent infections. In this review, we will discuss recent advances in our understanding of the mechanism controlling apoptotic cell recognition and removal from the lung in homeostasis and during inflammation, the contribution of defective efferocytosis to chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease, asthma and cystic fibrosis, and implications of the signals triggered by apoptotic cells in the susceptibility to pulmonary microbial infections. PMID:26957481

  7. Salivary apoptotic cells in oral (pre-) cancer as a potential diagnostic means

    OpenAIRE

    Kaur, Jasdeep; Politis, Constantinus; Jacobs, Reinhilde

    2015-01-01

    Background Apoptosis is a genetically programmed form of cell death which is indispensable for development and homeostasis of multi-cellular organism. Objectives The aim of this study was to find out the salivary apoptotic cells in oral precancerous and cancerous patients and furthermore to observe the potential diagnostic value of salivary apoptotic cells in detection of oral pre-cancer and cancer. Material and Methods Unsimulated saliva was collected from a group of 103 subjects diagnosed w...

  8. Effect of Transient Maternal Hypotension on Apoptotic Cell Death in Foetal Rat Brain

    OpenAIRE

    Özyürek, Hamit; Bayrak, Sibel; Pehlivanoğlu, Bilge; Atilla, Pergin; Balkancı, Zeynep Dicle; Çakar, Nur; Anlar, Banu

    2014-01-01

    Background: Intrauterine perfusion insufficiency induced by transient maternal hypotension has been reported to be associated with foetal brain malformations. However, the effects of maternal hypotension on apoptotic processes in the foetal brain have not been investigated experimentally during the intrauterine period. Aims: The aim of this study was to investigate the effects of transient maternal hypotension on apoptotic cell death in the intrauterine foetal brain. Study...

  9. Apoptotic-like programed cell death in fungi: the benefits in filamentous species

    OpenAIRE

    Shlezinger, Neta; Goldfinger, Nir; Sharon, Amir

    2012-01-01

    Studies conducted in the early 1990s showed for the first time that Saccharomyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well-documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologs of human apoptotic genes have been identified and characterized. These studies also revealed fund...

  10. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination.

    Directory of Open Access Journals (Sweden)

    Hiroshi Udo

    Full Text Available One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter. Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I-mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15 resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75% and directional cloning (99% by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.

  11. Another Facet to the Anticancer Response to Lamellarin D: Induction of Cellular Senescence through Inhibition of Topoisomerase I and Intracellular Ros Production

    Directory of Open Access Journals (Sweden)

    Caroline Ballot

    2014-01-01

    Full Text Available Lamellarin D (LamD is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2 and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytostatic effect have not yet been characterized. Here, we demonstrated that induction of cellular senescence (depicted by cell cycle arrest in G2 associated with β-galactosidase activity is a common response to subtoxic concentrations of LamD. Cellular senescence is observed in a large panel of cancer cells following in vitro or in vivo exposure to LamD. The onset of cellular senescence is dependent on the presence of intact topoisomerase I since topoisomerase I-mutated cells are resistant to senescence induced by LamD. LamD-induced senescence occurs without important loss of telomere integrity. Instead, incubation with LamD results in the production of intracellular reactive oxygen species (ROS, which are critical for senescence as demonstrated by the inhibitory effect of antioxidants. In addition, cancer cells lacking mitochondrial DNA also exhibit cellular senescence upon LamD exposure indicating that LamD can trigger senescence, unlike apoptosis, in the absence of functional mitochondria. Overall, our results identify senescence-associated growth arrest as a powerful effect of LamD and add compelling evidence for the pharmacological interest of lamellarins as potential anticancer agents.

  12. Topoisomerase 3alpha and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frank Hartung

    2008-12-01

    Full Text Available Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3alpha (TOP3alpha functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3alpha in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3alpha-2, which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3alpha-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3alpha, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3alpha is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3alpha mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3alpha and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.

  13. An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

    OpenAIRE

    Udo, Hiroshi

    2015-01-01

    One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as...

  14. Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort

    OpenAIRE

    David Hersi Smith; Ib Jarle Christensen; Niels Frank Jensen; Bo Markussen; Maria Unni Rømer; Sune Boris Nygård; Sven Müller; Hans Jørgen Nielsen; Nils Brünner; Kirsten Vang Nielsen

    2013-01-01

    BACKGROUND: Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH). METHODS: Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a re...

  15. The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα

    OpenAIRE

    Yalowich, Jack C.; Wu, Xing; Zhang, Rui; Kanagasabai, Ragu; Hornbaker, Marissa; Hasinoff, Brian B

    2012-01-01

    The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-957, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of as...

  16. Inhibition of DNA Topoisomerase Type IIα (TOP2A by Mitoxantrone and Its Halogenated Derivatives: A Combined Density Functional and Molecular Docking Study

    Directory of Open Access Journals (Sweden)

    Md. Abu Saleh

    2016-01-01

    Full Text Available In this study, mitoxantrone and its halogenated derivatives have been designed by density functional theory (DFT to explore their structural and thermodynamical properties. The performance of these drugs was also evaluated to inhibit DNA topoisomerase type IIα (TOP2A by molecular docking calculation. Noncovalent interactions play significant role in improving the performance of halogenated drugs. The combined quantum and molecular mechanics calculations revealed that CF3 containing drug shows better preference in inhibiting the TOP2A compared to other modified drugs.

  17. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation

    OpenAIRE

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2015-01-01

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded...

  18. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    OpenAIRE

    Hong Yan; Margaret Tammaro; Shuren Liao

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-prot...

  19. Characterization of DNA topoisomerase-1 in Spodoptera exigua for toxicity evaluation of camptothecin and hydoxy-camptothecin.

    Directory of Open Access Journals (Sweden)

    Lan Zhang

    Full Text Available Camptothecin (CPT, a plant alkaloid originally isolated from the native Chinese tree, Camptotheca acuminate, exerts the toxic effect by targeting eukaryotic DNA topoisomerase 1 (DNA Topo1. Besides as potent anti-cancer agents, CPT and its derivatives are now being explored as potential pesticides for insect control. In this study, we assessed their toxicity to an insect homolog, the Topo1 protein from beet armyworms (Spodoptera exigua Hübner, a worldwide pest of many important crops. The S. exigua Topo1 gene contains an ORF of 2790 base pairs that is predicted to encode a polypeptide of 930 amino acids. The deduced polypeptide exhibits polymorphism at residue sites V420, L530, A653 and T729 (numbered according to human Topo1 among insect species, which are predicted to confer sensitivity to CPT. The DNA relaxation activity of this protein was subsequently examined using a truncated form that contained the residues 337-930 and was expressed in bacteria BL21 cells. The purified protein retained the ability to relax double-stranded DNA and was susceptible to CPT and its derivative hydroxy-camptothecin (HCPT in a dose-dependent manner. The same inhibitory effect was also found on the native Topo1 extracted from IOZCAS-Spex-II cells, a cell line established from beet armyworms. Additionally, CPT and HCPT treatment reduced the steady accumulation of Topo1 protein despite the increased mRNA expression in response to the treatment. Our studies provide information of the S. exigua Topo1 gene and its amino acid polymorphism in insects and uncover some clues about potential mechanisms of CPT toxicity against insect pests. These results also are useful for development of more effective Topo1-targeted CPT insecticides in the future.

  20. Identification and characterization of the regions involved in the nuclear translocation of the heterodimeric leishmanial DNA topoisomerase IB.

    Directory of Open Access Journals (Sweden)

    Christopher F Prada

    Full Text Available Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique heterodimeric DNA-topoisomerase IB (LdTopIB. LdTopIB is a heterodimer made up of a large subunit and a small subunit that must interact with each other to build an active enzyme able to solve the topological tensions on the DNA. As LdTopIB is located within the nucleus, one or more nuclear localization signals (NLS should exist to ensure its nuclear translocation. In this report three novel NLS have been identified through a sequential deletion study of the genes encoding of both subunits fused to that encoding the green fluorescent protein (GFP. NLS1 is a highly basic sequence of 43 amino acids in the C-terminal extension of the large protomer. We found two well-defined sequences in the small protomer: NLS2 is a 10-amino acid motif located in the N-terminal extension of the protein; NLS3 consists of a complex region of 28 amino acids placed in the vicinity of the catalytic Tyr-222 included at the conserved SKINY signature within the C-terminal. Furthermore, by means of yeast cell viability assays, conducted with several LdTopIB chimeras lacking any of the NLS motives, we have revealed that both subunits are transported independently to the nucleus. There was no evidence of LdTopIB accumulation in mitochondria or association to the kinetoplast DNA network. The results rule out the former hypothesis, which attributes nucleocytoplasmic transport of LdTopIB entirely to the large subunit. The LdTopIB is localized to the nucleus only.

  1. Inhibitory effects of low molecular weight polyphenolics from Inonotus obliquus on human DNA topoisomerase activity and cancer cell proliferation.

    Science.gov (United States)

    Kuriyama, Isoko; Nakajima, Yuki; Nishida, Hiroshi; Konishi, Tetsuya; Takeuchi, Toshifumi; Sugawara, Fumio; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2013-08-01

    Low molecular weight (LMW) polyphenolics containing a polyhydroxylated benzyl moiety are abundant in medicinal plants. In the present study, we report on the activities of seven LMW polyphenolics isolated from Inonotus obliquus, a medicinal mushroom. The isolated compounds included caffeic acid (CA), 3,4-dihydroxybenzalacetone (DBL), gallic acid, syringic acid, protocatechuic acid, 3,4-dihydroxybenzaldehyde and 2,5-dihydroxyterephthalic acid. We analyzed their inhibitory effects on DNA polymerase (pol) and DNA topoisomerase (topo), and their effects on human cancer cell growth. All isolated compounds inhibited human topo II activity; the most potent were DBL and CA, which contain a catechol propanoid moiety. CA and DBL inhibited the activity of human topo I, whereas other compounds had no effect. No compound modulated the activities of 11 mammalian pol species or other DNA metabolic enzymes, including T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase and bovine deoxyribonuclease I. CA and DBL markedly suppressed the proliferation of human colon HCT116 carcinoma cells with an LD50 of 70.0 and 49.4 µM, respectively, and halted the cell cycle in the G2/M phase. The suppressive effect of these compounds on cancer cell growth correlated with their ability to inhibit topo II. These results suggest that CA- and DBL-dependent decreases in cell proliferation are due to the inhibition of cellular topo II. The mechanism of action of these catechol propanoid compounds and the implication for their use as anticancer agents are discussed. PMID:23799608

  2. Lack of topoisomerase copy number changes in patients with de novo and relapsed diffuse large B-cell lymphoma.

    Science.gov (United States)

    Pedersen, Mette Ø; Poulsen, Tim S; Gang, Anne O; Knudsen, Helle; Lauritzen, Anne F; Pedersen, Michael; Nielsen, Signe L; Brown, Peter; Høgdall, Estrid; Nørgaard, Peter

    2015-07-01

    Topoisomerase (TOP) gene copy number changes may predict response to treatment with TOP-targeting drugs in cancer treatment. This was first described in patients with breast cancer and is currently being investigated in other malignant diseases. TOP-targeting drugs may induce TOP gene copy number changes at relapse, with possible implications for relapse therapy efficacy. TOP gene alterations in lymphoma are poorly investigated. In this study, TOP1 and TOP2A gene alterations were investigated in patients with de novo diffuse large B-cell lymphoma (DLBCL) (n = 33) and relapsed DLBCL treated with chemotherapy regimens including TOP2-targeting drugs (n = 16). No TOP1 or TOP2A copy number changes were found. Polysomy of chromosomes 20 and 17 was seen in 3 of 25 patients (12%) and 2 of 32 patients (6%) with de novo DLBCL. Among relapsed patients, chromosome polysomy was more frequently observed in 5 of 13 patients (38%) and 4 of 16 patients (25%) harboring chromosome 20 and 17 polysomy, respectively; however, these differences only tended to be significant (p = 0.09 and p = 0.09, respectively). The results suggest that TOP gene copy number changes are very infrequent in DLBCL and not likely induced by TOP2-targeting drugs. Increased polyploidy of chromosomes 17 and 20 among patients with relapsed DLBCL may reflect genetic compensation in the tumor cells after TOP2 inhibition, but is more likely due to the increased genetic instability often seen in progressed cancers. Therefore, it is unlikely that TOP1 and TOP2A gene alterations can be used as predictive markers for response to treatment with TOP2-targeting drugs in patients with DLBCL.

  3. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Wei [Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Sa, Ke-Di; Zhang, Xiang; Jia, Lin-Tao; Zhao, Jing [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Yang, An-Gang [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Zhang, Rui, E-mail: ruizhang@fmmu.edu.cn [State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi' an (China); Fan, Jing, E-mail: jingfan@fmmu.edu.cn [Department of Vascular and Endocrine Surgery, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Bian, Ka, E-mail: kakamax85@hotmail.com [State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, 710032 Xi' an (China); Department of Otolaryngology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-08-07

    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells.

  4. HER2 and topoisomerase Ⅱα : possible predictors of response to neoadjuvant chemotherapy for breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    ZHU Li; LI Ya-fen; CHEN Wei-guo; HE Jian-rong; PENG Chen-hong; ZHU Zheng-gang; LI Hong-wei

    2008-01-01

    Background Surrogate markers may be used to assess the response to neoadjuvant treatment. The association between HER2 overexpression and favorable response to specific therapy in breast cancer is controversial, and the mechanism unclear. The purpose of the study was to evaluate HER2 and topoisomerase lla (Topo Ⅱα ) as candidates for predicting the response to neoadjuvant chemotherapy in breast cancer patients.Methods Between 1999 and 2006, seventy-six breast cancer patients who had received neoadjuvant chemotherapy were studied. Regimens including either CEF (cyclophosphamide, epirubicin, 5-fluorouracil) or CMF (cyclophosphamide, methotrexate, 5-fluorouracil) were given in more than three cycles to this group of patients. Protein expression of HER2 and Topo lla were determined by immunohistochemistry. The primary endpoint was pathological and clinical response. Results Of 76 primary breast cancer samples, 27 (35.5%) showed overexpression of either HER2 (25%) or Topo Ⅱα protein (10.5%), whereas in 7 tumors (9.2%) both proteins were found to be overexpressed. Ten patients (13.2%) had a clinical complete response and 21 (27.6%) had a clinical partial response. Five women (6.6%) had a pathological complete response, 5 (6.6%) had microscopic residual disease, and 46 (60.5%) had macroscopic residual disease. HER2 and Topo lla overexpression was significantly associated with a favorable response (P <0.001 and P=0.005 respectively).Conclusion Our study suggests that HER2 and Topo Ⅱα overexpression could be predictors of the response to neoadjuvant chemothrapy in both the CEF and CMF arms.

  5. MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

    International Nuclear Information System (INIS)

    The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells

  6. Salicylate, a catalytic inhibitor of topoisomerase II, inhibits DNA cleavage and is selective for the α isoform.

    Science.gov (United States)

    Bau, Jason T; Kang, Zhili; Austin, Caroline A; Kurz, Ebba U

    2014-02-01

    Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity. PMID:24220011

  7. Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

    Science.gov (United States)

    Attasart, Pongsopee; Boonma, Siriwan; Sunintaboon, Panya; Tanwilai, Dolpawan; Pothikasikorn, Jinrapa; Noonpakdee, Wilai Tienrungroj

    2016-05-01

    The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 μg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.

  8. Supraadditive apoptotic response of R3327-G rat prostate tumors to androgen ablation and radiation

    International Nuclear Information System (INIS)

    Purpose: Androgen ablation is often combined with radiation in the treatment of patients with prostate cancer, yet, the optimal sequencing and the mechanisms governing the interaction are not understood. The objectives were to determine if cell killing via apoptosis is enhanced when the combined treatment is administered and to define the relationship of changes in this form of cell killing to tumor volume growth delay. Materials and Methods: Dunning R3327-G rat prostate tumors, grown in the flanks of Copenhagen rats, were used at a volume of approximately 1 cc. Androgen ablation was initiated by castration, and androgen restoration was achieved with 0.5 cm silastic tube implants containing testosterone. 60Co was used for irradiation. The terminal deoxynucleotidyl transferase (TUNEL) histochemical assay was used to quantify apoptosis. Results: Tumors from intact and castrate unirradiated control rats had average apoptotic indices (percent of apoptotic cells) of 0.4 and 1.0%, respectively. The apoptotic index varied only slightly over time (3 h to 28 days) after castration (range 0.75-1.43%). Irradiation of intact rats to 7 Gy resulted in a peak apoptotic response at 6 h of 2.3%. A supra additive apoptotic response was seen when castration was initiated 3 days prior to 7 Gy radiation, with peak levels of about 10.1%. When the radiation was administered at increasing times beyond 3 days after castration, the apoptotic response gradually diminished and was back to levels seen in intact rats by 28 days after castration. Tumor volume growth delay studies were consistent with, but not conclusive proof of, a supra additive effect when the combination was used. Discussion: A supra additive apoptotic response was seen when androgen ablation and radiation were used to treat androgen sensitive R3327-G rat prostate tumors. This supra additive effect was dependent on the timing of the two treatments. Further studies are required to more fully define the optimal timing and

  9. Androstane derivatives induce apoptotic death in MDA-MB-231 breast cancer cells.

    Science.gov (United States)

    Jakimov, Dimitar S; Kojić, Vesna V; Aleksić, Lidija D; Bogdanović, Gordana M; Ajduković, Jovana J; Djurendić, Evgenija A; Penov Gaši, Katarina M; Sakač, Marija N; Jovanović-Šanta, Suzana S

    2015-11-15

    Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development.

  10. Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation.

    Science.gov (United States)

    Stuart, Lynda M; Lucas, Mark; Simpson, Cathy; Lamb, Jonathan; Savill, John; Lacy-Hulbert, Adam

    2002-02-15

    Dendritic cells (DCs) are the sentinels of the immune system, able to interact with both naive and memory T cells. The recent observation that DCs can ingest cells dying by apoptosis has raised the possibility that DCs may, in fact, present self-derived Ags, initiating both autoimmunity and tumor-specific responses, especially if associated with appropriate danger signals. Although the process of ingestion of apoptotic cells has not been shown to induce DC maturation, the exact fate of these phagocytosing DCs remains unclear. In this paper we demonstrate that DCs that ingest apoptotic cells are able to produce TNF-alpha but have a diminished ability to produce IL-12 in response to external stimuli, a property that corresponds to a failure to up-regulate CD86. By single-cell analysis we demonstrate that these inhibitory effects are restricted to those DCs that have engulfed apoptotic cells, with bystander DCs remaining unaffected. These changes were independent of the production of anti-inflammatory cytokines TGF-beta1 and IL-10 and corresponded with a diminished capacity to stimulate naive T cells. Thus, the ingestion of apoptotic cells is not an immunologically null event but is capable of modulating DC maturation. These results have important implications for our understanding of the role of clearance of dying cells by DCs not only in the normal resolution of inflammation but also in control of subsequent immune responses to apoptotic cell-derived Ags.

  11. Relaxin has anti-apoptotic effects on human trophoblast-derived HTR-8/SV neo cells.

    Science.gov (United States)

    Lodhi, Romana S Z; Nakabayashi, Koji; Suzuki, Kaho; Yamada, Ai Y; Hazama, Rhoichi; Ebina, Yasuhiko; Yamada, Hideto

    2013-12-01

    The study was conducted to evaluate the effects of human relaxin on apoptosis in the human trophoblast derived HTR-8/SV neo cell line, which is a possible model of human extravillous trophoblasts (EVTs). HTR-8/SV neo cells, cultured in phenol red free RPMI1640 medium, were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions. RT-PCR was used for evaluating relaxin receptor: RXFP1 and RXFP2 expression in HTR-8/SV neo cells. The cell death was examined by TUNEL assay. Furthermore, we investigated caspase-3, cleaved PARP and Bcl-2 expressions by Western blot analysis to recognize the translational effects of anti-apoptotic and pro-apoptotic proteins. RXFP1 and RXFP2 mRNA expression was observed in HTR-8/SV neo cells. Compared with untreated control cultures, treatment with rH2 relaxin, decreased TUNEL-positive rate in HTR-8/SV neo cells was observed. Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 and cleaved PARP, but in contrast increased Bcl-2 expression in those cells. These results suggest that rH2 relaxin has anti-apoptotic effects on HTR8/SV neo cells by decreasing pro-apoptotic caspase-3 and cleaved PARP expression and up-regulating anti-apoptotic Bcl-2 expression. PMID:24070111

  12. Selection of apoptotic cell specific human antibodies from adult bone marrow.

    Directory of Open Access Journals (Sweden)

    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  13. APOPTOTIC AND PROLIFERATIVE ACTIVITY IN OVARIAN BENIGN,BORDERLINE AND MALIGNANT TUMORS

    Institute of Scientific and Technical Information of China (English)

    刘爱军; 陈乐真; 颜婉嫦; 邱玮璇; 赵昀; 张雅贤

    2002-01-01

    Objective.To determine the apoptotic and proliferative activities in various ovarian epithelial tumors.Methods.Formalin fixed,paraffin embedded tissues of 86 ovarian epithelial tumors,including 52 adenocarcinomas,23 borderline tumors and 11 cystadenomas,were retrieved.Apoptotic (AI) and proliferative (PI) index were estimated using the monoclonal antibodies: M30,Ki 67 and Ki S1 in these tumors.Quantitative assessment of AI and PI was estimated by calculating the percentage of positive cells among no less than 1000 tumor cells.Results.Statistically significant difference in AI was found between benign and borderline tumors or carcinomas (P=0.028,0.001,respectively).Significant differences in PI,as assessed by both Ki 67 and topo IIα,were demonstrated between carcinomas and benign or borderline tumors (both P< 0.001).Benign tumors had both low PI and AI; borderline tumors had lower PI but higher AI,while adenocarcinomas had both high proliferative and high apoptotic rates.Among borderline tumors,serous tumors had significantly lower AI and higher PI than mucinous ones.Conclusions.The results suggest that apoptotic and proliferative activities play important roles in the pathogenesis and development of ovarian borderline and malignant tumors.The high apoptotic rate in borderline tumor may explain its relatively indolent behavior while the high proliferative rate in carcinomas tends to explain its aggressive behavior.

  14. Modafinil abrogates methamphetamine-induced neuroinflammation and apoptotic effects in the mouse striatum.

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    Mariana Raineri

    Full Text Available Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4 × 5 mg/kg, i.p., 2 h apart and modafinil co-administration (2 × 90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections on glial cells (microglia and astroglia. We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.

  15. Attenuation of cisplatin-induced acute renal failure is associated with less apoptotic cell death.

    Science.gov (United States)

    Zhou, H; Miyaji, T; Kato, A; Fujigaki, Y; Sano, K; Hishida, A

    1999-12-01

    To clarify the pathophysiologic role of apoptosis in acute renal failure (ARF), we examined whether the attenuation of cisplatin-induced ARF is associated with the change in the degree of apoptotic cell death. The administration of cisplatin (CDDP) (6 mg/kg body weight) in rats induced ARF at day 5, as manifested by a significant increase in serum creatinine (Scr) and tubular damage. CDDP-induced apoptotic cell death was confirmed by electron microscopic examination, agarose gel electrophoresis, and increased cells positive for TaT-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in the outer medulla of the kidney. Treatment with dimethylthiourea (DMTU)--a scavenger of hydroxyl radicals--or glycine abrogated CDDP-induced increases in Scr, the tubular damage score, and the number of TUNEL-positive cells. Pretreatment with uranyl acetate (UA) induced a significant expression of Bcl-2 in the kidney and ameliorated CDDP-induced increases in Scr, the tubular damage score, and TUNEL-positive cells in the outer stripe of the outer medulla. Our findings indicate (1) that the attenuation of CDDP-induced ARF was associated with less apoptotic cell death and (2) that the induction of the anti-apoptotic protein Bcl-2 attenuated apoptosis and tubular damage. Our results suggest that apoptotic cell death may play an important role in the development of cisplatin-induced ARF. PMID:10595794

  16. FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

    Science.gov (United States)

    Marsman, Gerben; Stephan, Femke; de Leeuw, Karina; Bulder, Ingrid; Ruinard, Jessica T; de Jong, Jan; Westra, Johanna; Bultink, Irene E M; Voskuyl, Alexandre E; Aarden, Lucien A; Luken, Brenda M; Kallenberg, Cees G M; Zeerleder, Sacha

    2016-03-01

    Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

  17. The Mannose Receptor Is Involved in the Phagocytosis of Mycobacteria-Induced Apoptotic Cells

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    Teresa Garcia-Aguilar

    2016-01-01

    Full Text Available Upon Mycobacterium tuberculosis infection, macrophages may undergo apoptosis, which has been considered an innate immune response. The pathways underlying the removal of dead cells in homeostatic apoptosis have been extensively studied, but little is known regarding how cells that undergo apoptotic death during mycobacterial infection are removed. This study shows that macrophages induced to undergo apoptosis with mycobacteria cell wall proteins are engulfed by J-774A.1 monocytic cells through the mannose receptor. This demonstration was achieved through assays in which phagocytosis was inhibited with a blocking anti-mannose receptor antibody and with mannose receptor competitor sugars. Moreover, elimination of the mannose receptor by a specific siRNA significantly diminished the expression of the mannose receptor and the phagocytosis of apoptotic cells. As shown by immunofluorescence, engulfed apoptotic bodies are initially located in Rab5-positive phagosomes, which mature to express the phagolysosome marker LAMP1. The phagocytosis of dead cells triggered an anti-inflammatory response with the production of TGF-β and IL-10 but not of the proinflammatory cytokines IL-12 and TNF-α. This study documents the previously unreported participation of the mannose receptor in the removal of apoptotic cells in the setting of tuberculosis (TB infection. The results challenge the idea that apoptotic cell phagocytosis in TB has an immunogenic effect.

  18. Treatment of lymphoid cells with the topoisomerase II poison etoposide leads to an increased juxtaposition of AML1 and ETO genes on the surface of nucleoli.

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    Razin S. V.

    2011-10-01

    Full Text Available AML1 and ETO genes are known partners in the t(8,21 translocation associated with the treatment-related leukaemias in the patients receiving chemotherapy with DNA-topoisomerase II (topo II poisons. Aim. To determine whether the genes AML1 and ETO are in close proximity either permanently or temporarily in the nucleus. Methods. 3D FISH. Results. We found that in 5 % of untreated cells, alleles of AML1 and ETO are in close proximity. This number increased two-fold in the cells treated with the topo II poison etoposide. Surprisingly, in more than 50 % of the cases observed, co-localization of the genes occurred at the nucleoli surface. We found also that the treatment of cells triggers preferential loading of RAD51 onto bcr of the AML1 and ETO genes. Conclusions. Our results suggest that the repair of DNA lesions introduced by topoisomerase II poisons may be mediated simultaneously by multiple mechanisms, which may be the cause of mistakes resulting in translocations.

  19. EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    李金华; 梁念慈; 莫丽儿; 张晓; 何承伟

    2001-01-01

    Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

  20. RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli.

    Science.gov (United States)

    Baaklini, Imad; Hraiky, Chadi; Rallu, Fabien; Tse-Dinh, Yuk-Ching; Drolet, Marc

    2004-10-01

    It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis. PMID:15458416

  1. Inhibitors of DNA topoisomerase or β-(but not α-) DNA polymerases alter the incision response of repair-deficient human cells after UV- or mnng-treatment

    International Nuclear Information System (INIS)

    Two types of repair-deficient human cells, those incapable of incision after UV irradiation (XPA or XPD) and those that fail to do so after incubation with MNNG (Al336 Mer/sup -/ cells), incised their DNA after treatment with UV or MNNG and incubation a) with inhibitors of DNA topoisomerase II (novobiocin) or b) with dideoxythymidine or at 450C (both inhibitory to β polymerase). This result was not produced by ara C or aphidicolin, specific inhibitors of α DNA polymerase. Thymidine incorporation in these cell lines was refractory to the β but not to the α polymerase inhibitors, although Al336 cells had normal in vitro β DNA polymerase activity with normal sensitivity to dideoxythymidine triphosphate. Thus, modulation of this activity in the cells prevented its inhibition. These results could be produced by allosteric interactions among components of an enzyme complex, similar to the ''replitase,'' that carries out DNA repair. The authors propose that DNA topoisomerase, β-DNA polymerase, a specific damage recognition protein (or proteins), and DNA nicking activity are part of this complex

  2. PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.

    Science.gov (United States)

    Kota, Swathi; Charaka, Vijaya K; Ringgaard, Simon; Waldor, Matthew K; Misra, Hari S

    2014-01-01

    PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s) in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB) as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal), an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA) in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.

  3. PprA contributes to Deinococcus radiodurans resistance to nalidixic acid, genome maintenance after DNA damage and interacts with deinococcal topoisomerases.

    Directory of Open Access Journals (Sweden)

    Swathi Kota

    Full Text Available PprA is known to contribute to Deinococcus radiodurans' remarkable capacity to survive a variety of genotoxic assaults. The molecular bases for PprA's role(s in the maintenance of the damaged D. radiodurans genome are incompletely understood, but PprA is thought to promote D. radiodurans's capacity for DSB repair. PprA is found in a multiprotein DNA processing complex along with an ATP type DNA ligase, and the D. radiodurans toposiomerase IB (DraTopoIB as well as other proteins. Here, we show that PprA is a key contributor to D. radiodurans resistance to nalidixic acid (Nal, an inhibitor of topoisomerase II. Growth of wild type D. radiodurans and a pprA mutant were similar in the absence of exogenous genotoxic insults; however, the pprA mutant exhibited marked growth delay and a higher frequency of anucleate cells following treatment with DNA-damaging agents. We show that PprA interacts with both DraTopoIB and the Gyrase A subunit (DraGyrA in vivo and that purified PprA enhances DraTopoIB catalysed relaxation of supercoiled DNA. Thus, besides promoting DNA repair, our findings suggest that PprA also contributes to preserving the integrity of the D. radiodurans genome following DNA damage by interacting with DNA topoisomerases and by facilitating the actions of DraTopoIB.

  4. A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4'-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation.

    Science.gov (United States)

    Zhao, Wei; Chen, Lu; Li, Hong-Mei; Wang, Duan-Ji; Li, Dong-Sheng; Chen, Tao; Yuan, Zhan-Peng; Tang, Ya-Jie

    2014-06-01

    A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4'-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential antitumor prodrug. Firstly, according to the structure-activity relationship, drug combination principle and bioisosterism, the -COO- and the -NH- bond substituents at the 4 position of cycloparaffin would be a great modification direction to improve antitumor activity of 4'-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view, the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a series of heterocyclic with pharmacological activity were chosen as module for improving antitumor activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-4'-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell line HeLa (i.e., the IC50 value of 0.60 ± 0.20 μM), A549 (i.e., the IC50 value of 3.83 ± 0.08 μM), HepG2 (i.e., the IC50 value of 1.21 ± 0.05 μM), and BGC-823 (i.e., the IC50 value of 4.15 ± 1.13 μM) was significantly improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer drug etoposide (i.e., the IC50 values of 15.32 ± 0.10, 59.38 ± 0.77, 67.25 ± 7.05, and 30.74 ± 5.13 μM), respectively. Compound 1 could arrest HeLa cell cycle G2/M and induce apoptosis by strongly diminishing the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly demonstrated by the

  5. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    OpenAIRE

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect ...

  6. Structural study of TTR-52 reveals the mechanism by which a bridging molecule mediates apoptotic cell engulfment

    OpenAIRE

    Kang, Yanyong; Zhao, Dongfeng; Liang, Huanhuan; Liu, Bin; Zhang, Yan; Liu, Qinwen; Wang, Xiaochen; Liu, Yingfang

    2012-01-01

    Apoptotic cells display various “eat me” signals that can be recognized through bridging molecules that cross-link the dying cells to phagocytes. This work illustrates the first full-length structure of such a bridging molecule, TTR-52. The study elucidates the binding of these bridging molecules with the apoptotic cell signals and phagocyte receptors, providing valuable new insight into the process of apoptotic cell recognition.

  7. Facile construction of pyrrolo[1,2-b]isoquinolin-10(5H)-ones via a redox-amination-aromatization-Friedel-Crafts acylation cascade reaction and discovery of novel topoisomerase inhibitors.

    Science.gov (United States)

    Wu, Shanchao; Liu, Na; Dong, Guoqiang; Ma, Lin; Wang, Shengzheng; Shi, Wencai; Fang, Kun; Chen, Shuqiang; Li, Jian; Zhang, Wannian; Sheng, Chunquan; Wang, Wei

    2016-07-21

    An efficient redox-amination-aromatization-Friedel-Crafts acylation cascade process from trans-4-hydroxyproline and 2-formylbenzoic acids has been developed for the synthesis of pyrrolo[1,2-b]isoquinolin-10(5H)-ones. Compound 3h was identified as a new potent dual topoisomerase I/II inhibitor. PMID:27400278

  8. To the nucleolar density and size in apoptotic human leukemic myeloblasts produced in vitro by Trichostatin A

    Directory of Open Access Journals (Sweden)

    K Smetana

    2009-08-01

    Full Text Available The present study was designed to provide more information on nucleoli in apoptotic cells, which were represented in the present study by cultured leukemic myeloblasts (Kasumi-1 cells. The apoptotic process in these cells was produced by trichostatin A (TSA that is a histone deacetylase inhibitor with strong cytostatic effects. The selected TSA concentration added to cultures facilitated to study apoptotic and notapoptotic cells in one and the same specimen. The nucleolar diameter and density were determined using computer assisted measurement and densitometry in specimens stained for RNA. In comparison with not-apoptotic cells, in apoptotic cells, nucleolar mean diameter did not change significantly and nucleolar RNA density was also not apparently different. On the other hand, the cytoplasmic RNA density in apoptotic cells was markedly reduced. Thus it seemed to be possible that the transcribed RNA remained “frozen” within the nucleolus but its transport to the cytoplasm decreased or stopped. However, the possibility of the RNA degradation in the cytoplasm of apoptotic cells based on the present study cannot be eliminated. At this occasion it should be added that AgNORs reflecting nucleolar biosynthetic and cell proliferation activity in apoptotic cells decreased in number or disappeared. The presented results also indicated that large nucleoli intensely stained for RNA need not be necessarily related to the high nucleolar biosynthetic or cell proliferation activity and may be also present in apoptotic cells responding to the cytostatic treatment.

  9. Cell-to-Cell stochastic fluctuations in apoptotic signaling can decide between life and death

    CERN Document Server

    Raychaudhuri, S; Nguyen, T; Khan, E M; Goldkorn, T

    2007-01-01

    Apoptosis, or genetically programmed cell death, is a crucial cellular process that maintains the balance between life and death in cells. The precise molecular mechanism of apoptosis signaling and how these two pathways are differentially activated under distinct apoptotic stimuli is poorly understood. We developed a Monte Carlo-based stochastic simulation model that can characterize distinct signaling behaviors in the two major pathways of apoptotic signaling using a novel probability distribution-based approach. Specifically, we show that for a weak death signal, such as low levels of death ligand Fas (CD95) binding or under stress conditions, the type 2 mitochondrial pathway dominates apoptotic signaling. Our results also show signaling in the type 2 pathway is stochastic, where the population average over many cells does not capture the cell-to-cell fluctuations in the time course (~1 - 10 hours) of downstream caspase-3 activation. On the contrary, the probability distribution of caspase-3 activation for...

  10. Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells

    DEFF Research Database (Denmark)

    Grunnet, Lars G; Aikin, Reid; Tonnesen, Morten F;

    2009-01-01

    OBJECTIVE: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the...... intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells. RESEARCH DESIGN AND METHODS: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis...... factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to...

  11. Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

    Directory of Open Access Journals (Sweden)

    Moreira Maria Elisabete C.

    2004-01-01

    Full Text Available Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.

  12. Impaired clearance of apoptotic cells in chronic inflammatory diseases: therapeutic implications

    Directory of Open Access Journals (Sweden)

    Zsuzsa eSzondy

    2014-08-01

    Full Text Available In healthy individuals billions of cells die by apoptosis every day. Removal of the dead cells by phagocytosis (a process called efferocytosis must be efficient to prevent secondary necrosis and the consequent release of proinflammatory cell contents that damages the tissue environment and provokes autoimmunity. In addition, detection and removal of apoptotic cells generally induces an anti-inflammatory response. As a consequence improper clearance of apoptotic cells, being the result of either genetic anomalies and /or a persistent disease state, contributes to the establishment and progression of a number of human chronic inflammatory diseases such as autoimmune and neurological disorders, inflammatory lung diseases, obesity, type 2 diabetes or atherosclerosis. During the past decade our knowledge about the mechanism of efferocytosis has significantly increased, providing therapeutic targets through which impaired phagocytosis of apoptotic cells and the consequent inflammation could be influenced in these diseases.

  13. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  14. Ultrastructural observation of human neutrophils during apoptotic cell death triggered by Entamoeba histolytica.

    Science.gov (United States)

    Sim, Seobo; Kim, Kyeong Ah; Yong, Tai-Soon; Park, Soon-Jung; Im, Kyung-il; Shin, Myeong Heon

    2004-12-01

    Neutrophils are important effector cells against protozoan extracellular parasite Entamoeba histolytica, which causes amoebic colitis and liver abscess in human beings. Apoptotic cell death of neutrophils is an important event in the resolution of inflammation and parasite's survival in vivo. This study was undertaken to investigate the ultrastructural aspects of apoptotic cells during neutrophil death triggered by Entamoeba histolytica. Isolated human neutrophils from the peripheral blood were incubated with or without live trophozoites of E. histolytica and examined by transmission electron microscopy (TEM). Neutrophils incubated with E. histolytica were observed to show apoptotic characteristics, such as compaction of the nuclear chromatin and swelling of the nuclear envelop. In contrast, neutrophils incubated in the absence of the amoeba had many protrusions of irregular cell surfaces and heterogenous nuclear chromatin. Therefore, it is suggested that Entamoeba-induced neutrophil apoptosis contribute to prevent unwanted tissue inflammation and damage in the amoeba-invaded lesions in vivo.

  15. Anti-apoptotic peptides protect against radiation-induced cell death

    International Nuclear Information System (INIS)

    The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15 Gy radiation. In mice exposed to 5 Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues

  16. Anti-apoptotic signaling and failure of apoptosis in the ischemic rat hippocampus

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Lassmann, Hans; Johansen, Flemming Fryd

    2007-01-01

    colchicine injection severed as a reference for classical apoptosis. Heat shock protein 70 (Hsp70), neuronal apoptosis inhibitory protein (NAIP) and manganese superoxide dismutase (MnSOD) were upregulated in the majority of intact CA1 neurons paralleling the occurrence of CA1 neuronal death (days 3-7) as...... well as in a proportion of apoptosis-(<50%) and necrosis-like (<30%) CA1 neurons. Colchicine did not provoke an anti-apoptotic response in DGC at all. In addition, more than 70% of apoptosis- and necrosis-like CA1 neurons had completely lost their RCC subunits suggesting bioenergetic failure; by...... contrast, following colchicine injection, 88% of all apoptotic DGC presented RCC subunits. Thus, anti-apoptotic proteins may, in a subset of ischemic CA1 neurons, prevent cell death, while in others, affected by pronounced energy failure, they may cause secondary necrosis....

  17. Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Limin Zhang; Yunhe Zhang; Xifeng Zou; Qunxi Li; Yun Li; Jun Zhu; Jianmin Li; Aijun Fu; Qingjun Liu; Tong Chen; Zelin Sun; Zhiyong Zhang

    2011-01-01

    The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury (DAI) at 33 °C. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

  18. Regulation of apoptotic signal transduction pathways by the heat shock proteins

    Institute of Scientific and Technical Information of China (English)

    LI; Zhengyu; ZHAO; Xia; WEI; Yuquan

    2004-01-01

    The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.

  19. Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP,which could induce tumor cell apoptosis. To further explore the function of N37,we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database,the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apopto...

  20. Synthesis of apoptotic chalcone analogues in HepG2 human hepatocellular carcinoma cells.

    Science.gov (United States)

    Park, Cheon-Soo; Ahn, Yongchel; Lee, Dahae; Moon, Sung Won; Kim, Ki Hyun; Yamabe, Noriko; Hwang, Gwi Seo; Jang, Hyuk Jai; Lee, Heesu; Kang, Ki Sung; Lee, Jae Wook

    2015-12-15

    Eight chalcone analogues were prepared and evaluated for their cytotoxic effects in human hepatoma HepG2 cells. Compound 5 had a potent cytotoxic effect. The percentage of apoptotic cells was significantly higher in compound 5-treated cells than in control cells. Exposure to compound 5 for 24h induced cleavage of caspase-8 and -3, and poly (ADP-ribose) polymerase (PARP). Our findings suggest that compound 5 is the active chalcone analogue that contributes to cell death in HepG2 cells via the extrinsic apoptotic pathway. PMID:26564263

  1. Staphylococcus aureus alpha-toxin-induced cell death : predominant necrosis despite apoptotic caspase activation

    NARCIS (Netherlands)

    Essmann, F; Bantel, H; Totzke, G; Engels, I H; Sinha, B; Schulze-Osthoff, K; Jänicke, R U

    2003-01-01

    Recent data suggest that alpha-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation,

  2. Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk

    OpenAIRE

    Smith, Alyson J.; Nelson, Natalie G.; Oommen, Saji; Hartjes, Katherine A.; Folmes, Clifford D.; Terzic, Andre; Nelson, Timothy J.

    2012-01-01

    The pluripotent cell-purging assay validated herein demonstrates that pluripotent cells are selectively hypersensitive to DNA damage-induced apoptosis as a function of the specific apoptotic inducer protein Puma. Risk of dysregulated growth is decreased and the safety profile of transplant-ready, bioengineered progenitor cells is augmented.

  3. Modulation of apoptotic pathways of macrophages by surface-functionalized multi-walled carbon nanotubes.

    Directory of Open Access Journals (Sweden)

    Yuanqin Jiang

    Full Text Available Biomedical applications of carbon nanotubes (CNTs often involve improving their hydrophilicity and dispersion in biological media by modifying them through noncovalent or covalent functionalization. However, the potential adverse effects of surface-functionalized CNTs have not been well characterized. In this study, we functionalized multi-walled CNTs (MWCNTs via carboxylation, to produce MWCNTs-COOH, and via poly (ethylene glycol linking, to produce MWCNTs-PEG. We used these functionalized MWCNTs to study the effect of surface functionalization on MWCNTs-induced toxicity to macrophages, and elucidate the underlying mechanisms of action. Our results revealed that MWCNTs-PEG were less cytotoxic and were associated with less apoptotic cell death of macrophages than MWCNTs-COOH. Additionally, MWCNTs-PEG induced less generation of reactive oxygen species (ROS involving less activation of NADPH oxidase compared with MWCNTs-COOH, as evidenced by membrane translocation of p47(phox and p67(phox in macrophages. The less cytotoxic and apoptotic effect of MWCNTs-PEG compared with MWCNTs-COOH resulted from the lower cellular uptake of MWCNTs-PEG, which resulted in less activation of oxidative stress-responsive pathways, such as p38 mitogen-activated protein kinases (MAPK and nuclear factor (NF-κB. These results demonstrate that surface functionalization of CNTs may alter ROS-mediated cytotoxic and apoptotic response by modulating apoptotic signaling pathways. Our study thus provides new insights into the molecular basis for the surface properties affecting CNTs toxicity.

  4. Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex.

    Science.gov (United States)

    Petrenko, Volodymyr; Mihhailova, Jevgenia; Salmon, Patrick; Kiss, Jozsef Z

    2015-11-01

    Apoptotic cell death is the leading cause of neuronal loss after neonatal brain injury. Little is known about the intrinsic capacity of the immature cerebral cortex for replacing dead cells. Here we test the hypothesis that neuronal apoptosis is able to trigger compensatory proliferation in surrounding cells. In order to establish a "pure" apoptotic cell death model and to avoid the confounding effects of broken blood-brain barrier and inflammatory reactions, we used a diphtheria toxin (DT) and diphtheria toxin receptor (DTR) system to induce ablation of layer IV neurons in the rodent somatosensory cortex during the early postnatal period. We found that DT-triggered apoptosis is a slowly progressing event lasting about for 7 days. While dying cells expressed the morphological features of apoptosis, we could not detect immunoreactivity for activated caspase-3 in these cells. Microglia activation and proliferation represented the earliest cellular responses to apoptotic cell death. In addition, we found that induced apoptosis triggered a massive proliferation of undifferentiated progenitor cell pool including Sox2 as well as NG2 cells. The default differentiation pattern of proliferating progenitors appears to be the glial phenotype; we could not find evidence for newly generated neurons in response to apoptotic neuronal death. These results suggest that mitotically active progenitor populations are intrinsically capable to contribute to the repair process of injured cortical tissue and may represent a potential target for neuronal replacement strategies.

  5. ImmunoPET imaging of phosphatidylserine in pro-apoptotic therapy treated tumor models

    International Nuclear Information System (INIS)

    An immunoPET imaging probe for the detection of phosphatidylserine was developed and tested in animal models of human cancer treated with pro-apoptotic therapy. We hypothesized that the relatively long plasma half-life of a probe based on a full-length antibody coupled with a residualizing radionuclide would be able to catch the wave of drug-induced apoptosis and lead to a specific accumulation in apoptotic tumor tissue. Methods: The imaging probe is based on a 89Zr-labeled monoclonal antibody PGN635 targeting phosphatidylserine. The probe was evaluated pre-clinically in four tumor xenograft models: one studied treatment with paclitaxel to trigger the intrinsic apoptotic pathway, and three others interrogated treatment with an agonistic death-receptor monoclonal antibody to engage the extrinsic apoptotic pathway. Results: High accumulation of 89Zr-PGN635 was observed in treated tumors undergoing apoptosis reaching 30 %ID/g and tumor-to-blood ratios up to 13. The tumor uptake in control groups treated with vehicle or imaged with a non-binding antibody probe was significantly lower. Conclusions: The results demonstrate the ability of 89Zr-PGN635 to image drug-induced apoptosis in animal models and corroborate our hypothesis that radiolabeled antibodies binding to intracellular targets transiently exposed on the cell surface during apoptosis can be employed for detection of tumor response to therapy.

  6. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Hee, E-mail: leedneo@gmail.com [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Kim, Dong-Wook [Department of Microbiology, Immunology, and Cancer Biology, University of VA (United States); Jung, Chang-Hwa [Division of Metabolism and Functionality Research, Korea Food Research Institute (Korea, Republic of); Lee, Yong J. [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Park, Daeho, E-mail: daehopark@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  7. The c-Myc Transactivation Domain Is a Direct Modulator of Apoptotic versus Proliferative Signals

    Science.gov (United States)

    Chang, David W.; Claassen, Gisela F.; Hann, Stephen R.; Cole, Michael D.

    2000-01-01

    We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals. PMID:10825194

  8. Reversal of Apoptotic Resistance by Lycium barbarum Glycopeptide 3 in Aged T Cells

    Institute of Scientific and Technical Information of China (English)

    LONG-GUO YUAN; HONG-BIN DENG; LI-HUI CHEN; DIAN-DONG LI; QI-YANG HE

    2008-01-01

    Objective To study whether Lycium barbarian glycopeptide 3 (LBGP3) affects T cell apeptosis in aged mice. Methods LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic "sub-Gl peak" was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-γ, and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. Resdts LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide.Treatment with 200 μg/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T ceils. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. Conclusion Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.

  9. Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

    Institute of Scientific and Technical Information of China (English)

    Yan-xia Bai; Qing-yong Ma; Guang-xiao Yang

    2009-01-01

    Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP, which could induce tumor cell apoptosis. To further explore the function of N37, we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database, the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis, p53 (N37) gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www. ncbi. him. nih. gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid, we could further investigate the function of N37 peptide.

  10. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    Science.gov (United States)

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer. PMID:20032400

  11. Apoptotic and free radical scavenging properties of the methanolic extract of Gentianella alborosea.

    Science.gov (United States)

    Acero, Nuria; Llinares, Francisco; Galán de Mera, Antonio; Oltra, Beatriz; Muñoz-Mingarro, Dolores

    2006-09-01

    Gentianella alborosea ("Hercampure") is a Peruvian species used in folk medicine for the treatment of a variety of health disorders. We tested the free radical scavenging (DPPH) and induction of apoptosis on a human uterus tumor cell line (HeLa) by its methanolic extract. The results showed a noticeable radical scavenging activity and a dose-dependent apoptotic effect. PMID:16814959

  12. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats.

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-08-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  13. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats☆

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-01-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  14. Antiproliferative and pro-apoptotic effects of Uncaria tomentosa in human medullary thyroid carcinoma cells.

    Science.gov (United States)

    Rinner, Beate; Li, Zeng Xia; Haas, Helga; Siegl, Veronika; Sturm, Sonja; Stuppner, Hermann; Pfragner, Roswitha

    2009-11-01

    Medullary thyroid carcinoma (MTC), a rare calcitonin-producing tumor, is derived from parafollicular C-cells of the thyroid and is characterized by constitutive Bcl-2 overexpression. The tumor is relatively insensitive to radiation therapy as well as conventional chemotherapy. To date, the only curative treatment is the early and complete surgical removal of all neoplastic tissue. In this study, the antiproliferative and pro-apoptotic effects of fractions obtained from Uncaria tomentosa (Willd.) DC, commonly known as uña de gato or cat's claw were investigated. Cell growth of MTC cells as well as enzymatic activity of mitochondrial dehydrogenase was markedly inhibited after treatment with different fractions of the plant. Furthermore, there was an increase in the expressions of caspase-3 and -7 and poly(ADP-ribose) polymerase (PARP) fraction, while bcl-2 overexpression remained constant. In particular, the alkaloids isopterpodine and pteropodine of U. tomentosa exhibited a significant pro-apoptotic effect on MTC cells, whereas the alkaloid-poor fraction inhibited cell proliferation but did not show any pro-apoptotic effects. These promising results indicate the growth-restraining and apoptotic potential of plant extracts against neuroendocrine tumors, which may add to existing therapies for cancer.

  15. Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

    International Nuclear Information System (INIS)

    To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle

  16. Developmental toxicity of toluene in male rats: effects on semen quality, testis morphology, and apoptotic neurodegeneration

    DEFF Research Database (Denmark)

    Dalgaard, M.; Hossaini, A.; Hougaard, K.S.;

    2001-01-01

    differences between toluene-exposed animals and control animals. In the hippocampus! almost no apoptosis was observed in any age group, and there were no differences in apoptotic neurodegeneration between male rats exposed to 1800 ppm and control animals at PND 11, 21 or 90. Generally. a marked increase...

  17. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    International Nuclear Information System (INIS)

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway

  18. The calcimimetic R-568 induces apoptotic cell death in prostate cancer cells

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    Cheng Guangming

    2009-07-01

    Full Text Available Abstract Background Increased serum level of parathyroid hormone (PTH was found in metastatic prostate cancers. Calcimimetic R-568 was reported to reduce PTH expression, to suppress cell proliferation and to induce apoptosis in parathyroid cells. In this study, we investigated the effect of R-568 on cellular survival of prostate cancer cells. Methods Prostate cancer cell lines LNCaP and PC-3 were used in this study. Cellular survival was determined with MTT, trypan blue exclusion and fluorescent Live/Death assays. Western blot assay was utilized to assess apoptotic events induced by R-568 treatment. JC-1 staining was used to evaluate mitochondrial membrane potential. Results In cultured prostate cancer LNCaP and PC-3 cells, R-568 treatment significantly reduced cellular survival in a dose- and time-dependent manner. R-568-induced cell death was an apoptotic event, as evidenced by caspase-3 processing and PARP cleavage, as well as JC-1 color change in mitochondria. Knocking down calcium sensing receptor (CaSR significantly reduced R-568-induced cytotoxicity. Enforced expression of Bcl-xL gene abolished R-568-induced cell death, while loss of Bcl-xL expression led to increased cell death in R-568-treated LNCaP cells,. Conclusion Taken together, our data demonstrated that calcimimetic R-568 triggers an intrinsic mitochondria-related apoptotic pathway, which is dependent on the CaSR and is modulated by Bcl-xL anti-apoptotic pathway.

  19. Expression of defender against apoptotic death (DAD-1) in iris and dianthus petals

    NARCIS (Netherlands)

    Kop, van der D.A.M.; Ruys, G.; Dees, D.; Schoot, van der C.; Boer, de A.D.; Doorn, van W.G.

    2003-01-01

    The gene defender against apoptotic death (DAD-1) prevents programmed cell death in animal cells. We investigated the expression pattern of DAD-1 in petals of iris (Iris x hollandica cv. Blue Magic) and carnation (Dianthus caryophyllus cv. Etarro). DAD-1 expression in Iris petals was strongly reduce

  20. Different Sensitivities to Apoptotic Induction by Camptothecin between Normal and Senescent Lens Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Haike Guo; Haiying Jin; Liya Wang; Hongyang Zhang; Xin Yang

    2002-01-01

    Purpose: To investigate whether normal and senescent lens epithelial cells have different defense abilities to apoptotic induction factor in vitro.Methods: Rabbit lens epithelial cells were cultured, passed. When reaching confluence, cells from the first and seventh passage were stained by x-gal staining to detect cell senescence. Cell apoptosis was detected by TUNEL(Roche).10μmol/L camptothecin was used to induce cell apoptosis from the lens epithelial cells of the first and seventh passage to distinguish different sensitivities to apoptotic induction factor between normal and senescent cells.Results: The senescent cells (41.17% ± 5.24% ) were detected in the lens epithelial cell culture of the seventh passage, which are higher than those of the first passage (0.98% ±0. 39% ). There was no apoptotic cell detected in the cell cultures undisturbed. Exposure of the first passage cells to camptothecin resulted in death of approximately 23.87% ± 3.45% of the cells during a 36 hour exposure period. In contrast, significantly more lens epithelial cells died through the apoptosis (38.29% ±4. 01% ) from the seventh passage.Conclusion: Senescent cells increased with cell passage. Senescence lens epithelial cells do not undergo apoptosis if they were not disturbed. But the vulnerabilities to apoptotic induction between health and senescence cells were different.

  1. Cloning and analysis of a defender against apoptotic cell death (DAD1) homologue from tomato

    NARCIS (Netherlands)

    Hoeberichts, F.A.; Woltering, E.J.

    2001-01-01

    A cDNA clone homologous to the human defender against apoptotic cell death (DAD1) gene, which is believed to be a conserved inhibitor of programmed cell death, was isolated from tomato (Lycopersicon esculentum cv. Prisca). The 351 basepairs open reading frame predicted a 116 amino acid protein seque

  2. SorCS2 Regulates Dopaminergic Wiring and Is Processed into an Apoptotic Two-Chain Receptor in Peripheral Glia

    DEFF Research Database (Denmark)

    Glerup, Simon; Olsen, Ditte; Vægter, Christian Bjerggaard;

    2014-01-01

    Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75NTR. CNS neurons, but not glia, express SorCS2 as a single-cha...

  3. Pro‑apoptotic effects of pycnogenol on HT1080 human fibrosarcoma cells.

    Science.gov (United States)

    Harati, Kamran; Slodnik, Pawel; Chromik, Ansgar Michael; Behr, Björn; Goertz, Ole; Hirsch, Tobias; Kapalschinski, Nicolai; Klein-Hitpass, Ludger; Kolbenschlag, Jonas; Uhl, Waldemar; Lehnhardt, Marcus; Daigeler, Adrien

    2015-04-01

    Complete surgical resection with clear margins remains the mainstay of therapy for localised fibrosarcomas. Nevertheless, metastatic fibrosarcomas still represent a therapeutic dilemma. Commonly used chemotherapeutic agents like doxorubicin have proven to be effective in pycnogenol and its constituents on human fibrosarcoma cells (HT1080). Ten healthy subjects (six females, four males, mean age 24.8 ± 6 years) received a single dose of 300 mg pycnogenol orally. Blood plasma samples were obtained before and 6 h after intake of pycnogenol. HT1080 cells were treated with these plasma samples. Additionally, HT1080 were incubated separately with catechin, epicatechin and taxifolin that are known as the main constituents of pycnogenol. Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Gene expression was analyzed by RNA microarray. The results showed that single application of taxifolin, catechin and epicatechin reduced cell viability of HT1080 cells only moderately. A single dose of 300 mg pycnogenol given to 10 healthy adults produced plasma samples that led to significant apoptotic cell death ex vivo whereas pycnogenol-negative serum displayed no apoptotic activity. Microarray analysis revealed remarkable expression changes induced by pycnogenol in a variety of genes, which are involved in different apoptotic pathways of cancer cells [Janus kinase 1 (JAK1), DUSP1, RHOA, laminin γ1 (LAMC1), fibronectin 1 (FN1), catenin α1 (CTNNA1), ITGB1]. In conclusion, metabolised pycnogenol induces apoptosis in human fibrosarcoma cells. Pycnogenol exhibits its pro-apoptotic activity as a mixture and is more effective than its main constituents catechin, epicatechin and taxifolin indicating that the metabolised components interact synergistically. These results provide experimental support for in vivo trials assessing the effect of the pine bark extract pycnogenol. PMID:25625225

  4. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

    Science.gov (United States)

    Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-01-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  5. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  6. New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Alejandro Romero

    Full Text Available Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

  7. Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics

    Institute of Scientific and Technical Information of China (English)

    Ina O Specht; Marcello Spanò; Karin S Hougaard; Gian C Manicardi; Davide Bizzaro; Gunnar Toft; Aleksander Giwercman; Jens-Peter E Bonde

    2012-01-01

    Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ ceil population to numbers that can be supported by the Sertoli cells,and,possibly,selective depletion of meiotic and postmeiotic abnormal germ cells.We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men.Six hundred and four men from Greenland,Poland and Ukraine were consecutively enrolled during their pregnant wife's antenatal visits.Semen analysis was performed as recommended by the World Health Organization.Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell.DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.The percentage of Fas-positive sperm cells was higher in men with high total sperm count (P<O.01),more motile sperms (P=O.04) and fewer sperm head defects (P=O.05).These associations were consistent within and across study regions.Furthermore,testosterone,follicle-stimulating hormone (FSH) and sexual hormone-binding globulin (SHBG) were significantly negatively correlated with Fas within and across regions as well.The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics.The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarifv the biological significance of sperm apoptotic markers.

  8. Abl kinase inhibits the engulfment of apoptotic [corrected] cells in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Michael E Hurwitz

    2009-04-01

    Full Text Available The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.

  9. Biomolecular structure manipulation using tailored electromagnetic radiation: a proof of concept on a simplified model of the active site of bacterial DNA topoisomerase.

    Science.gov (United States)

    Jarukanont, Daungruthai; Coimbra, João T S; Bauerhenne, Bernd; Fernandes, Pedro A; Patel, Shekhar; Ramos, Maria J; Garcia, Martin E

    2014-10-21

    We report on the viability of breaking selected bonds in biological systems using tailored electromagnetic radiation. We first demonstrate, by performing large-scale simulations, that pulsed electric fields cannot produce selective bond breaking. Then, we present a theoretical framework for describing selective energy concentration on particular bonds of biomolecules upon application of tailored electromagnetic radiation. The theory is based on the mapping of biomolecules to a set of coupled harmonic oscillators and on optimal control schemes to describe optimization of temporal shape, the phase and polarization of the external radiation. We have applied this theory to demonstrate the possibility of selective bond breaking in the active site of bacterial DNA topoisomerase. For this purpose, we have focused on a model that was built based on a case study. Results are given as a proof of concept.

  10. TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo.

    Directory of Open Access Journals (Sweden)

    Fernando Gómez-Herreros

    Full Text Available Anticancer topoisomerase "poisons" exploit the break-and-rejoining mechanism of topoisomerase II (TOP2 to generate TOP2-linked DNA double-strand breaks (DSBs. This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2, an enzyme that hydrolyses 5'-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.

  11. c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1

    Directory of Open Access Journals (Sweden)

    Jézéquel Pascal

    2011-09-01

    Full Text Available Abstract Background Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. Methods We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. Results We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. Conclusions This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of

  12. Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

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    Ayelén Rayen Toro

    Full Text Available Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1 was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle

  13. Elevated Levels of Uterine Anti-Apoptotic Signaling May Activate NFKB and Potentially Confer Resistance to Caspase 3-Mediated Apoptotic Cell Death During Pregnancy in Mice1

    Science.gov (United States)

    Jeyasuria, Pancharatnam; Subedi, Kalpana; Suresh, Arvind; Condon, Jennifer C.

    2011-01-01

    Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis. This current study defines the mechanism whereby the pregnant uterine myocyte may harness the tocolytic activity of active caspases while avoiding apoptotic cell death. Using the pregnant mouse model, we have analyzed the uterus for changes in pro- and antiapoptotic signaling patterns associated with the advancing stages of pregnancy. Briefly, we have found that members of the IAP family, such as SURVIVIN and XIAP, and the Bcl2 family members, such as MCL1, are elevated in the uterine myocyte during late gestation. The IAP family members are the only endogenous inhibitors of active caspase 3, and MCL1 limits activation of caspase 3 by suppressing proapoptotic signaling. Elevated XIAP levels partner with SURVIVIN, resulting in increased levels of the antiapoptotic MCL1 via NFKB activation; these together have the potential to limit both the activity and level of active caspase 3 in the pregnant uterus as term approaches. We propose that modification of these antiapoptotic signaling partners allows the pregnant uterus to escape the apoptotic action of elevated active caspase 3 levels but also functions to limit the levels of active uterine caspase 3 near term. PMID:21566000

  14. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

    Energy Technology Data Exchange (ETDEWEB)

    Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp; Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp

    2015-07-17

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.

  15. CX3CL1(+ Microparticles Mediate the Chemoattraction of Alveolar Macrophages toward Apoptotic Acute Promyelocytic Leukemic Cells

    Directory of Open Access Journals (Sweden)

    Wen-Hui Tsai

    2014-02-01

    Full Text Available Background/Aims: During the resolution phase of inflammation, release of “find-me” signals by apoptotic cells is crucial in the chemoattraction of macrophages toward apoptotic cells for subsequent phagocytosis, in which microparticles derived from apoptotic cells (apo-MPs are involved. A recent study reports that CX3CL1 is released from apoptotic cells to stimulate macrophages chemotaxis. In this study, we investigated the role of CX3CL1 in the apo-MPs in the cell-cell interaction between alveolar macrophage NR8383 cells and apoptotic all-trans retinoic acid-treated NB4 (ATRA-NB4 cells. Methods/Results: Apoptotic ATRA-NB4 cells and their conditioning medium (CM enhanced the chemoattraction of NR8383 cells as well as their phagocytosis activity in engulfing apoptotic ATRA-NB4 cells. The levels of CX3CL1(+ apo-MPs and CX3CL1 were rapidly elevated in the CM of ATRA-NB4 cell culture after induction of apoptosis. Both exogenous CX3CL1 and apo-MPs enhanced the transmigration of NR8383 cells toward apoptotic ATRA-NB4 cells. This pro-transmigratory activity was able to be partially inhibited either by blocking the CX3CR1 (CX3CL1 receptor of NR8383 cells with its specific antibody or by blocking the surface CX3CL1 of apo-MPs with its specific antibody before incubating these apo-MPs with NR8383 cells. Conclusion: CX3CL1(+ apo-MPs released by apoptotic cells mediate the chemotactic transmigration of alveolar macrophages.

  16. The apoptotic effects of escin in the H-Ras transformed 5RP7 cell line.

    Science.gov (United States)

    Güney, G; Kutlu, H M; Işcan, A

    2013-06-01

    Extracts of Aesculus hippocastanum L. (horse chestnut) seed have been used in the treatment of chronic venous insufficiency, edema and hemorrhoids. Most of the beneficial effects of horse chestnut are attributed to its principal component β-escin or escin. We have evaluated the cytotoxic and apoptotic effects of escin in the H-Ras 5RP7 cell line by analyzing cell growth inhibition, apoptosis and caspase-3 dependent activity. We have also shown structural and ultrastructural changes in these cell using confocal and transmission electron microscopy. The results indicated that escin has significant inhibitory effects on cell growth and the percentage of apoptotic cells increased after treatment with escin, and the micrographs confirmed that escin damaged these cells and induced apoptosis. PMID:22911540

  17. Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

    Directory of Open Access Journals (Sweden)

    Maria Dolores Boyano

    2011-03-01

    Full Text Available Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies.

  18. Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

    Science.gov (United States)

    Ishaq, M.; Bazaka, K.; Ostrikov, K.

    2015-11-01

    Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

  19. Nucleo-cytoplasmic communication in apoptotic response to genotoxic and inflammatory stress

    Institute of Scientific and Technical Information of China (English)

    Jean Y. J. WANG

    2005-01-01

    Genotoxic agents or inflammatory cytokines activate cellular stress responses and trigger programmed cell death.We have identified a signal transduction module, including three nuclear proteins that participate in the regulation of cell death induced by chemotherapeutic agents and tumor necrosis factor (TNF). In this nuclear signaling module, retinoblastoma protein (Rb) functions as an inhibitor of apoptotic signal transduction. Inactivation of Rb by phosphorylation or caspase-dependent cleavage/degradation is required for cell death to occur. Rb inhibits the Abl tyrosine kinase. Thus,Rb inactivation is a pre-requisite for Abl activation by DNA damage or TNF. Activation of nuclear Abl and its downstream effector p73 induces mitochondriadependent cell death. The involvement of these nuclear signal transducers in TNF induced apoptosis, which does not require new gene expression, indicates that nuclear events other than transcription can contribute to extrinsic apoptotic signal transduction.

  20. Apoptotic gene expression in the neural tube during early human embryonic development

    Institute of Scientific and Technical Information of China (English)

    Guifang Chen; Tiandong Li; Peipei Ding; Ping Yang; Xiao Zhang

    2011-01-01

    Neural tube development comprises neural induction,neural epithelial cell proliferation,and apoptosis,as well as migration of nerve cells.Too much or too little apoptosis leads to abnormal nervous system development.The present study analyzed expression and distribution of apoptotic-related factors,including Fas,FasL,and caspase-3,during human embryonic neural tube development.Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondriai-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development.Subsequently,Fas and FasL expression increased during embryonic development.The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways.Therefore,neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.

  1. The nuclear γ-H2AX apoptotic ring: implications for cancers and autoimmune diseases.

    Science.gov (United States)

    Solier, Stéphanie; Pommier, Yves

    2014-06-01

    Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers and to the outcome of cancer chemotherapies, as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by "death receptors" and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, which microscopically looks like a nuclear annular staining early in apoptosis. This ring is, in three-dimensional space, a thick intranuclear shell consisting of epigenetic modifications including histone H2AX and DNA damage response (DDR) proteins. It excludes the DNA repair factors usually associated with γ-H2AX in the DDR nuclear foci. Here, we summarize our knowledge of the apoptotic ring, and discuss its biological and pathophysiological relevance, as well as its value as a potential pharmacodynamic biomarker for anticancer therapies. PMID:24448903

  2. Tuning the anticancer activity of a novel pro-apoptotic peptide using gold nanoparticle platforms

    Science.gov (United States)

    Akrami, Mohammad; Balalaie, Saeed; Hosseinkhani, Saman; Alipour, Mohsen; Salehi, Fahimeh; Bahador, Abbas; Haririan, Ismaeil

    2016-01-01

    Pro-apoptotic peptides induce intrinsic apoptosis pathway in cancer cells. However, poor cellular penetration of the peptides is often associated with limited therapeutic efficacy. In this report, a series of peptide-gold nanoparticle platforms were developed to evaluate the anticancer activity of a novel alpha-lipoic acid-peptide conjugate, LA-WKRAKLAK, with respect to size and shape of nanoparticles. Gold nanoparticles (AuNPs) were found to enhance cell internalization as well as anticancer activity of the peptide conjugates. The smaller nanospheres showed a higher cytotoxicity, morphological change and cellular uptake compared to larger nanospheres and nanorods, whereas nanorods showed more hemolytic activity compared to nanospheres. The findings suggested that the anticancer and biological effects of the peptides induced by intrinsic apoptotic pathway were tuned by peptide-functionalized gold nanoparticles (P-AuNPs) as a function of their size and shape. PMID:27491007

  3. Proposed Pharmacological Countermeasures Against Apoptotic Cell Death in Experimental Models Mimicking Space Environment Damage

    Science.gov (United States)

    Lulli, Matteo; Papucci, Laura; Witort, Ewa; Donnini, Martino; Lapucci, Andrea; Lazzarano, Stefano; Mazzoni, Tiziano; Simoncini, Madine; Falciani, Piergiuseppe; Capaccioli, Sergio

    2008-06-01

    Several damaging agents have been suggested to affect human vision during long term space travels. Recently, apoptosis induced by DNA-damaging agents has emerged as frequent pathogenetic mechanism of ophthalmologic pathologies. Here, we propose two countermeasures: coenzyme Q10 and bcl-2 downregulation preventing antisense oligoribonucleotides (ORNs), aimed to inhibit cellular apoptotic death. Our studies have been carried out on retina and neuronal cultured cells treated with the following apoptotic stimuli mimicking space environment: a several-day exposure to either 3H-labeled tymidine or to the genotoxic drug doxorubicin, UV irradiation, hypoxia and glucose/growth factor starvation (Locke medium). The preliminary results clearly indicate that CoQ10, as well as bcl-2 down-regulation preventing ORNs, significantly counteract apoptosis in response to different DNA damaging agents in cultured eye and in neuronal cells. This supports the possibility that both could be optimal countermeasures against ophthalmologic lesions during space explorations.

  4. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    Directory of Open Access Journals (Sweden)

    Lucilia B. Lepsch

    2015-01-01

    Full Text Available To study cocaine’s toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2 and/or neuronal nucleus protein (NeuN staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine.

  5. Thapsigargin increases apoptotic cell death in human hepatoma BEL—7404 cells

    Institute of Scientific and Technical Information of China (English)

    GUJUN; HELIU

    1995-01-01

    Effects of thapsigargin,an inhibitor of Ca2+-ATPase in surface of endoplasmic reticulum,on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron microscopy.Propidium iodide staining and flow cytometry revealed that in the serum-free condition,thapsigargin increased the rate of apoptosis of BEL-7404 cells in a dose-dependent manner.Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment.Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation,apoptotic bodies existed in TG-treated cells,supporting that thapsigargin is a potent activator of apoptosis in the cells.

  6. The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens

    DEFF Research Database (Denmark)

    Straten, Per thor; Andersen, Mads Hald

    2010-01-01

    Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti......, spontaneous cellular immune responses against the Bcl-2 family proteins have been identified as frequent features in cancer patients underscoring that these proteins are natural targets for the immune system. Thus, Bcl-2 family may serve as an important and widely applicable target for anti......-cancer immunotherapeutic strategies, alone or in the combination with conventional therapy. Here, we summarize the current knowledge of Bcl-2 family proteins as T-cell antigens, which has set the stage for the first explorative trial using these antigens in therapeutic vaccinations against cancer, and discuss future...

  7. Sulbutiamine counteracts trophic factor deprivation induced apoptotic cell death in transformed retinal ganglion cells.

    Science.gov (United States)

    Kang, Kui Dong; Majid, Aman Shah Abdul; Kim, Kyung-A; Kang, Kyungsu; Ahn, Hong Ryul; Nho, Chu Won; Jung, Sang Hoon

    2010-11-01

    Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B(1) and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture. PMID:20809085

  8. The gastroprotective effect of menthol: involvement of anti-apoptotic, antioxidant and anti-inflammatory activities.

    Directory of Open Access Journals (Sweden)

    Ariane Leite Rozza

    Full Text Available The aim of this research was to investigate the anti-apoptotic, antioxidant and anti-inflammatory properties of menthol against ethanol-induced gastric ulcers in rats. Wistar rats were orally treated with vehicle, carbenoxolone (100 mg/kg or menthol (50 mg/kg and then treated with ethanol to induce gastric ulcers. After euthanasia, stomach samples were prepared for histological slides and biochemical analyses. Immunohistochemical analyses of the cytoprotective and anti-apoptotic heat-shock protein-70 (HSP-70 and the apoptotic Bax protein were performed. The neutrophils were manually counted. The activity of the myeloperoxidase (MPO was measured. To determine the level of antioxidant functions, the levels of glutathione (GSH, glutathione peroxidase (GSH-Px, glutathione reductase (GR and superoxide dismutase (SOD were measured using ELISA. The levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 and the anti-inflammatory cytokine interleukin-10 (IL-10 were assessed using ELISA kits. The menthol treated group presented 92% gastroprotection compared to the vehicle-treated group. An increased immunolabeled area was observed for HSP-70, and a decreased immunolabeled area was observed for the Bax protein in the menthol treated group. Menthol treatment induced a decrease in the activity of MPO and SOD, and the protein levels of GSH, GSH-Px and GR were increased. There was also a decrease in the levels of TNF-α and IL-6 and an increase in the level of IL-10. In conclusion, oral treatment with menthol displayed a gastroprotective activity through anti-apoptotic, antixidant and anti-inflammatory mechanisms.

  9. Reemergence of apoptotic cells between fractionated doses in irradiated murine tumors

    International Nuclear Information System (INIS)

    The purpose of this investigation was to follow up our previous studies on the development of apoptosis in irradiated murine tumors by testing whether an apoptotic subpopulation of cells reemerges between fractionated exposures. Mice bearing a murine ovarian carcinoma, OCa-I, were treated in vivo with two fractionation protocols: two doses of 12.5 Gy separated by various times out to 5 days and multiple daily fractions of 2.5 Gy. Animals were killed 4 h after the last dose in each protocol, and the percent apoptosis was scored from stained histological sections made from the irradiated tumors according to the specific features characteristic of this mode of cell death. The 12.5+12.5 Gy protocol yielded a net total percent apoptosis of about 45% when the two doses were separated by 5 days (total dose = 25 Gy), whereas the 2.5 Gy per day protocol yielded about 50% net apoptotic cells when given for 5 days (total dose = 12.5 Gy). These values are to be compared to the value of 36% apoptotic cells that is yielded by large single doses (> 25 Gy). Thus, these results indicate that an apoptotic subpopulation of cells reemerged between the fractions in both protocols, but the kinetics appeared to be delayed in the 12.5+12.5 Gy vs. the multiple 2.5 Gy protocol. This reemergence of cells with the propensity for radiation-induced apoptosis between fractionated exposures is consistent with a role for this mode of cell death in the response of tumors to radiotherapy and may represent the priming of a new subpopulation of tumor cells for apoptosis as part of normal tumor homeostasis to counterbalance cell division. 25 refs., 3 figs., 1 tab

  10. Oncogenic Properties of Apoptotic Tumor Cells in Aggressive B Cell Lymphoma

    Science.gov (United States)

    Ford, Catriona A.; Petrova, Sofia; Pound, John D.; Voss, Jorine J.L.P.; Melville, Lynsey; Paterson, Margaret; Farnworth, Sarah L.; Gallimore, Awen M.; Cuff, Simone; Wheadon, Helen; Dobbin, Edwina; Ogden, Carol Anne; Dumitriu, Ingrid E.; Dunbar, Donald R.; Murray, Paul G.; Ruckerl, Dominik; Allen, Judith E.; Hume, David A.; van Rooijen, Nico; Goodlad, John R.; Freeman, Tom C.; Gregory, Christopher D.

    2015-01-01

    Summary Background Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. Results Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. Conclusions In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy. PMID:25702581

  11. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    OpenAIRE

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Bur...

  12. The Nuclear γ-H2AX Apoptotic Ring: Implications for Cancers and Autoimmune Diseases

    OpenAIRE

    Solier, Stéphanie; Pommier, Yves

    2014-01-01

    Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers, and the outcome of cancer chemotherapies as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by “death receptors” and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, whi...

  13. Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

    OpenAIRE

    Sesso, A.; Belizário, JE; Marques, MM; Higuchi, ML; Schumacher, RI; Colquhoun, A; Ito, E.; Kawakami, J.

    2012-01-01

    Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM ru...

  14. Apoptotic effects of the 'designer drug' methylenedioxypyrovalerone (MDPV) on the neonatal mouse brain.

    Science.gov (United States)

    Adám, Agota; Gerecsei, László István; Lepesi, Nikolett; Csillag, András

    2014-09-01

    The designer drug of cathinone family, methylenedioxypyrovalerone (MDPV), is a cheap and frequently used psychoactive drug of abuse. However, its mechanism of action, particularly its potential detrimental effect on the developing brain, is largely unknown, despite the fact that pregnant females may occur among the users. The objective of our study was to identify the brain areas sensitive for a possible apoptotic effect of the widely abused MDPV on the developing brain. To this end, we used a mouse model which can be compared with the human fetus of third trimester, considering the developmental stage of the brain. Litters of 7-day-old C57BL/6J mice were treated either with i.p. injection of 10mg/kg b.wt.of MDPV or vehicle (saline), and sacrificed after 24h. Similar dose of MDPV enhanced locomotor activity of pups. The brains were processed for anti-caspase 3 (Casp3) immunohistochemistry and the apoptotic cells were identified and counted. We found prominent increase in the number of apoptotic cells in the piriform cortex, retrosplenial area, hippocampus CA1 and nucleus accumbens, whereas the overall density of cells did not change significantly in these regions. The neurons of the nucleus accumbens appeared to be especially sensitive to MDPV: Casp3-immunoreactive cells marked out the core and shell regions of the accumbens. Highest percentage of apoptotic cells as compared to total cell density was also found in the nucleus accumbens. However, we did not observe the same effect on the brain of adult mice. Thus, MDPV did not seem to increase apoptosis in the mature nervous system. The results are in agreement with the assumption that cathinones (in particular MDPV) may adversely affect neural integrity in the developing CNS.

  15. Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation

    OpenAIRE

    Shepelev, Mikhail V; Chernoff, Jonathan; Korobko, Igor V

    2011-01-01

    Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by...

  16. Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

    Science.gov (United States)

    Deegan, Shane; Saveljeva, Svetlana; Logue, Susan E; Pakos-Zebrucka, Karolina; Gupta, Sanjeev; Vandenabeele, Peter; Bertrand, Mathieu J M; Samali, Afshin

    2014-01-01

    Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells. PMID:25470234

  17. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    OpenAIRE

    Lepsch, Lucilia B; Planeta, Cleopatra S.; Critoforo Scavone

    2015-01-01

    To study cocaine’s toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mec...

  18. Effect of ethanol on pro-apoptotic mechanisms in polarized hepatic cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover,it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcoholinduced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.

  19. Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Baeza-Squiban Armelle

    2010-04-01

    Full Text Available Abstract Background Increasing environmental and occupational exposures to nanoparticles (NPs warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB and titanium dioxide (TiO2 NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm and TiO2(15 nm NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation and biochemical (caspase activation and DNA fragmentation features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2 production was only involved in apoptosis induction by CB NPs. Conclusions Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis, the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

  20. Sculpting Skin Appendages Out of Epidermal Layers Via Temporally and Spatially Regulated Apoptotic Events

    OpenAIRE

    Chang, Chung-Hsing; Yu, Mingke; Wu, Ping; Jiang, Ting-Xin; Yu, Hsin-Su; Widelitz, Randall B.; Chuong, Cheng-Ming

    2004-01-01

    Complex skin appendages are built from the epidermal cells through induction, cell fate specification, proliferation, apoptosis, and differentiation, etc. Here we used the TUNEL assay and caspase-3 immuno-localization to examine apoptotic events in different stages of feather morphogenesis. We deduced three modes through which apoptosis may impact morphogenesis. In Mode 1A, apoptosis occurs within the localized growth zone to regulate growth as seen in growing buds. In Mode 1B, morphogen secr...

  1. Apoptotic factors in physiological and pathological processes of teeth and periodontal tissues – literature review

    Directory of Open Access Journals (Sweden)

    Orzedala-Koszel Urszula

    2014-12-01

    Full Text Available Apoptosis is a physiological process that occurs in the human body throughout the entire life span. This process can be seen in the tissues of the stomatognathic system. A disorder in such programmed cell death processes leads to the development of pathological lesions. Among these are inflammation, osteolytic lesions and neoplastic hyperplasia. We put forward that future studies should concentrate on how to use the knowledge of apoptotic processes and their inhibitors in therapeutic processes involving the stomatognathic system.

  2. BAG1: the guardian of anti-apoptotic proteins in acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Sanja Aveic

    Full Text Available BCL2 associated Athano-Gene 1 (BAG1 is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.

  3. Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

    Institute of Scientific and Technical Information of China (English)

    XIU Meihong; PENG Jianxin; HONG Huazhu

    2005-01-01

    Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (△Ψm) began to decrease in SL-1 cells at 4 h post infection and △Ψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was down- regulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrion-mediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.

  4. Antitumor effects of traditional Chinese medicine targeting the cellular apoptotic pathway

    Directory of Open Access Journals (Sweden)

    Xu HL

    2015-05-01

    Full Text Available Huanli Xu,1 Xin Zhao,2 Xiaohui Liu,1 Pingxiang Xu,1 Keming Zhang,2 Xiukun Lin11Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, 2Department of Hepatobiliary Surgery, 302 Hospital of Chinese People’s Liberation Army, Beijing, People’s Republic of ChinaAbstract: Defects in apoptosis are common phenomena in many types of cancer and are also a critical step in tumorigenesis. Targeting the apoptotic pathway has been considered an intriguing strategy for cancer therapy. Traditional Chinese medicine (TCM has been used in the People’s Republic of China for thousands of years, and many of the medicines have been confirmed to be effective in the treatment of a number of tumors. With increasing cancer rates worldwide, the antitumor effects of TCMs have attracted more and more attention globally. Many of the TCMs have been shown to have antitumor activity through multiple targets, and apoptosis pathway-related targets have been extensively studied and defined to be promising. This review focuses on several antitumor TCMs, especially those with clinical efficacy, based on their effects on the apoptotic signaling pathway. The problems with and prospects of development of TCMs as anticancer agents are also presented.Keywords: traditional Chinese medicine, antitumor effects, apoptotic pathway

  5. Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

    Science.gov (United States)

    Bhat, Vikas; Kurouski, Dmitry; Olenick, Max B; McDonald, Caleb B; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2012-12-01

    Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues. PMID:22960132

  6. Apoptotic cell death, detected ex vivo in peripheral blood lymphocytes of HIV-1 infected persons

    Directory of Open Access Journals (Sweden)

    L. F. te Velde

    1996-01-01

    Full Text Available In HIV-1 infection the ongoing depletion of CD4+ T-lymphocytes is believed, to a large extent, to be due to apoptosis. Until now quantitative information about in vivo apoptosis of lymphocytes in HIV-patients is scarce because of the very nature of the apoptotic process. Successful detection of apoptosis ex vivo requires the recognition of the initial phase of this process, because at a later stage the cells may not remain any longer in the circulation. We measured quantitatively the amount of early apoptotic peripheral blood lymphocytes directly ex vivo in HIV-1 infected patients using a recently described flow cytometric assay. With this method we observed in an unselected heterogenous group of twelve HIV-infected individuals a median percentage of apoptotic lymphocytes to be significantly higher than in ten healthy controls. To the best of our knowledge this is the first report of ex vivo observed increased apoptosis of peripheral blood lymphocytes in HIV-infected persons.

  7. p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

    Directory of Open Access Journals (Sweden)

    Paramita Banerjee Sawant

    Full Text Available Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD, leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD, ultimately resulting in significant (p<0.05 embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

  8. Isolation of apoptotic mouse fetal oocytes by AnnexinV assay.

    Science.gov (United States)

    Lobascio, Anna-Maria; Klinger, Francesca-Gioia; De Felici, Massimo

    2007-01-01

    Expression of phosphotidylserine by fetal oocytes in culture renders significant numbers of such cells able to bind AnnexinV-coated microbeads and allows their separation from Annexin V-negative oocytes on a Magnetic Cell Separation (MACS) column in a magnetic field. The majority of oocytes (> or =75%) which bound Annexin V-coated microbeads were viable, as indicated by their propidium iodine (PI) negativity. However, they showed apoptotic morphologies and were found to be TUNEL-positive. On the other hand, AnnexinV-negative oocytes, besides being PI negative, appeared morphologically healthy and TUNEL negative. Moreover, AnnexinV-positive oocytes showed a marked lower ratio of Bcl-xL/Bax transcripts in comparison to AnnexinV-negative oocytes. We conclude that the present method is able to separate fetal oocytes in two distinct populations: AnnexinV-positive oocytes showing features typical of apoptotic cells and AnnexinV-negative oocytes comprising for the most part viable non-apoptotic cells. This procedure should greatly facilitate studies aimed to identify the currently poorly understood molecular pathways governing apoptosis in mammalian fetal oocytes. PMID:17294366

  9. The proteasomal and apoptotic phenotype determine bortezomib sensitivity of non-small cell lung cancer cells

    Directory of Open Access Journals (Sweden)

    Chęcińska Agnieszka

    2007-11-01

    Full Text Available Abstract Bortezomib is a novel anti-cancer agent which has shown promising activity in non-small lung cancer (NSCLC patients. However, only a subset of patients respond to this treatment. We show that NSCLC cell lines are differentially sensitive to bortezomib, IC50 values ranging from 5 to 83 nM. The apoptosis-inducing potential of bortezomib in NSCLC cells was found to be dependent not only on the apoptotic phenotype but also on the proteasomal phenotype of individual cell lines. Upon effective proteasome inhibition, H460 cells were more susceptible to apoptosis induction by bortezomib than SW1573 cells, indicating a different apoptotic phenotype. However, exposure to a low dose of bortezomib did only result in SW1573 cells, and not in H460 cells, in inhibition of proteasome activity and subsequent apoptosis. This suggests a different proteasomal phenotype as well. Additionally, overexpression of anti-apoptotic protein Bcl-2 in H460 cells did not affect the proteasomal phenotype of H460 cells but did result in decreased bortezomib-induced apoptosis. In conclusion, successful proteasome-inhibitor based treatment strategies in NSCLC face the challenge of having to overcome apoptosis resistance as well as proteasomal resistance of individual lung cancer cells. Further studies in NSCLC are warranted to elucidate underlying mechanisms.

  10. Gene therapy for carcinoma of the breast: Pro-apoptotic gene therapy

    International Nuclear Information System (INIS)

    The dysregulation of apoptosis contributes in a variety of ways to the malignant phenotype. It is increasingly recognized that the alteration of pro-apoptotic and anti-apoptotic molecules determines not only escape from mechanisms that control cell cycle and DNA damage, but also endows the cancer cells with the capacity to survive in the presence of a metabolically adverse milieu, to resist the attack of the immune system, to locally invade and survive despite a lack of tissue anchorage, and to evade the otherwise lethal insults induced by drugs and radiotherapy. A multitude of apoptosis mediators has been identified in the past decade, and the roles of several of them in breast cancer have been delineated by studying the clinical correlates of pathologically documented abnormalities. Using this information, attempts are being made to correct the fundamental anomalies at the genetic level. Fundamental to this end are the design of more efficient and selective gene transfer systems, and the employment of complex interventions that are tailored to breast cancer and that are aimed concomitantly towards different components of the redundant regulatory pathways. The combination of such genetic modifications is most likely to be effective when combined with conventional treatments, thus robustly activating several pro-apoptotic pathways

  11. Proliferative and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line

    Institute of Scientific and Technical Information of China (English)

    LI Shu-guang; WANG Yuan-yu; YE Zai-yuan; SHAO Qing-shu; TAO Hou-quan; SHU Li-sha; ZHAO Yi-feng

    2013-01-01

    Background Andrographolide has been shown to have anticancer activity on diverse cancer cell lines representing different types of human cancers.The aim of this research was to investigate the anticancer and apoptotic effects of andrographolide on the BGC-823 human gastric cancer cell line.Methods Cell proliferation and IC50 were evaluated using MTT assay,cell-cycle analysis with flow cytometry apoptotic effects with Annexin-V/propidium iodide double-staining assay,and morphologic structure with transmission electron microscopy.Immunohistochemistry and reverse-transcription PCR was used to analyze Bcl-2,Bax,and caspase-3 expressions.Results Andrographolide showed a time-and concentration-dependent inhibitory effects on BGC-823 cell growth.Compared to controls,the number of cells in the G0-G1-phase increased significantly,S and G2-M-phase cells decreased after 48 hours of treatment with andrographolide,and both early and late apoptotic rates increased significantly compared to the controls,all in a concentration-dependent manner.Bax and caspase-3 expressions were markedly increased,and Bcl-2 expression was decreased.Conclusions Andrographolide inhibits BGC-823 cell growth and induces BGC-823 cell apoptosis by up-regulating Bax and caspase-3 expressions and down-regulating Bcl-2 expression.Andrographolide may be useful as a potent and selective agent in the treatment of human gastric cancers.

  12. Increased ratio of anti-apoptotic to pro-apoptotic Bcl2 gene-family members in lithium-responders one month after treatment initiation

    Directory of Open Access Journals (Sweden)

    Lowthert Lori

    2012-09-01

    Full Text Available Abstract Background Lithium is considered by many as the gold standard medication in the management of bipolar disorder (BD. However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response. Methods Illumina Sentrix Beadchip (Human-6v2 microarrays containing > 48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8 weeks of open-label treatment with lithium. Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more and non-responders. Pathway analysis was conducted using GeneGO Metacore software. Results 127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2 were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1 and BCL2-associated agonist of cell death (BAD, were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point. Conclusions These results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of

  13. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B;

    1999-01-01

    -AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change......Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m...... demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost...

  14. Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

    Science.gov (United States)

    Lindenboim, L; Yuan, J; Stein, R

    2000-03-30

    Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by coexpression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants deltaC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 ml-3 or deltaC22 mutants. These

  15. Investigation of the apoptotic pathway induced by benzimidazole-oxindole conjugates against human breast cancer cells MCF-7.

    Science.gov (United States)

    Lakshma Nayak, Vadithe; Nagaseshadri, Bobburi; Vishnuvardhan, M V P S; Kamal, Ahmed

    2016-07-15

    In our previous studies, benzimidazole-oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP. PMID:27262596

  16. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop.

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V; Greene, Lloyd A

    2005-11-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  17. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  18. Altered expression of apoptotic genes in response to OCT4B1 suppression in human tumor cell lines.

    Science.gov (United States)

    Mirzaei, Mohammad Reza; Najafi, Ali; Arababadi, Mohammad Kazemi; Asadi, Malek Hosein; Mowla, Seyed Javad

    2014-10-01

    OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1. PMID:25008565

  19. The roles and acting mechanism of Caenorhabditis elegans DNase II genes in apoptotic dna degradation and development.

    Directory of Open Access Journals (Sweden)

    Huey-Jen Lai

    Full Text Available DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3' OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3' OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation.

  20. In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

    LENUS (Irish Health Repository)

    Murphy, Therese M

    2011-01-01

    Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

  1. In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus.

    Science.gov (United States)

    Lepak, A J; Seiler, P; Surivet, J P; Ritz, D; Kohl, C; Andes, D R

    2016-06-01

    ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be

  2. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zeilstra, Jurrit; Joosten, Sander P.J. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Wensveen, Felix M. [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Dessing, Mark C.; Schuetze, Denise M. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Eldering, Eric [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Spaargaren, Marcel [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Pals, Steven T., E-mail: s.t.pals@amc.uva.nl [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)

    2011-03-04

    Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causes constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which

  3. HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

    Science.gov (United States)

    Vasaikar, Suhas V; Ghosh, Sourish; Narain, Priyam; Basu, Anirban; Gomes, James

    2015-01-01

    Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NFκB determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.

  4. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    International Nuclear Information System (INIS)

    Research highlights: → Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. → Expression profiling of apoptosis-related genes in ApcMin/+ mice revealed the differential expression of pro-apoptotic Bok and Bax. → APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. → Blocking of β-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or β-catenin causes constitutively active β-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the ApcMin/+ mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of β-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which uncontrolled epithelial cell proliferation in the stem

  5. Autoantibodies against complement C1q specifically target C1q bound on early apoptotic cells.

    Science.gov (United States)

    Bigler, Cornelia; Schaller, Monica; Perahud, Iryna; Osthoff, Michael; Trendelenburg, Marten

    2009-09-01

    Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q. PMID:19648280

  6. Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

    Science.gov (United States)

    Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

    2016-01-01

    Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

  7. Expression of leptin and its receptor in female breast cancer in relation with selected apoptotic markers.

    Directory of Open Access Journals (Sweden)

    Stanislaw Sulkowski

    2008-04-01

    Full Text Available Leptin and its receptor may be engaged in pathogenesis of breast cancer among various human tumors. In vitro investigations showed leptin-mediated escalation of estrogen synthesis and boosted activity of estrogen receptor ERalpha. Furthermore, leptin induced growth of malignant cells, counteracted apoptosis and stimulated cell migration as well as overexpression of angiogenic factors and degrading enzymes that split network of intercellular matrix. On the other side, leptin has been reported to favor apoptosis, lately. Proapoptotic effect of leptin action was revealed in interstitial cells of bone marrow and adipocytes. Our past reports provide evidences for overexpression of leptin and its receptor in breast cancer in comparison with benign mammary lesions. In current study we aimed at assessment of eventual relationships between leptin, leptin receptor and selected protein regulators of apoptosis in breast cancer. We applied immunohistochemistry for leptin, leptin receptor, anti-apoptotic Bcl-2 and Bcl-xL as well as pro-apoptotic Bak and Bax expression assessment in 106 cases of human breast cancers. The immunoreaction was graded and statistically evaluated. Expression of leptin was positively correlated with Bcl-xL, Bak and Bax (p<0.001, r=0.614; p<0.001, r=0.518; p<0.001, r=0.511, respectively. Statistical significances were noted between expression of leptin receptor and Bcl-xL or Bax (p=0.011, r=0.210; p<0.001, r=0.313, respectively. No correlation was encountered between leptin and Bcl-2, either leptin receptor and Bcl-2 or leptin receptor and Bak. On the basis of obtained results, leptin system could interfere in balance among expressions of pro- and anti-apoptotic proteins and regulate cell turnover and--by means of it--facilitate breast cancer progression.

  8. Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia

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    Roytrakul Sittiruk

    2011-06-01

    Full Text Available Abstract Background Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. Methods The phosphoproteome of bone marrow HSCs/CD34+ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34+ cells were compared with HbE/β-thalassemia and normal HSCs. Results A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia. Conclusions Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.

  9. Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

    Science.gov (United States)

    Rodríguez-Hernández, A; Navarro-Villarán, E; González, R; Pereira, S; Soriano-De Castro, L B; Sarrias-Giménez, A; Barrera-Pulido, L; Álamo-Martínez, J M; Serrablo-Requejo, A; Blanco-Fernández, G; Nogales-Muñoz, A; Gila-Bohórquez, A; Pacheco, D; Torres-Nieto, M A; Serrano-Díaz-Canedo, J; Suárez-Artacho, G; Bernal-Bellido, C; Marín-Gómez, L M; Barcena, J A; Gómez-Bravo, M A; Padilla, C A; Padillo, F J; Muntané, J

    2015-12-01

    Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

  10. Methane attenuates retinal ischemia/reperfusion injury via anti-oxidative and anti-apoptotic pathways.

    Science.gov (United States)

    Liu, Lin; Sun, Qinglei; Wang, Ruobing; Chen, Zeli; Wu, Jiangchun; Xia, Fangzhou; Fan, Xian-Qun

    2016-09-01

    Retinal ischemia/reperfusion injury (IRI) may cause incurable visual impairment due to neural regeneration limits. Methane was shown to exert a protective effect against IRI in many organs. This study aims to explore the possible protective effects of methane-rich saline against retinal IRI in rat. Retinal IRI was performed on the right eyes of male Sprague-Dawley rats, which were immediately injected intraperitoneally with methane-saturated saline (25ml/kg). At one week after surgery, the number of retinal ganglion cells (RGCs), total retinal thickness, visual function were measured by hematoxylin and eosin staining, FluoroGold anterograde labeling and flash visual evoked potentials. The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-Hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), caspase-3, caspase-9, B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in retinas were assessed by immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. As expected, methane treatment significantly improved the retinal IRI-induced RGC loss, total retinal layer thinning and visual dysfunction. Moreover, methane treatment significantly reduced the levels of oxidative stress biomarkers (8-OHdG, 4-HNE, MDA) and increased the antioxidant enzyme activities (SOD, CAT, GPx) in the retinas with IRI. Meanwhile, methane treatment significantly increased the anti-apoptotic gene (Bcl-2) expression and decreased the pro-apoptotic gene (Bax) expression, accompanied by the suppression of caspase-3 and caspase-9 activity. Thus, these data demonstrated that methane can exert a neuroprotective role against retinal IRI through anti-oxidative and anti-apoptotic pathways. PMID:27208496

  11. Apoptotic-like programmed cell death in fungi: the benefits in filamentous species

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    Neta eShlezinger

    2012-08-01

    Full Text Available Studies conducted in the early 1990's showed for the first time that Saccahromyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologues of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with ageing and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programmed cell death (PCD instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts and multi-cellular (filamentous species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis.

  12. Clinical significance of expression of apoptotic signal proteins in gastric carcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    Xin-Han Zhao; Shan-Zhi Gu; Hong-Gang Tian; Ping Quan; Bo-Rong Pan

    2005-01-01

    AIM: To evaluate the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFκB in gastric carcinoma tissues and their clinical significance.METHODS: Western blot immune trace method was adopted to detect the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFκB in 55 tissue specimens of gastric carcinoma.RESULTS: Five apoptotic signal proteins had different expressions in the gastric carcinoma samples and their expressions were not correlated to age (P = 0.085).Expressions of the FADD, FasL, Fas, and NFκB proteins reduced with increase of the volume of tumor with the exception of increased expression the TRADD protein (64.7-71.1%, P = 0.031). With gradual increase of the malignancy of gastric carcinoma tissues, expressions of the FADD, FasL, and Fas proteins decreased (78.6-28.0%,P= 0.008; 78.6-65.9%, P= 0.071; 100.0-46.3%, P= 0.014),while expressions of the TRADD and NFκB proteins increased (42.9-78.1%, P= 0.063; 78.6-79.1%, P= 0.134).With gradual increase of serum CEA, expression of the FADD protein decreased (62.5-34.0%, P = 0.073), but expressions of the TRADD, FasL, Fas, and NFκB proteins increased (0.0-80.8%, P = 0.005; 62.5-70.2%, P = 0.093;0.0-70.2%, P = 0.003; 62.5-80.9%, P = 0.075). When compared to the tissues of gastric carcinoma without metastasis, the positive rate of expressions of the FADD and FasL proteins increased, whereas expressions of the TRADD, FADD, and NFκB proteins decreased. There was no significant difference between them (P = 0.095).CONCLUSION: Gastric carcinoma is endurable to Fasrelated apoptosis and apoptotic signal proteins are differently expressed in gastric carcinoma.

  13. The role of PGC-1alpha on mitochondrial function and apoptotic susceptibility in muscle

    DEFF Research Database (Denmark)

    Adhihetty, Peter J; Uguccioni, Giulia; Leick, Lotte;

    2009-01-01

    Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome...... oxidative heart, indicating a change in mitochondrial composition. A change in muscle organelle composition was also evident from the alterations in subsarcolemmal and intermyofibrillar mitochondrial respiration, which was impaired in the absence of PGC-1alpha. However, endurance-trained KO animals did...

  14. Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

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    Rossella Crescitelli

    2013-09-01

    Full Text Available Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and

  15. Anti-apoptotic effects of aspirin following cerebral ischemia-reperfusion injury in rats

    Institute of Scientific and Technical Information of China (English)

    Liying Qiu; Bin Du; Ying Li; Hongbin Fan; Zhiyong Yang

    2008-01-01

    BACKGROUND: The pharmacological effects of aspirin on apoptosis are complex. The underlying mechanisms have not been properly defined. OBJECTIVE: To observe the effect of different doses of aspirin on brain cell apoptosis following focal cerebral ischemia-reperfusion injury (CIRI) in rats. DESING, TIME AND SETTING: A randomized, controlled, animal experiment, performed at the School of Medicine and Pharmaceutics, Jiangnan University between June and October 2006. MATERIALS: Twenty-six male, adult, Sprague Dawley rats (grade II), weighing 240-290 g, were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Aspirin was provided by Sigma (USA). METHODS: The rats were randomly divided into four groups: sham-operation (SO), CIRI+ vehicle, CIRI+ aspirin (6 mg/kg), and CIRI + aspirin (60 mg/kg). Rats in the lesion groups were intragastrically administrated saline, aspirin (6 mg/kg), or aspirin (60 mg/kg), respectively. MAIN OUTCOME MEASURES: The number of pyramidal neurons with normal appearance in the cerebral cortex at 2-4 mm from the midline; apoptotic cell death as measured by TUNEL; Bcl-2 and Bax protein localization was determined by immunohistochemistry; maiondiaidehyde (MDA) and super oxidation (SOD) content were determined by biochemistry method; adenosine triphosphate (ATP) content measured by capillary electrophoresis. RESULTS: Following CIRI, the following parameters were altered compared with sham-operated animals: the number of neurons with normal appearance was significantly reduced in the cerebral cortex; the number of apoptotic cells increased; Bax protein expression was enhanced; and the ratio between Bcl-2 and Bax decreased. In addition, MDA content increased significantly, whereas ATP content decreased (P < 0.01 ). Aspirin ameliorated the loss of healthy pyramidal neurons. Both 6 and 60 mg/kg aspirin increased the ratio between Bcl-2 and Bax, with no significant difference between the treatment groups. In addition, 60 mg

  16. Long noncoding RNA-mediated anti-apoptotic activity in murine erythroid terminal differentiation.

    Science.gov (United States)

    Hu, Wenqian; Yuan, Bingbing; Flygare, Johan; Lodish, Harvey F

    2011-12-15

    Long noncoding RNAs (lncRNAs) are differentially expressed under both normal and pathological conditions, implying that they may play important biological functions. Here we examined the expression of lncRNAs during erythropoiesis and identified an erythroid-specific lncRNA with anti-apoptotic activity. Inhibition of this lncRNA blocks erythroid differentiation and promotes apoptosis. Conversely, ectopic expression of this lncRNA can inhibit apoptosis in mouse erythroid cells. This lncRNA represses expression of Pycard, a proapoptotic gene, explaining in part the inhibition of programmed cell death. These findings reveal a novel layer of regulation of cell differentiation and apoptosis by a lncRNA.

  17. Copper(II) complexes with highly water-soluble L- and D-proline-thiosemicarbazone conjugates as potential inhibitors of Topoisomerase IIα.

    Science.gov (United States)

    Bacher, Felix; Enyedy, Éva A; Nagy, Nóra V; Rockenbauer, Antal; Bognár, Gabriella M; Trondl, Robert; Novak, Maria S; Klapproth, Erik; Kiss, Tamás; Arion, Vladimir B

    2013-08-01

    Two proline-thiosemicarbazone bioconjugates with excellent aqueous solubility, namely, 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [L-Pro-FTSC or (S)-H2L] and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [D-Pro-FTSC or (R)-H2L], have been synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, and electrospray ionization mass spectrometry. The complexation behavior of L-Pro-FTSC with copper(II) in an aqueous solution and in a 30% (w/w) dimethyl sulfoxide/water mixture has been studied via pH potentiometry, UV-vis spectrophotometry, electron paramagnetic resonance, (1)H NMR spectroscopy, and spectrofluorimetry. By the reaction of copper(II) acetate with (S)-H2L and (R)-H2L in water, the complexes [Cu(S,R)-L] and [Cu(R,S)-L] have been synthesized and comprehensively characterized. An X-ray diffraction study of [Cu(S,R)-L] showed the formation of a square-pyramidal complex, with the bioconjugate acting as a pentadentate ligand. Both copper(II) complexes displayed antiproliferative activity in CH1 ovarian carcinoma cells and inhibited Topoisomerase IIα activity in a DNA plasmid relaxation assay. PMID:23829568

  18. Solvent-Free Synthesis, DNA-Topoisomerase II Activity and Molecular Docking Study of New Asymmetrically N,N'-Substituted Ureas

    Directory of Open Access Journals (Sweden)

    Aurea Echevarria

    2012-11-01

    Full Text Available A new series of asymmetrically N,N'-substituted ureas 20–25 was prepared using solvent free conditions, which is an eco-friendly methodology, starting with Schiff bases derived from cinnamaldehyde and p-substituted anilines, which are subsequently submitted to reduction reactions that afford the corresponding asymmetric secondary amines. All of the intermediates were prepared using solvent free reactions, which were compared to traditional methodologies. All of the reactions required a remarkably short amount of time and provided good yields when solvent free conditions were employed compared to other methodologies. The DNA-topoisomerase II-α (topo II-α activity was evaluated in relaxation assays, which showed that all of the compounds inhibited the enzyme activity at 10 μM, except for urea 24. Furthermore, a molecular docking study indicated that the compounds 20–25 binding to the topo II-α are able to interact with the same binding site as the anticancer drug etoposide, suggesting that the ureas could inhibit the enzyme by the same mechanism of action observed for etoposide, which prevents re-ligation of the DNA strands.

  19. Quinolino[3,4-b]quinoxalines and pyridazino[4,3-c]quinoline derivatives: Synthesis, inhibition of topoisomerase IIα, G-quadruplex binding and cytotoxic properties.

    Science.gov (United States)

    Palluotto, Fausta; Sosic, Alice; Pinato, Odra; Zoidis, Grigoris; Catto, Marco; Sissi, Claudia; Gatto, Barbara; Carotti, Angelo

    2016-11-10

    The quinoline motif fused with other heterocyclic systems plays an important role in the field of anticancer drug development. An extensive series of tetracyclic quinolino[3,4-b]quinoxalines N-5 or C-6 substituted with basic side chain and a limited number of tricyclic pyridazino[4,3-c]quinolines N-6 substituted were designed, synthesized and evaluated for topoisomerase IIα (Topo IIα) inhibitory activity, ability to bind and stabilize G-quadruplex structures and cytotoxic properties against two human cancer cell lines (HeLa and MCF-7). Almost all of the tested agents showed a high activity as Topo IIα inhibitors and G-quadruplex stabilizers. Among all the derivatives studied, the quinolino[3,4-b]quinoxalines 11 and 23, N-5 and C-6 substituted respectively, stand out as the most promising compounds. Derivative 11 resulted a selective binder to selected G-quadruplex sequences, while derivative 23 displayed the most interesting Topo IIα inhibitory activity (IC50 = 5.14 μM); both showed high cytotoxic activity (IC50 HeLa = 2.04 μM and 2.32 μM, respectively).

  20. Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

    Science.gov (United States)

    Duca, Maria; Guianvarc'h, Dominique; Oussedik, Kahina; Halby, Ludovic; Garbesi, Anna; Dauzonne, Daniel; Monneret, Claude; Osheroff, Neil; Giovannangeli, Carine; Arimondo, Paola B

    2006-01-01

    Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning. PMID:16598074

  1. The G-quadruplex ligand telomestatin impairs binding of topoisomerase IIIalpha to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.

    Directory of Open Access Journals (Sweden)

    Nassima Temime-Smaali

    Full Text Available In Alternative Lengthening of Telomeres (ALT cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIalpha (Topo III is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.

  2. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

    Science.gov (United States)

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J; Low, Nicola; Shafer, William M; Althaus, Christian L; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment. PMID:26696986

  3. Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery

    Science.gov (United States)

    Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W; Palle, Komaraiah

    2016-01-01

    Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB. PMID:26871286

  4. Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

    Science.gov (United States)

    Onodera, Takefumi; Takenaka, Yukiko; Kozaki, Sachiko; Tanahashi, Takao; Mizushina, Yoshiyuki

    2016-03-01

    We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent.

  5. Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

    Science.gov (United States)

    Onodera, Takefumi; Takenaka, Yukiko; Kozaki, Sachiko; Tanahashi, Takao; Mizushina, Yoshiyuki

    2016-03-01

    We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent. PMID:26781450

  6. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5′ Adducts

    Directory of Open Access Journals (Sweden)

    Hong Yan

    2016-07-01

    Full Text Available Topoisomerase 2 (Top2 is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5′ end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc. In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ. Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by etoposide, the unique features of such DSBs and how they are repaired. Implications for the improvement of cancer therapy will be discussed.

  7. Collision of Trapped Topoisomerase 2 with Transcription and Replication: Generation and Repair of DNA Double-Strand Breaks with 5' Adducts.

    Science.gov (United States)

    Yan, Hong; Tammaro, Margaret; Liao, Shuren

    2016-01-01

    Topoisomerase 2 (Top2) is an essential enzyme responsible for manipulating DNA topology during replication, transcription, chromosome organization and chromosome segregation. It acts by nicking both strands of DNA and then passes another DNA molecule through the break. The 5' end of each nick is covalently linked to the tyrosine in the active center of each of the two subunits of Top2 (Top2cc). In this configuration, the two sides of the nicked DNA are held together by the strong protein-protein interactions between the two subunits of Top2, allowing the nicks to be faithfully resealed in situ. Top2ccs are normally transient, but can be trapped by cancer drugs, such as etoposide, and subsequently processed into DSBs in cells. If not properly repaired, these DSBs would lead to genome instability and cell death. Here, I review the current understanding of the mechanisms by which DSBs are induced by etoposide, the unique features of such DSBs and how they are repaired. Implications for the improvement of cancer therapy will be discussed. PMID:27376333

  8. Genetic resistance determinants, in vitro time-kill curve analysis and pharmacodynamic functions for the novel topoisomerase II inhibitor ETX0914 (AZD0914 in Neisseria gonorrhoeae

    Directory of Open Access Journals (Sweden)

    Sunniva eFoerster

    2015-12-01

    Full Text Available Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914 is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914. ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections, should be considered for phase III clinical trials and future gonorrhea treatment.

  9. Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae

    Science.gov (United States)

    Foerster, Sunniva; Golparian, Daniel; Jacobsson, Susanne; Hathaway, Lucy J.; Low, Nicola; Shafer, William M.; Althaus, Christian L.; Unemo, Magnus

    2015-01-01

    Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment. PMID:26696986

  10. Integrin α PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hsiao-Han Hsieh

    Full Text Available Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180/CED-12 (ELMO or CED-6 (GULP respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.

  11. Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes.

    Science.gov (United States)

    Warzych, E; Peippo, J; Szydlowski, M; Lechniak, D

    2007-02-01

    The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.

  12. GalNAc-T14 may be involved in regulating the apoptotic action of IGFBP-3

    Indian Academy of Sciences (India)

    Chen Wu; Yaojun Shan; Xinxia Liu; Wenqian Song; Jiali Wang; Minji Zou; Min Wang; Donggang Xu

    2009-09-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to induce apoptosis in an insulin-like growth factor (IGF)-dependent and IGF-independent manner, but the mechanism underlying the IGF-independent effects remains unclear. Polypeptide -acetylgalactosaminyltransferase 14 (GalNAc-T14) is a novel IGFBP-3 binding partner. In this paper, small interference RNA (siRNA) targeting GalNAc-T14 was used to examine whether GalNAc-T14 affects the apoptotic action of IGFBP-3. Using semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, we determined that GalNAc-T14 expression was downregulated by the siRNA directed against GalNAc-T14. Apoptosis analysis of IGFBP-3-overexpressing cells treated with siRNA against GalNAc-T14 was performed to determine if GalNAc-T14 was specifically involved in IGFBP-3 signalling. The results, as determined by flow cytometric analysis and caspase-3 assay, showed that the extent of apoptosis induced by IGFBP-3 increased with RNA interference (RNAi) knockdown of GalNAc-T14. Our data suggest that GalNAc-T14 influences the apoptotic action of IGFBP-3 and might mediate the signalling pathway of IGFBP-3. Experiments to determine the role of GalNAc-T14 in the regulation of apoptosis induced by IGFBP-3 are under way.

  13. Two independent positive feedbacks and bistability in the Bcl-2 apoptotic switch.

    Directory of Open Access Journals (Sweden)

    Jun Cui

    Full Text Available BACKGROUND: The complex interplay between B-cell lymphoma 2 (Bcl-2 family proteins constitutes a crucial checkpoint in apoptosis. Its detailed molecular mechanism remains controversial. Our former modeling studies have selected the 'Direct Activation Model' as a better explanation for experimental observations. In this paper, we continue to extend this model by adding interactions according to updating experimental findings. METHODOLOGY/PRINCIPAL FINDINGS: Through mathematical simulation we found bistability, a kind of switch, can arise from a positive (double negative feedback in the Bcl-2 interaction network established by anti-apoptotic group of Bcl-2 family proteins. Moreover, Bax/Bak auto-activation as an independent positive feedback can enforce the bistability, and make it more robust to parameter variations. By ensemble stochastic modeling, we also elucidated how intrinsic noise can change ultrasensitive switches into gradual responses. Our modeling result agrees well with recent experimental data where bimodal Bax activation distributions in cell population were found. CONCLUSIONS/SIGNIFICANCE: Along with the growing experimental evidences, our studies successfully elucidate the switch mechanism embedded in the Bcl-2 interaction network and provide insights into pharmacological manipulation of Bcl-2 apoptotic switch as further cancer therapies.

  14. p53 contributes to T cell homeostasis through the induction of pro-apoptotic SAP.

    Science.gov (United States)

    Madapura, Harsha S; Salamon, Daniel; Wiman, Klas G; Lain, Sonia; Klein, George; Klein, Eva; Nagy, Noémi

    2012-12-15

    Lack of functional SAP protein, due to gene deletion or mutation, is the cause of X-linked lymphoproliferative disease (XLP), characterized by functionally impaired T and NK cells and a high risk of lymphoma development. We have demonstrated earlier that SAP has a pro-apoptotic function in T and B cells. Deficiency of this function might contribute to the pathogenesis of XLP. We have also shown that SAP is a target of p53 in B cell lines. In the present study, we show that activated primary T cells express p53, which induces SAP expression. p53 is functional as a transcription factor in activated T cells and induces the expression of p21, PUMA and MDM2. PARP cleavage in the late phase of activation indicates that T cells expressing high levels of SAP undergo apoptosis. Modifying p53 levels using Nutlin-3, which specifically dissociates the MDM2-p53 interaction, was sufficient to upregulate SAP expression, indicating that SAP is a target of p53 in T cells. We also demonstrated p53's role as a transcription factor for SAP in activated T cells by ChIP assays. Our result suggests that p53 contributes to T cell homeostasis through the induction of the pro-apoptotic SAP. A high level of SAP is necessary for the activation-induced cell death that is pivotal in termination of the T cell response.

  15. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

    Science.gov (United States)

    de Castro, M A G; Bunt, G; Wouters, F S

    2016-01-01

    The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis. PMID:27551506

  16. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  17. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells].

    Science.gov (United States)

    Qiu, Qiang; Wang, Zhen; Jiang, Jian-ming; Li, Dian-dong

    2007-02-01

    Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities. PMID:17518039

  18. An anti-apoptotic peptide improves survival in lethal total body irradiation.

    Science.gov (United States)

    McDunn, Jonathan E; Muenzer, Jared T; Dunne, Benjamin; Zhou, Anthony; Yuan, Kevin; Hoekzema, Andrew; Hilliard, Carolyn; Chang, Katherine C; Davis, Christopher G; McDonough, Jacquelyn; Hunt, Clayton; Grigsby, Perry; Piwnica-Worms, David; Hotchkiss, Richard S

    2009-05-15

    Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

  19. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    Science.gov (United States)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  20. GalNAc-T14 may be involved in regulating the apoptotic action of IGFBP-3.

    Science.gov (United States)

    Wu, Chen; Shan, Yaojun; Liu, Xinxia; Song, Wenqian; Wang, Jiali; Zou, Minji; Wang, Min; Xu, Donggang

    2009-09-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to induce apoptosis in an insulin-like growth factor (IGF)-dependent and IGF-independent manner, but the mechanism underlying the IGF-independent effects remains unclear. Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14) is a novel IGFBP-3 binding partner. In this paper, small interference RNA (siRNA) targeting GalNAc-T14 was used to examine whether GalNAc-T14 affects the apoptotic action of IGFBP-3. Using semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, we determined that GalNAc-T14 expression was downregulated by the siRNA directed against GalNAc-T14. Apoptosis analysis of IGFBP-3-overexpressing cells treated with siRNA against GalNAc-T14 was performed to determine if GalNAc-T14 was specifically involved in IGFBP-3 signalling. The results, as determined by flow cytometric analysis and caspase-3 assay, showed that the extent of apoptosis induced by IGFBP-increased with RNA interference (RNAi) knockdown of GalNAc-T14. Our data suggest that GalNAc-T14 influences the apoptotic action of IGFBP-3 and might mediate the signalling pathway of IGFBP-3. Experiments to determine the role of GalNAc-T14 in the regulation of apoptosis induced by IGFBP-3 are under way.

  1. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease.

    Science.gov (United States)

    Wang, Yan; Cai, Biao; Shao, Jing; Wang, Ting-Ting; Cai, Run-Ze; Ma, Chang-Ju; Han, Tao; Du, Jun

    2016-07-01

    Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer's disease. A rat model of Alzheimer's disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25-35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer's disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genistein in Alzheimer's disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c. PMID:27630702

  2. ORF3 of duck circovirus: a novel protein with apoptotic activity.

    Science.gov (United States)

    Xiang, Qi-Wang; Wang, Xin; Xie, Zhi-Jing; Sun, Ya-Ni; Zhu, Yan-Li; Wang, Shu-Jing; Liu, Hong-Jie; Jiang, Shi-Jin

    2012-09-14

    Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (Pbaculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity. PMID:22537707

  3. Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal?

    Science.gov (United States)

    Lokhmatikov, Alexey V.; Voskoboynikova, Natalia; Cherepanov, Dmitry A.; Skulachev, Maxim V.; Steinhoff, Heinz-Jürgen; Skulachev, Vladimir P.; Mulkidjanian, Armen Y.

    2016-01-01

    Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane—liposomes of pure bovine heart CL—we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature. PMID:27313834

  4. Protective natural autoantibodies to apoptotic cells: evidence of convergent selection of recurrent innate-like clones.

    Science.gov (United States)

    Silverman, Gregg J

    2015-12-01

    During murine immune development, recurrent B cell clones arise in a predictable fashion. Among these B cells, an archetypical clonotypic set that recognizes phosphorylcholine (PC) antigens and produces anti-PC IgM, first implicated for roles in microbial protection, was later found to become expanded in hyperlipidemic mice and in response to an increased in vivo burden of apoptotic cells. These IgM natural antibodies can enhance clearance of damaged cells and induce intracellular blockade of inflammatory signaling cascades. In clinical populations, raised levels of anti-PC IgM correlate with protection from atherosclerosis and may also downmodulate the severity of autoimmune disease. Human anti-PC-producing clones without hypermutation have been isolated that can similarly discriminate apoptotic from healthy cells. An independent report on unrelated adults has described anti-PC-producing B cells with IgM genes that have conserved CDR3 motifs, similar to stereotypic clonal sets of B cell chronic lymphocytic leukemia. Taken together, emerging evidence suggests that, despite the capacity to form an effectively limitless range of Ig receptors, the human immune system may often recurrently generate lymphocytes expressing structurally convergent B cell receptors with protective and homeostatic roles.

  5. Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice

    Directory of Open Access Journals (Sweden)

    Debajit Bhowmick

    2016-01-01

    Full Text Available Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP, generation of reactive oxygen species (ROS, and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma.

  6. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer’s disease

    Institute of Scientific and Technical Information of China (English)

    Yan Wang; Biao Cai; Jing Shao; Ting-ting Wang; Run-ze Cai; Chang-ju Ma; Tao Han; Jun Du

    2016-01-01

    Genistein is effective against amyloid-βtoxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer’s disease. A rat model of Alzheimer’s disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-βpeptide (25–35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer’s dis-ease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer’s disease rats. These ifndings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  7. Local Augmented Angiotensinogen Secreted from Apoptotic Vascular Endothelial Cells Is a Vital Mediator of Vascular Remodelling.

    Directory of Open Access Journals (Sweden)

    Shyh-Jong Wu

    Full Text Available Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs and vascular smooth muscle cells (VSMCs through serum starvation (SS. After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II were found in a serum-free medium conditioned by HUVECs (SSC. The increased Ang II was suppressed using lisinopril (an ACE inhibitor treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

  8. Apoptotic effects of non-edible parts of Punica granatum on human multiple myeloma cells.

    Science.gov (United States)

    Kiraz, Yağmur; Neergheen-Bhujun, Vidushi S; Rummun, Nawraj; Baran, Yusuf

    2016-02-01

    Multiple myeloma is of great concern since existing therapies are unable to cure this clinical condition. Alternative therapeutic approaches are mandatory, and the use of plant extracts is considered interesting. Punica granatum and its derived products were suggested as potential anticancer agents due to the presence of bioactive compounds. Thus, polypenolic-rich extracts of the non-edible parts of P. granatum were investigated for their antiproliferative and apoptotic effects on U266 multiple myeloma cells. We demonstrated that there were dose-dependent decreases in the proliferation of U266 cells in response to P. granatum extracts. Also, exposure to the extracts triggered apoptosis with significant increases in loss of mitochondrial membrane potential in U266 cells exposed to the leaves and stem extracts, while the flower extract resulted in slight increases in loss of MMP. These results were confirmed by Annexin-V analysis. These results documented the cytotoxic and apoptotic effects of P. granatum extracts on human U266 multiple myeloma cells via disruption of mitochondrial membrane potential and increasing cell cycle arrest. The data suggest that the extracts can be envisaged in cancer chemoprevention and call for further exploration into the potential application of these plant parts. PMID:26318303

  9. Potential for Modulation of the Fas Apoptotic Pathway by Epidermal Growth Factor in Sarcomas

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    David E. Joyner

    2011-01-01

    Full Text Available One important mechanism by which cancer cells parasitize their host is by escaping apoptosis. Thus, selectively facilitating apoptosis is a therapeutic mechanism by which oncotherapy may prove highly advantageous. One major apoptotic pathway is mediated by Fas ligand (FasL. The death-inducing signaling Ccmplex (DISC and subsequent death-domain aggregations are created when FasL is bound by its receptor thereby enabling programmed cell death. Conceptually, if a better understanding of the Fas pathway can be garnered, an oncoselective prodeath therapeutic approach can be tailored. Herein, we propose that EGF and CTGF play essential roles in the regulation of the Fas apoptotic pathway in sarcomas. Tumor and in vitro data suggest viable cells counter the prodeath signal induced by FasL by activating EGF, which in turn induces prosurvival CTGF. The prosurvival attributes of CTGF ultimately predominate over the death-inducing FasL. Cells destined for elimination inhibit this prosurvival response via a presently undefined pathway. This scenario represents a novel role for EGF and CTGF as regulators of the Fas pathway in sarcomas.

  10. Gene Expression Profiling in Apoptotic K562 Cells Treated by Homoharringtonine

    Institute of Scientific and Technical Information of China (English)

    Wei JIN; Jiong WU; Zhigang ZHUANG; Junjie Li; Fei FEI; Genhong DI; Ying CHEN; Ming YAO; Zhimin SHAO

    2007-01-01

    Gene chip technology was used to determine the gene expression profiles in apoptotic K562 cells induced by homoharringtonine. The expression of forty-four mRNAs was found to be changed significantly were identified after screening with a gene chip capable of detecting 14,218 different human mRNA species simultaneously. Of these genes, 17 were up-regulated and 27 were down-regulated.Most of them were found to be related to apoptosis, oncogenes, or tumor suppression. Several genes with altered gene expression, such as human transforming growth factor-beta inducible early protein gene (TIEG), vitamin D3 upregulated protein 1 gene (VDUP1), RNA binding motif protein 4 gene (RBM4) and v-myc myelocytomatosis viral oncogene homolog (C-MYC), were confirmed by Northern blot analysis.According to the dynamic gene expression pattern in these apoptotic cells, the activated transforming growth factor-β and tumor necrosis factor signaling pathways play an important role in homoharringtonine-induced apoptosis. TIEG was significantly altered after apoptosis induction, it should be critical for apoptosis signal transmission.

  11. The Anti-apoptotic Effect of Ghrelin on Restraint Stress-Induced Thymus Atrophy in Mice.

    Science.gov (United States)

    Lee, Jun Ho; Kim, Tae-Jin; Kim, Jie Wan; Yoon, Jeong Seon; Kim, Hyuk Soon; Lee, Kyung-Mi

    2016-08-01

    Thymic atrophy is a complication that results from exposure to many environmental stressors, disease treatments, and microbial challenges. Such acute stress-associated thymic loss can have a dramatic impact on the host's ability to replenish the necessary naïve T cell output to reconstitute the peripheral T cell numbers and repertoire to respond to new antigenic challenges. We have previously reported that treatment with the orexigenic hormone ghrelin results in an increase in the number and proliferation of thymocytes after dexamethasone challenge, suggesting a role for ghrelin in restraint stress-induced thymic involution and cell apoptosis and its potential use as a thymostimulatory agent. In an effort to understand how ghrelin suppresses thymic T cell apoptosis, we have examined the various signaling pathways induced by receptor-specific ghrelin stimulation using a restraint stress mouse model. In this model, stress-induced apoptosis in thymocytes was effectively blocked by ghrelin. Western blot analysis demonstrated that ghrelin prevents the cleavage of pro-apoptotic proteins such as Bim, Caspase-3, and PARP. In addition, ghrelin stimulation activates the Akt and Mitogen-activated protein kinases (MAPK) signaling pathways in a time/dose-dependent manner. Moreover, we also revealed the involvement of the FoxO3a pathway in the phosphorylation of Akt and ERK1/2. Together, these findings suggest that ghrelin inhibits apoptosis by modulating the stress-induced apoptotic signal pathway in the restraint-induced thymic apoptosis. PMID:27574503

  12. Anti-apoptotic role of retinoic acid in the inner ear of noise-exposed mice

    International Nuclear Information System (INIS)

    Exposure to loud noise can induce temporary or permanent hearing loss, and acoustic trauma is the major cause of hearing impairment in industrial nations. However, the mechanisms underlying the death of hair cells after acoustic trauma remain unclear. In addition to its involvement in cellular stress and apoptosis, the c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is involved in cell survival, transformation, embryonic morphogenesis, and differentiation. JNK is primarily activated by various environmental stresses including noise, and the phenotypic result appears be to cell death. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A that regulates a wide range of biological processes, including cell proliferation, differentiation, and morphogenesis. We evaluated the role of ATRA in preserving hearing in mice exposed to noise that can induce permanent hearing loss. Mice fed with ATRA before and during 3 consecutive days of noise exposure had a more preserved hearing threshold than mice fed sesame oil or saline. Histological and TUNEL staining of the cochlea showed significantly enhanced preservation of the organ of Corti, including outer hair cells and relatively low apoptotic nuclei, in mice-fed ATRA than in mice-fed sesame oil or saline. Phospho-JNK immunohistochemistry showed that ATRA inhibited the activation of JNK. These results suggest that ATRA has an anti-apoptotic effect on cochleae exposed to noise

  13. Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice.

    Science.gov (United States)

    Bhowmick, Debajit; Bhar, Kaushik; Mallick, Sanjaya K; Das, Subhadip; Chatterjee, Nabanita; Sarkar, Tuhin Subhra; Chakrabarti, Rajarshi; Das Saha, Krishna; Siddhanta, Anirban

    2016-01-01

    Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma. PMID:27293892

  14. Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease

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    Yan Wang

    2016-01-01

    Full Text Available Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer's disease. A rat model of Alzheimer's disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25–35. In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg was given prior to establishing Alzheimer's disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genist-ein in Alzheimer's disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

  15. Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin.

    Science.gov (United States)

    Huang, Fu-Jen; Chan, Wen-Hsiung

    2016-10-01

    Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo. PMID:26712108

  16. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation.

    Science.gov (United States)

    Haka, Abigail S; Barbosa-Lorenzi, Valéria C; Lee, Hyuek Jong; Falcone, Domenick J; Hudis, Clifford A; Dannenberg, Andrew J; Maxfield, Frederick R

    2016-06-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  17. Molecular mechanism of PDT-induced apoptotic cells stimulation NO production in macrophages

    Science.gov (United States)

    Song, Sheng; Zhou, Fei-fan; Yang, Si-hua; Chen, Wei R.

    2011-03-01

    It is well known that apoptotic cells (AC) participate in immune response. The immune response induced by AC, either immunostimulatory or immunosuppressive, have been extensively studied. However, the molecular mechanisms of the immunostimulatory effects induced by PDT-treated AC remain unclear. Nitric oxide (NO) is an important signal transduction molecule and has been implicated in a variety of functions. It has also been found to play an important role not only as a cytotoxic effector but an immune regulatory mediator. In this study, we demonstrate that the PDT-induced apoptotic tumor cells stimulate the production of NO in macrophages by up-regulating expression of inducible nitric oxide synthase (iNOS). In addition, we show that AC, through toll-like receptors (TLRs), can activate myeloid differentiation factor-88 (MyD88), indicating that AC serves as an intercellular signal to induce iNOS expression in immune cells after PDT treatment. This study provided more details for understanding the molecular mechanism of the immune response induced by PDT-treated AC.

  18. Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

    Science.gov (United States)

    Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

    2016-08-01

    Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. PMID:25691005

  19. Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.

    Science.gov (United States)

    Smith, Alyson J; Nelson, Natalie G; Oommen, Saji; Hartjes, Katherine A; Folmes, Clifford D; Terzic, Andre; Nelson, Timothy J

    2012-10-01

    Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.

  20. Apoptotic phosphorylation of histone H3 on Ser-10 by protein kinase Cδ.

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    Choon-Ho Park

    Full Text Available Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new 'apoptotic histone code' mediated by PKCδ.