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

NASA Astrophysics Data System (ADS)

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.

Phytochemicals as Anticancer and Chemopreventive Topoisomerase II Poisons

PubMed Central

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

Virtual Screening for the Development of Dual-Inhibitors Targeting Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1.

PubMed

Cardamone, Francesca; Pizzi, Simone; Iacovelli, Federico; Falconi, Mattia; Desideri, Alessandro

2017-01-01

Human topoisomerase IB is an important target in cancer therapy and drugs selectively stabilizing the topoisomerase IB-DNA covalent complex are in clinical use for several cancer types. Tyrosyl- DNA phosphodiesterase 1 is involved in the DNA repair resolving the topoisomerase IB-DNA covalent complex that is extremely dangerous for the survival of the cells since it produces an irreversible DNA damage. Given the close biological relationship between these two enzymes, the development of synergistic inhibitors, called dual-inhibitors, is an important challenge in cancer therapy and computer-aided drug design may help in the identification of the best compounds. In this review, an overview of the compounds inhibiting one of the two enzymes or acting as dual inhibitors is provided. Moreover, the general procedures of the virtual screening approach, providing a description of two widely used opensource programs, namely AutoDock4 and AutoDock Vina, are described. Finally, an application of the two programs on a selected number of dual inhibitors for tyrosyl-DNA phosphodiesterase 1 and topoisomerase IB and their performance is briefly discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The topoisomerase II-Hsp90 complex: a new chemotherapeutic target?

PubMed

Barker, Catherine R; Hamlett, Jane; Pennington, Stephen R; Burrows, Francis; Lundgren, Karen; Lough, Rachel; Watson, Alastair J M; Jenkins, John R

2006-06-01

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIalpha-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

HU-331, a novel cannabinoid-based anticancer topoisomerase II inhibitor.

PubMed

Kogan, Natalya M; Schlesinger, Michael; Priel, Esther; Rabinowitz, Ruth; Berenshtein, Eduard; Chevion, Mordechai; Mechoulam, Raphael

2007-01-01

Anthracyclines, a large group of quinonoid compounds, are used to treat some forms of cancer. Although highly effective in cancer therapy, the mechanism of action of these compounds is not specific; they act on cancer and other cells by numerous mechanisms. A new anticancer quinone (HU-331) was synthesized from cannabidiol. It shows significant high efficacy against human cancer cell lines in vitro and against in vivo tumor grafts in nude mice. In this study, we investigated its mode of action and present evidence on its unique mechanism. HU-331 does not cause cancer cell cycle arrest, cell apoptosis, or caspase activation. HU-331-caused cell death of human cancer cell lines is not mediated by reactive oxygen intermediates/species, as exposure to HU-331 failed to elicit the generation of reactive oxygen species. HU-331 inhibits DNA topoisomerase II even at nanomolar concentrations but has only a slight nonsignificant effect on DNA topoisomerase I action. The cannabinoid quinone HU-331 is a highly specific inhibitor of topoisomerase II, compared with most known anticancer quinones. It might represent a new potent anticancer drug.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

PubMed

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

Genotoxicity of topoisomerase II inhibitors: an anti-infective perspective.

PubMed

Smart, Daniel J

2008-12-30

At present, an inevitable consequence of a chemical's inhibitory activity on key regulators of DNA topology in bacteria, the type II topoisomerases, is a less pronounced effect on their eukaryotic counterparts. In the context of anti-infectives drug development, this may pose a risk to patient safety as inhibition of eukaryotic type II topoisomerases (TOPO II) can result in the generation of DNA double-strand breaks (DSBs), which have the potential to manifest as mutations, chromosome breakage or cell death. The biological effects of several TOPO II inhibitors in mammalian cells are described herein; their modulation of DSB damage response parameters is examined and evidence for the existence of a threshold concept for genotoxicity and its relevance in safety assessment is discussed. The potential utility of gammaH2AX, a promising and highly sensitive molecular marker for DSBs, in a novel genotoxicity 'pre-screen' to conventional assays is also highlighted.

A Fluorescence-Based Assay for Identification of Bacterial Topoisomerase I Poisons.

PubMed

Annamalai, Thirunavukkarasu; Cheng, Bokun; Keswani, Neelam; Tse-Dinh, Yuk-Ching

2018-01-01

Bacterial Topoisomerase I is a potential target for the identification of novel topoisomerase poison inhibitors that could provide leads for a new class of antibacterial compounds. Here we describe in detail a fluorescence-based cleavage assay that is successfully used in HTS for the discovery of bacterial topoisomerase Ι poisons.

Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones.

PubMed

Jeannot, Frédéric; Taillier, Thomas; Despeyroux, Pierre; Renard, Stéphane; Rey, Astrid; Mourez, Michaël; Poeverlein, Christoph; Khichane, Imène; Perrin, Marc-Antoine; Versluys, Stéphanie; Stavenger, Robert A; Huang, Jianzhong; Germe, Thomas; Maxwell, Anthony; Cao, Sha; Huseby, Douglas L; Hughes, Diarmaid; Bacqué, Eric

2018-04-26

In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

University of Texas Southwestern: SW04428 is a Novel Topoisomerase 1 Inhibitor | Office of Cancer Genomics

Cancer.gov

This study shows the changes in gene expression in response to SW044248, a compound that displays selective toxicity for some NSCLC cell lines. This data led to the discovery that SW044248 is an inhibitor of topoisomerase 1 (Top1) different from other Top1 inhibitors such as camptothecin1. Read the abstract

Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

PubMed Central

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-01-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

Hsp90 inhibitors sensitise human colon cancer cells to topoisomerase I poisons by depletion of key anti-apoptotic and cell cycle checkpoint proteins.

PubMed

McNamara, Anne V; Barclay, Monica; Watson, Alastair J M; Jenkins, John R

2012-02-01

Hsp90 and topoisomerase I are both targets for chemotherapeutic agents. Topoisomerase I poisons are standard clinical treatments, whilst Hsp90 inhibitors are progressing through clinical trials. We have demonstrated that when an Hsp90 inhibitor and topoisomerase I poison are combined they produce a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 human colon cancer cells. Lack of p53 is associated with an increase in sensitivity to the combination treatment; p53⁺/⁺ cells treated with the topoisomerase I poison topotecan (TPT) arrest at G2, whereas in p53⁻/⁻ cells the additional presence of the Hsp90 inhibitor geldanamycin (GA) selectively abrogates the G2M checkpoint. More importantly we report that there is a common underlying p53-independent mechanism behind the observed synergistic combined drug effect. We show that concurrent treatment with GA and TPT is able to reverse TPT induced up-regulation of the anti-apoptotic protein Bcl2 in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells. The data suggests that inhibition of Hsp90 mediates down-regulation of Bcl2 following the combination treatment and cause a synergistic increase in apoptosis in both p53⁺/⁺ and p53⁻/⁻ HCT116 cells; p53⁻/⁻ HCT116 cells are more sensitive to the treatment because they also fail to arrest at G2 in the cell cycle. Copyright © 2011 Elsevier Inc. All rights reserved.

University of Texas Southwestern Medical Center (UTSW): SW04428 is a Novel Topoisomerase 1 Inhibitor | Office of Cancer Genomics

Cancer.gov

This study shows the changes in gene expression in response to SW044248, a compound that displays selective toxicity for some NSCLC cell lines. This data led to the discovery that SW044248 is an inhibitor of topoisomerase 1 (Top1) different from other Top1 inhibitors such as camptothecin1. Read the abstract

Synthesis and topoisomerase II inhibitory and cytotoxic activity of oxiranylmethoxy- and thiiranylmethoxy-chalcone derivatives.

PubMed

Na, Younghwa; Nam, Jung-Min

2011-01-01

In order to find potential anticancer drug candidate targeting topoisomerases enzyme, we have designed and synthesized oxiranylmethoxy- and thiiranylmethoxy-retrochalcone derivatives and evaluated their pharmacological activity including topoisomerases inhibitory and cytotoxic activity. Of the compounds prepared compound 25 showed comparable or better cytotoxic activity against cancer cell lines tested. Compound 25 inhibited MCF7 (IC(50): 0.49 ± 0.21 μM) and HCT15 (IC(50): 0.23 ± 0.02 μM) carcinoma cell growth more efficiently than references. In the topoisomerases inhibition test, all the compounds were inactive to topoisomerase I but moderate inhibitors to topoisomerase II enzyme. Especially, compound 25 inhibited topoisomerase II activity with comparable extent to etoposide at 100 μM concentrations. Correlation between cytotoxicity and topoisomerase II inhibitory activity implies that compound 25 can be a possible lead compound for anticancer drug impeding the topoisomerase II function. Copyright © 2010 Elsevier Ltd. All rights reserved.

Oxabicyclooctane-Linked Novel Bacterial Topoisomerase Inhibitors as Broad Spectrum Antibacterial Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Sheo B.; Kaelin, David E.; Wu, Jin

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIsmore » with reduced off-target activity (hERG IC50 > 18 μM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC50 = 1.02 μM) and topo IV (IC50 = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.« less

Development of New ADC Technology with Topoisomerase I Inhibitor.

PubMed

Agatsuma, Toshinori

2017-01-01

Antibody-drug conjugates (ADCs) selectively deliver large amounts of antitumor drugs to tumor tissue and show significant antitumor effects with a wide therapeutic window. We developed a new linker-drug technology platform with an exatecan derivative, which is a highly potent topoisomerase I inhibitor. The major advantages of the technology are: 1) high and homogeneous drug-to-antibody ratio (DAR) availability; 2) potent antitumor activity in conjunction with bystander killing; 3) few safety concerns because of the stable linker limiting release of free drug; and 4) a wide application to therapeutic antibodies. Using this linker-drug technology, we generated an anti-HER2 ADC, namely DS-8201a. DS-8201a, in which almost all eight cysteine residues of the antibody are bound to drug, was effective against trastuzumab DM1 (T-DM1)-insensitive patient-derived xenograft (PDX) models with high HER2 expression and also demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. DS-8201a was well tolerated in rats and monkeys following repeated administration. These results suggest that DS-8201a may be efficacious in a broader population of HER2-positive cancer patients and also confirm the importance of this new class of novel topoisomerase I inhibitor-based ADC technology.

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

PubMed

Lin, Ru-Wei; Yang, Chia-Ning; Ku, ShengYu; Ho, Cheng-Jung; Huang, Shih-Bo; Yang, Min-Chi; Chang, Hsin-Wen; Lin, Chun-Mao; Hwang, Jaulang; Chen, Yeh-Long; Tzeng, Cherg-Chyi; Wang, Chihuei

2014-01-01

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) 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.

Synthesis and biological activities of new furo[3,4-b]carbazoles: potential topoisomerase II inhibitors.

PubMed

Hajbi, Youssef; Neagoie, Cléopatra; Biannic, Bérenger; Chilloux, Aurélie; Vedrenne, Emeline; Baldeyrou, Brigitte; Bailly, Christian; Mérour, Jean-Yves; Rosca, Sorin; Routier, Sylvain; Lansiaux, Amélie

2010-11-01

New 1,5-dihydro-4-(substituted phenyl)-3H-furo[3,4-b]carbazol-3-ones were synthesised via a key step Diels-Alder reaction under microwave irradiation. 3-Formylindole was successfully used in a 6-step synthesis to obtain those complex heterocycles. The Diels-Alder reaction generating the carbazole ring was optimised under thermal conditions or microwave irradiation. After cleavage of functional groups, DNA binding, topoisomerase inhibition and cytotoxic properties of the new-formed furocarbazoles were investigated. These carbazoles do not present a strong interaction with the DNA, and do not modify the relaxation of the DNA in the presence of topoisomerase I or II except for one promising compound. This compound is a potent topoisomerase II inhibitor, and its cellular activity is not moderated compared to etoposide. The synthesis of these molecules allowed the generalisation of the method using indole and 5-OBn indole and several benzaldehydes. The synthesis of these molecules produced chemical structures endowed with promising cytotoxic and topoisomerase II inhibition activities. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Synthesis and Biological Evaluation of Indenoisoquinolines that Inhibit both Tyrosyl-DNA-Phosphodiesterase I (Tdp1) and Topoisomerase I (Top1)

PubMed Central

Conda-Sheridan, Martin; Narasimha Reddy, P. V.; Morrell, Andrew; Cobb, Brooklyn T.; Marchand, Christophe; Agama, Keli; Chergui, Adel; Renaud, Amélie; Stephen, Andrew G.; Pommier, Yves; Cushman, Mark

2013-01-01

Tyrosyl-DNA-phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationship. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 μM to 111 μM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean-graph midpoints ranged from 0.02 to 2.34 μM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents. PMID:23259865

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

PubMed Central

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

2013-01-01

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models. PMID:24386374

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

PubMed

Singh, Badri Nath; Achary, V Mohan Murali; Panditi, Varakumar; Sopory, Sudhir K; Reddy, Malireddy K

2017-08-01

The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Epidermal Growth Factor Receptor (EGFR) and its Cross-Talks with Topoisomerases: Challenges and Opportunities for Multi-Target Anticancer Drugs.

PubMed

Chauhan, Monika; Sharma, Gourav; Joshi, Gaurav; Kumar, Raj

2016-01-01

The interactions of Epidermal Growth Factor Receptor (EGFR) and topoisomerases have been seen in various cancer including brain, breast, ovarian, colorectal, gastric, etc. The studies in adenocarcinoma patients, chromogenic in situ hybridization, western blotting, receptor binding assay and electromobility shift assays, etc. threw light on the biophysical and biochemical features of EGFR and Topoisomerase cross-talks. It has been revealed that both the isomers of topoisomerase (Topo I and Topo II) interact via different mechanisms with EGFR. Topo II and HER2 share the same location i.e. 17q12-21 regions which could be a possible cause of predominant interactions seen between them. Topo I and EGFR interactions are mechanically related to the nucleolar translocation of heparenase by EGF and c-Jun. We compiled literature findings including the mechanistic interventions, signaling pathways, patents, in vitro and in vivo data of tested inhibitors and combinations in clinical trials, which provide convincing confirmations for the interactions of EGFR and topoisomerases. These interactions may be used for deriving a consistent route of mechanism, design and development of standard drug combinations and dual or multi inhibitors.

Reduced Smad4 expression and DNA topoisomerase inhibitor chemosensitivity in non-small cell lung cancer.

PubMed

Ziemke, Michael; Patil, Tejas; Nolan, Kyle; Tippimanchai, Darinee; Malkoski, Stephen P

2017-07-01

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules. We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets. We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules. Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair. Copyright © 2017 Elsevier B.V. All rights reserved.

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

DOE PAGES

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; ...

2013-12-26

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani.

PubMed

Carballeira, Néstor M; Cartagena, Michelle; Sanabria, David; Tasdemir, Deniz; Prada, Christopher F; Reguera, Rosa M; Balaña-Fouce, Rafael

2012-10-01

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7μM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1μM) and Trypanosoma brucei rhodesiense (IC(50)=64.5μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibition of human TDP2 by deazaflavins | Center for Cancer Research

Cancer.gov

Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively

DNA topoisomerase I and II expression in drug resistantgerm cell tumours

PubMed Central

Berney, D M; Shamash, J; Gaffney, J; Jordan, S; Oliver, R T D

2002-01-01

A small number of testicular germ cell tumours are refractory to current chemotherapy regimens. DNA topoisomerase I is the target for several new drugs and a potential candidate treatment for chemorefractory germ cell tumours. DNA topoisomerase IIα is the target for etoposide, which is currently used regularly in germ cell tumour treatment. The expression of DNA topoisomerase I and IIα were therefore assessed immunohistochemically in a range of testicular tumours, especially those with persistent malignant elements on retroperitoneal lymph node dissection. Pre-chemotherapy orchidectomy specimens were matched with post-chemotherapy retroperitoneal lymph node dissections to examine changes in expression. There was considerable variation in the expression of topoisomerase I in different tumour types. Both yolk sac tumours and teratoma, mature showed universal expression of topoisomerase I, while 38% of seminomas and 30% of embryonal carcinomas were positive. Strong topoisomerase IIα expression was found in embryonal carcinoma. There was a negative correlation between topoisomerase I and IIα expression (P=0.004) and downregulation of topoisomerase IIα after chemotherapy (P=0.02). Topoisomerase I expression appears to increase in those cases with residual teratoma, mature, but is largely unchanged in those cases remaining as embryonal carcinoma. These results suggest that topoisomerase I inhibitors may be useful in chemorefractory germ cell tumours, especially yolk sac tumours and where there are unresectable residual teratoma, mature deposits. British Journal of Cancer (2002) 21, 624–629. doi:10.1038/sj.bjc.6600472 www.bjcancer.com © 2002 Cancer Research UK PMID:12237772

The selective effect of glycyrrhizin and glycyrrhetinic acid on topoisomerase IIα and apoptosis in combination with etoposide on triple negative breast cancer MDA-MB-231 cells.

PubMed

Cai, Yun; Zhao, Boxin; Liang, Qianying; Zhang, Yunqi; Cai, Jieying; Li, Guofeng

2017-08-15

Triple negative breast cancer(TNBC) has generated growing interests due to its aggressive biologic behavior and absence of targeted therapy approach. Glycyrrhizin(GL) from licorice root and its metabolite, glycyrrhetinic acid(GA) have shown extensive bioactivities in clinic. Here, we demonstrate that GL and GA have contrary anti-cancer effect on TNBC MDA-MB-231 cells. Beside its inhibition of cell proliferation, GA at non-cytotoxic concentration showed synergistic effect in combination with anti-cancer drug, etoposide(VP-16). Specifically, GA enhanced cytotoxicity through regulating topoisomerase IIα(TOPO 2A) targeted by etoposide. GA sensitized the cells to etoposide through elevating TOPO 2A with a 2.4 fold rate at 12h. From 12 to 48h, GA halved the expression of TOPO 2A and stimulated apoptosis, which exhibited its antineoplastic effect. Our experiments showed that GSH depletion, modulation of MAPK and AKT pathways accounted for the regulation of topoisomerase IIα and apoptosis. However, GL showed protection and detoxication by decreasing reactive oxygen species generation, maintaining GSH and differentially modulating apoptosis, AKT pathway, ERK and JNK of MAPK pathway. Collectively, our results demonstrate that GA, instead of GL, is a better candidate for TNBC treatment because of its anti-cancer effect and sensitization of topoisomerase IIα inhibitor. Copyright © 2017 Elsevier B.V. All rights reserved.

[Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

PubMed

Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

1996-03-01

Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

Novel human topoisomerase I inhibitors, topopyrones A, B, C and D. I. Producing strain, fermentation, isolation, physico-chemical properties and biological activity.

PubMed

Kanai, Y; Ishiyama, D; Senda, H; Iwatani, W; Takahashi, H; Konno, H; Tokumasu, S; Kanazawa, S

2000-09-01

In the course of a screening program for specific inhibitors of human topoisomerase I using a recombinant yeast, we have discovered four new active compounds. All four compounds were isolated from the culture broth of a fungus, Phoma sp. BAUA2861, and two of them were isolated from the culture broth of a fungus, Penicillium sp. BAUA4206. We designated these compounds as topopyrones A, B, C and D. Topopyrones A, B, C and D selectively inhibited recombinant yeast growth dependent on expression of human topoisomerase I with IC50 values of 1.22, 0.15, 4.88 and 19.63 ng/ml, respectively. The activity and selectivity of topopyrone B were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however they did not inhibit human DNA topoisomerase II. Topopyrones A, B, C and D were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella zoster virus (VZV). It inhibited VZV growth with EC50 value of 0.038 microg/ml, which is 24-fold stronger than that of acyclovir (0.9 microg/ml). Topopyrones A, B, and C were inhibitory to Gram-positive bacteria.

Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan

PubMed Central

Meisenberg, Cornelia; Ashour, Mohamed E.; El-Shafie, Lamia; Liao, Chunyan; Hodgson, Adam; Pilborough, Alice; Khurram, Syed A.; Downs, Jessica A.; Ward, Simon E.

2017-01-01

Abstract The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance. PMID:28180300

The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine

PubMed Central

Walsby, Elisabeth J.; Coles, Steven J.; Knapper, Steven; Burnett, Alan K.

2011-01-01

Background Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study. Design and Methods Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II. Results The mean lethal dose 50% (LD50) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 μM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. Conclusions Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor. PMID:21134979

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

PubMed Central

Das, Aditi; Mandal, Chhabinath; Dasgupta, Arindam; Sengupta, Tanushri; Majumder, Hemanta K.

2002-01-01

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. PMID:11809893

Context-Dependent Antagonism between Akt Inhibitors and Topoisomerase Poisons

PubMed Central

Gálvez-Peralta, Marina; Flatten, Karen S.; Loegering, David A.; Peterson, Kevin L.; Schneider, Paula A.; Erlichman, Charles

2014-01-01

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized. PMID:24569089

Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

PubMed

Gálvez-Peralta, Marina; Flatten, Karen S; Loegering, David A; Peterson, Kevin L; Schneider, Paula A; Erlichman, Charles; Kaufmann, Scott H

2014-05-01

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.

Digesting dietary miRNA therapeutics

USDA-ARS?s Scientific Manuscript database

Hippocrates famously advised, "Let food be thy medicine and thy medicine be thy food." Numerous plant-derived compounds are used as cancer therapeutics including antimitotics, topoisomerase inhibitors, and kinase inhibitors. Here we will review emerging evidence suggesting that diet derived small RN...

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

PubMed

Gerasyuto, Aleksey I; Arnold, Michael A; Wang, Jiashi; Chen, Guangming; Zhang, Xiaoyan; Smith, Sean; Woll, Matthew G; Baird, John; Zhang, Nanjing; Almstead, Neil G; Narasimhan, Jana; Peddi, Srinivasa; Dumble, Melissa; Sheedy, Josephine; Weetall, Marla; Branstrom, Arthur A; Prasad, J V N; Karp, Gary M

2018-05-14

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC 90 values against Escherichia coli (0.5-1 μg/mL) and Acinetobacter baumannii (8-16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα.

PubMed

Chen, Jingjing; Zheng, Zhouyi; Chen, Yi; Li, Jiaqi; Qian, Shanhu; Shi, Yifen; Sun, Lan; Han, Yixiang; Zhang, Shenghui; Yu, Kang

2016-01-01

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.

How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor.

PubMed

Zuma, Aline Araujo; Mendes, Isabela Cecília; Reignault, Lissa Catherine; Elias, Maria Carolina; de Souza, Wanderley; Machado, Carlos Renato; Motta, Maria Cristina M

2014-02-01

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects approximately 8 million people in Latin America. This parasite contains a single nucleus and a kinetoplast, which harbors the mitochondrial DNA (kDNA). DNA topoisomerases act during replication, transcription and repair and modulate DNA topology by reverting supercoiling in the DNA double-strand. In this work, we evaluated the effects promoted by camptothecin, a topoisomerase I inhibitor that promotes protozoan proliferation impairment, cell cycle arrest, ultrastructure alterations and DNA lesions in epimastigotes of T. cruzi. The results showed that inhibition of cell proliferation was reversible only at the lowest drug concentration (1μM) used. The unpacking of nuclear heterochromatin and mitochondrion swelling were the main ultrastructural modifications observed. Inhibition of parasite proliferation also led to cell cycle arrest, which was most likely caused by nuclear DNA lesions. Following camptothecin treatment, some of the cells restored their DNA, whereas others entered early apoptosis but did not progress to late apoptosis, indicating that the protozoa stay alive in a "senescence-like" state. This programmed cell death may be associated with a decrease in mitochondrial membrane potential and an increase in the production of reactive oxygen species. Taken together, these results indicate that the inhibition of T. cruzi proliferation is related to events capable of affecting cell cycle, DNA organization and mitochondrial activity. Copyright © 2014. Published by Elsevier B.V.

Discovery and Characterization of a Water-Soluble Prodrug of a Dual Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV.

PubMed

O'Dowd, Hardwin; Shannon, Dean E; Chandupatla, Kishan R; Dixit, Vaishali; Engtrakul, Juntyma J; Ye, Zhengqi; Jones, Steven M; O'Brien, Colleen F; Nicolau, David P; Tessier, Pamela R; Crandon, Jared L; Song, Bin; Macikenas, Dainius; Hanzelka, Brian L; Le Tiran, Arnaud; Bennani, Youssef L; Charifson, Paul S; Grillot, Anne-Laure

2015-07-09

Benzimidazole 1 is the lead compound resulting from an antibacterial program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With the goal of improving key drug-like properties, namely, the solubility and the formulability of 1, an effort to identify prodrugs was undertaken. This has led to the discovery of a phosphate ester prodrug 2. This prodrug is rapidly cleaved to the parent drug molecule upon both oral and intravenous administration. The prodrug achieved equivalent exposure of 1 compared to dosing the parent in multiple species. The prodrug 2 has improved aqueous solubility, simplifying both intravenous and oral formulation.

High-throughput microtitre plate-based assay for DNA topoisomerases.

PubMed

Taylor, James A; Burton, Nicolas P; Maxwell, Anthony

2012-01-01

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of DNA topoisomerases. The assay utilizes intermolecular triplex formation between an immobilized triplex-forming oligo (TFO) and a triplex-forming region inserted into the plasmid substrate (pNO1), and capitalizes on the observation that supercoiled DNA forms triplexes more readily than relaxed DNA. Thus, supercoiled DNA is preferentially retained by the TFO under triplex-forming conditions while relaxed DNA can be washed away. Due to its high speed of sample analysis and reduced sample handling over conventional gel-based techniques, this assay can be used to screen chemical libraries for novel inhibitors of topoisomerases.

XPO1 Inhibition Using Selinexor Synergizes With Chemotherapy in Acute Myeloid Leukemia (AML) by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus

PubMed Central

Ranganathan, Parvathi; Kashyap, Trinayan; Yu, Xueyan; Meng, Xiaomei; Lai, Tzung-Huei; McNeil, Betina; Bhatnagar, Bhavana; Shacham, Sharon; Kauffman, Michael; Dorrance, Adrienne M.; Blum, William; Sampath, Deepa; Landesman, Yosef; Garzon, Ramiro

2016-01-01

Purpose Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore we asked whether adding already established effective drugs such as Topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. Experimental Design The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. Results Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared to each single therapy. Conclusions Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of AML patients that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression, and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. PMID:27358488

Synthesis and biological evaluation of indeno[1,5]naphthyridines as topoisomerase I (TopI) inhibitors with antiproliferative activity.

PubMed

Alonso, Concepción; Fuertes, María; González, María; Rubiales, Gloria; Tesauro, Cinzia; Knudsen, Birgitta R; Palacios, Francisco

2016-06-10

In an effort to establish new candidates with improved anticancer activity, we report here the synthesis of various series of 7H-indeno[2,1-c][1,5]-naphthyridines and novel 7H-indeno[2,1-c][1,5]-naphthyridine-7-ones and 7H-indeno[2,1-c][1,5]-naphthyridine-7-ols. Most of the products which were synthesized were able to inhibit Topoisomerase I activity. Moreover, in vitro testing demonstrated that a subset of the products exhibited a cytotoxic effect on cell lines derived from human breast cancer (BT 20), human lung adenocarcinoma (A 549), or human ovarian carcinoma (SKOV3). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Synthesis and Biological Evaluation of the First Dual Tyrosyl-DNA Phosphodiesterase I (Tdp1) - Topoisomerase I (Top1) Inhibitors

PubMed Central

Nguyen, Trung Xuan; Morrell, Andrew; Conda-Sheridan, Martin; Marchand, Christophe; Agama, Keli; Bermingam, Alun; Stephen, Andrew G.; Chergui, Adel; Naumova, Alena; Fisher, Robert; O’Keefe, Barry R.; Pommier, Yves; Cushman, Mark

2012-01-01

Substances with dual tyrosyl-DNA phosphodiesterase I - topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC50 = 1.52 ± 0.05 μM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors, even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors. PMID:22536944

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria.

PubMed

Charrier, Cédric; Salisbury, Anne-Marie; Savage, Victoria J; Duffy, Thomas; Moyo, Emmanuel; Chaffer-Malam, Nathan; Ooi, Nicola; Newman, Rebecca; Cheung, Jonathan; Metzger, Richard; McGarry, David; Pichowicz, Mark; Sigerson, Ralph; Cooper, Ian R; Nelson, Gary; Butler, Hayley S; Craighead, Mark; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

2017-05-01

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC 90 values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli , respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10 -8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC 50 ], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents. Copyright © 2017 American Society for Microbiology.

Molecular docking studies of curcumin natural derivatives with DNA topoisomerase I and II-DNA complexes.

PubMed

Kumar, Anil; Bora, Utpal

2014-12-01

DNA topoisomerase I (topo I) and II (topo II) are essential enzymes that solve the topological problems of DNA by allowing DNA strands or double helices to pass through each other during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Their critical roles make topoisomerases an attractive drug target against cancer. The present molecular docking study provides insights into the inhibition of topo I and II by curcumin natural derivatives. The binding modes suggested that curcumin natural derivatives docked at the site of DNA cleavage parallel to the axis of DNA base pairing. Cyclocurcumin and curcumin sulphate were predicted to be the most potent inhibitors amongst all the curcumin natural derivatives docked. The binding modes of cyclocurcumin and curcumin sulphate were similar to known inhibitors of topo I and II. Residues like Arg364, Asn722 and base A113 (when docked to topo I-DNA complex) and residues Asp479, Gln778 and base T9 (when docked to topo II-DNA complex) seem to play important role in the binding of curcumin natural derivatives at the site of DNA cleavage.

Evaluating the genotoxicity of topoisomerase-targeted antibiotics

PubMed Central

Smart, Daniel J.; Lynch, Anthony M.

2012-01-01

Antibiotics like fluoroquinolones (FQs) that target bacterial type II topoisomerases pose a potential genotoxic risk due to interactions with mammalian topoisomerase II (TOPO II) counterparts. Inhibition of TOPO II can lead to the generation of clastogenic DNA double-strand breaks (DSBs) that can in turn manifest in mutagenesis. Thus, methods that allow early identification of drugs that present the greatest hazard are warranted. A rapid, medium-throughput and predictive genotoxicity screen that can be applied to bacterial type II topoisomerase inhibitors is described herein. Maximal induction of the DSB biomarker serine139-phosphorylated histone H2AX (γH2AX) in L5178Y cells was quantified via flow cytometry and correlated with data derived from the mouse lymphoma screen (MLS), a default assay used to rank genotoxic potential. When applied to a class of novel bacterial type II topoisomerase inhibitors (NBTIs) in lead-optimisation, maximal γH2AX induction >1.4-fold (relative to controls) identified 22/27 NBTIs that induced >6-fold relative mutation frequency (MF) in MLS. Moreover, response signatures comprising of γH2AX induction and G2M cell cycle arrest elucidated using this approach suggested that these NBTIs, primarily of the H class, operated via a TOPO II poison-like mechanism of action (MoA) similar to FQs. NBTIs that induced ≤6-fold relative MF, which were mainly A class-derived, had less impact on γH2AX (≤1.4-fold) and also evoked G1 arrest, indicating that their cytotoxic effects were likely mediated through a non-poison MoA. Concordance between assays was 86% (54/63) when 1.4- and 6-fold ‘cut offs’ were applied. These findings were corroborated through inspection of human TOPO IIα IC50 data as NBTIs exhibiting equivalent inhibitory capacities had differing genotoxic potencies. Deployed in an early screening capacity, the γH2AX by flow assay coupled with structure–activity relationship evaluation can provide insight into MoA and impact medicinal chemistry efforts, ultimately leading to the production of inherently safer molecules. PMID:22155972

Total Synthesis and Biological Evaluation of 22-Hydroxyacuminatine

PubMed Central

Xiao, Xiangshu; Antony, Smitha; Pommier, Yves; Cushman, Mark

2008-01-01

A total synthesis of 22-hydroxyacuminatine, a cytotoxic alkaloid isolated from Camptotheca acuminata, is reported. The key step in the synthesis involves the reaction of 2,3-dihydro-1H-pyrrolo[3,4-b]quinoline with a brominated phthalide to generate a substituted pentacyclic 12H-5,11a-diazadibenzo[b,h]fluoren-11-one intermediate. Despite its structural resemblance to camptothecin and luotonin A, a biological evaluation of 22-hydroxyacuminatine in a topoisomerase I-deficient cell line P388/CPT45 has confirmed that the observed cytotoxicity is not due to topoisomerase I inhibition. This result is consistent with the hypothesis that π-π stacking is more important than hydrogen bonding interactions in determining topoisomerase I inhibitor binding in the ternary cleavage complex. PMID:16480276

Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

PubMed Central

Chen, Yi; Li, Jiaqi; Qian, Shanhu; Shi, Yifen; Sun, Lan; Han, Yixiang; Zhang, Shenghui; Yu, Kang

2016-01-01

Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity. PMID:27058040

New cancer-treatment model of photodynamic therapy combined with a type I topoisomerase inhibitor, CPT-11, against HeLa cell tumors in nude mice used by OPO parametric tunable laser

NASA Astrophysics Data System (ADS)

Yoshida, Takato O.; Matsuzawa, Eiji; Matsuo, Tetsumichi; Koide, Yukio; Terakawa, Susumu; Yokokura, Teruo; Hirano, Toru

1995-03-01

A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.

Etoposide damages female germ cells in the developing ovary.

PubMed

Stefansdottir, Agnes; Johnston, Zoe C; Powles-Glover, Nicola; Anderson, Richard A; Adams, Ian R; Spears, Norah

2016-08-11

As with many anti-cancer drugs, the topoisomerase II inhibitor etoposide is considered safe for administration to women in the second and third trimesters of pregnancy, but assessment of effects on the developing fetus have been limited. The purpose of this research was to examine the effect of etoposide on germ cells in the developing ovary. Mouse ovary tissue culture was used as the experimental model, thus allowing us to examine effects of etoposide on all stages of germ cell development in the same way, in vitro. Fetal ovaries from embryonic day 13.5 CD1 mice or neonatal ovaries from postnatal day 0 CD1 mice were cultured with 50-150 ng ml(-1) or 50-200 ng ml(-1) etoposide respectively, concentrations that are low relative to that in patient serum. When fetal ovaries were treated prior to follicle formation, etoposide resulted in dose-dependent damage, with 150 ng ml(-1) inducing a near-complete absence of healthy follicles. In contrast, treatment of neonatal ovaries, after follicle formation, had no effect on follicle numbers and only a minor effect on follicle health, even at 200 ng ml(-1). The sensitivity of female germ cells to etoposide coincided with topoisomerase IIα expression: in the developing ovary of both mouse and human, topoisomerase IIα was expressed in germ cells only prior to follicle formation. Exposure of pre-follicular ovaries, in which topoisomerase IIα expression was germ cell-specific, resulted in a near-complete elimination of germ cells prior to follicle formation, with the remaining germ cells going on to form unhealthy follicles by the end of culture. In contrast, exposure to follicle-enclosed oocytes, which no longer expressed topoisomerase IIα in the germ cells, had no effect on total follicle numbers or health, the only effect seen specific to transitional follicles. Results indicate the potential for adverse effects on fetal ovarian development if etoposide is administered to pregnant women when germ cells are not yet enclosed within ovarian follicles, a process that starts at approximately 17 weeks gestation and is only complete towards the end of pregnancy.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

PubMed Central

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

2014-01-01

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. PMID:25534741

AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.

PubMed

Yang, Jing; Ikezoe, Takayuki; Nishioka, Chie; Tasaka, Taizo; Taniguchi, Ayuko; Kuwayama, Yoshio; Komatsu, Naoki; Bandobashi, Kentaro; Togitani, Kazuto; Koeffler, H Phillip; Taguchi, Hirokuni; Yokoyama, Akihito

2007-09-15

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac.

PubMed

Wartlick, Friedrich; Bopp, Anita; Henninger, Christian; Fritz, Gerhard

2013-12-01

Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons. © 2013.

Synthesis and biological evaluation of N-(carbobenzyloxy)-l-phenylalanine and N-(carbobenzyloxy)-l-aspartic acid-β-benzyl ester derivatives as potent topoisomerase IIα inhibitors.

PubMed

Han, Xiaoyan; Zhong, Yifan; Zhou, Guan; Qi, Hui; Li, Shengbin; Ding, Qiang; Liu, Zhenming; Song, Yali; Qiao, Xiaoqiang

2017-06-15

A new series of thirteen N-(carbobenzyloxy)-l-phenylalanine and N-(carbobenzyloxy)-l-aspartic acid-β-benzyl ester compounds were synthesized and evaluated for antiproliferative activity against four different human cancer cell lines: cervical cancer (HeLa), lung cancer (A549), gastric cancer (MGC-803) and breast cancer (MCF-7) as well as topoisomerase I and IIα inhibitory activity. Compounds (5a, 5b, 5e, 8a, 8b) showed significant antiproliferative activity with low IC 50 values against the four cancer cell lines. Equally, compounds 5a, 5b, 5e, 5f, 8a, 8d, 8e and 8f showed topoisomerase IIα inhibitory activity at 100μM with 5b, 5e, 8f exhibiting potential topoisomerase IIα inhibitory activity compared to positive control at 100μM and 20μM, respectively. Conversely compounds 5e, 5f, 5g and 8a showed weaker topoisomerase I inhibitory activity compared to positive control at 100μM. Compound 5b exhibited the most potent topoisomerase IIα inhibitory activity at low concentration and better antiproliferative activity against the four human cancer cell lines. The molecular interactions between compounds 5a-5g, 8a-8f and the topoisomerase IIα (PDB ID: 1ZXM) were further investigated through molecular docking. The results indicated that these compounds could serve as promising leads for further optimization as novel antitumor agents. Copyright © 2017 Elsevier Ltd. All rights reserved.

NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy

PubMed Central

Dougherty, Thomas J.; Nayar, Asha; Newman, Joseph V.; Hopkins, Sussie; Stone, Gregory G.; Johnstone, Michele; Shapiro, Adam B.; Cronin, Mark; Reck, Folkert

2014-01-01

The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections. PMID:24566174

A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

PubMed

Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

2010-01-01

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors.

PubMed

Luniewski, Wojciech; Wietrzyk, Joanna; Godlewska, Joanna; Switalska, Marta; Piskozub, Malgorzata; Peczynska-Czoch, Wanda; Kaczmarek, Lukasz

2012-10-01

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

A comparison of the cytogenetic alterations and global DNA hypomethylation induced by the benzene metabolite, hydroquinone, with those induced by melphalan and etoposide

PubMed Central

Ji, Z; Zhang, L; Peng, V.; Ren, X; McHale, CM; Smith, MT

2015-01-01

Specific cytogenetic alterations and changes in DNA methylation are involved in leukemogenesis. Benzene, an established human leukemogen, is known to induce cytogenetic changes through its active metabolites including hydroquinone (HQ), but the specific alterations have not been fully characterized. Global DNA hypomethylation was reported in a population exposed to benzene, but has not been confirmed in vitro. In this study, we examined cytogenetic changes in chromosomes 5, 7, 8, 11 and 21, and global DNA methylation in human TK6 lymphoblastoid cells treated with HQ for 48 h, and compared the HQ-induced alterations with those induced by two well-known leukemogens, melphalan, an alkylating agent, and etoposide, a DNA topoisomerase II inhibitor. We found that rather than inducing cytogenetic alterations distinct from those induced by melphalan and etoposide, HQ induced alterations characteristic of each agent. HQ induced global DNA hypomethylation at a level intermediate to melphalan (no effect) and etoposide (potent effect). These results suggest that HQ may act similar to an alkylating agent and also similar to a DNA topoisomerase II inhibitor in living cells, both of which may be potential mechanisms of benzene toxicity. In addition to cytogenetic changes, global DNA hypomethylation may be another mechanism underlying the leukemogenicity of benzene. PMID:20339439

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Anchim, Tomasz; Wołczyński, Sławomir

2005-06-01

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.

Topoisomerase II Inhibitors and Poisons, and the Influence of Cell Cycle Checkpoints.

PubMed

D Arcy, Nicholas; Gabrielli, Brian

2017-01-01

Interactions between the decatenation checkpoint and Topoisomerase II (TopoII) are vital for maintaining integrity of the genome. Agents that target this enzyme have been in clinical use in cancer therapy for over 30 years with great success. The types of compounds that have been developed to target TopoII are broadly divided into poisons and catalytic inhibitors. The TopoII poisons are in clinical use as anti-cancer therapies, although in common to most chemotherapeutic agents, they display considerable normal tissue toxicity. Inhibition of the TopoIIb isoform has been implicated in this cytotoxicity. Response to TopoII active agents is determined by several factors, but cell cycle checkpoints play a large role in sensitivity and resistance. The G2/M phase checkpoints are of particular importance in considering the effectiveness of these drugs and are reviewed in this article. Functionality of the ATM dependent decatenation checkpoint may represent a new avenue for selective cancer therapy. Here we review the function of TopoII, the anti-cancer mechanisms and limitations of current catalytic inhibitors and poisons, and their influence on cell cycle checkpoints. We will also assess potential new mechanisms for targeting this enzyme to limit normal tissue toxicity, and how the cell cycle checkpoint triggered by these drugs may provide an alternative and possibly better target for novel therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Convection-enhanced delivery of nanoliposomal CPT-11 (irinotecan) and PEGylated liposomal doxorubicin (Doxil) in rodent intracranial brain tumor xenografts

PubMed Central

Krauze, Michal T.; Noble, Charles O.; Kawaguchi, Tomohiro; Drummond, Daryl; Kirpotin, Dmitri B.; Yamashita, Yoji; Kullberg, Erika; Forsayeth, John; Park, John W.; Bankiewicz, Krystof S.

2007-01-01

We have previously shown that convection-enhanced delivery (CED) of highly stable nanoparticle/liposome agents encapsulating chemotherapeutic drugs is effective against intracranial rodent brain tumor xenografts. In this study, we have evaluated the combination of a newly developed nanoparticle/liposome containing the topoisomerase I inhibitor CPT-11 (nanoliposomal CPT-11 [nLs-CPT-11]), and PEGylated liposomal doxorubicin (Doxil) containing the topoisomerase II inhibitor doxorubicin. Both drugs were detectable in the CNS for more than 36 days after a single CED application. Tissue half-life was 16.7 days for nLs-CPT-11 and 10.9 days for Doxil. The combination of the two agents produced synergistic cytotoxicity in vitro. In vivo in U251MG and U87MG intracranial rodent xenograft models, CED of the combination was also more efficacious than either agent used singly. Analysis of the parameters involved in this approach indicated that tissue pharmacokinetics, tumor microanatomy, and biochemical interactions of the drugs all contributed to the therapeutic efficacy observed. These findings have implications for further clinical applications of CED-based treatment of brain tumors. PMID:17652269

Convection-enhanced delivery of a topoisomerase I inhibitor (nanoliposomal topotecan) and a topoisomerase II inhibitor (pegylated liposomal doxorubicin) in intracranial brain tumor xenografts1

PubMed Central

Yamashita, Yoji; Krauze, Michal T.; Kawaguchi, Tomohiro; Noble, Charles O.; Drummond, Daryl C.; Park, John W.; Bankiewicz, Krystof S.

2007-01-01

Despite multimodal treatment options, the response and survival rates for patients with malignant gliomas remain dismal. Clinical trials with convection-enhanced delivery (CED) have recently opened a new window in neuro-oncology to the direct delivery of chemotherapeutics to the CNS, circumventing the blood-brain barrier and reducing systemic side effects. Our previous CED studies with liposomal chemotherapeutics have shown promising antitumor activity in rodent brain tumor models. In this study, we evaluated a combination of nanoliposomal topotecan (nLs-TPT) and pegylated liposomal doxorubicin (PLD) to enhance efficacy in our brain tumor models, and to establish a CED treatment capable of improving survival from malignant brain tumors. Both liposomal drugs decreased key enzymes involved in tumor cell replication in vitro. Synergistic effects of nLs-TPT and PLD on U87MG cell death were found. The combination displayed excellent efficacy in a CED-based survival study 10 days after tumor cell implantation. Animals in the control group and those in single-agent groups had a median survival of less than 30 days, whereas the combination group experienced a median survival of more than 90 days. We conclude that CED of two liposomal chemotherapeutics (nLs-TPT and PLD) may be an effective treatment option for malignant gliomas. PMID:17018695

Development of purely structure-based pharmacophores for the topoisomerase I-DNA-ligand binding pocket

NASA Astrophysics Data System (ADS)

Drwal, Malgorzata N.; Agama, Keli; Pommier, Yves; Griffith, Renate

2013-12-01

Purely structure-based pharmacophores (SBPs) are an alternative method to ligand-based approaches and have the advantage of describing the entire interaction capability of a binding pocket. Here, we present the development of SBPs for topoisomerase I, an anticancer target with an unusual ligand binding pocket consisting of protein and DNA atoms. Different approaches to cluster and select pharmacophore features are investigated, including hierarchical clustering and energy calculations. In addition, the performance of SBPs is evaluated retrospectively and compared to the performance of ligand- and complex-based pharmacophores. SBPs emerge as a valid method in virtual screening and a complementary approach to ligand-focussed methods. The study further reveals that the choice of pharmacophore feature clustering and selection methods has a large impact on the virtual screening hit lists. A prospective application of the SBPs in virtual screening reveals that they can be used successfully to identify novel topoisomerase inhibitors.

[The significance of extravasation in oncological care].

PubMed

Zatkóné Puskás, Gabriella

2008-03-01

The treatment of cancer may be associated with various chemotherapy-induced mucocutaneous reactions. One of the mucocutaneous adverse effects of antineoplastic drugs is the toxic local tissue reaction, the extravasation, which occurs in less than 1-2% of cytotoxic infusions. The standard management of vesicant extravasation includes: discontinuing all local infusions, aspiration of any residual drug, elevating the involved limb, local cooling or warm compresses, local anesthesia, antidotes (sodium thiosulfate for alkylating agents, dimethylsulfoxide (DMSO) for anthracyclines and mitomycin, and hyaluronidase for the vinca alkaloids), and finally surgical debridement with plastic surgery reconstruction. Because the anthracyclines are topoisomerase II poisons that are antagonized by topoisomerase II catalytic inhibitors such as dexrazoxane, it seems to be the treatment of choice immediately after extravasation of doxorubicin, epirubicin, daunorubicin, etc. One systemic dose of dexrazoxane after the accident may significantly reduce the toxic tissue lesions. Repeated intralesional injections of GM-CSF may accelerate the wound healing without the need of skin grafts.

Synthesis and evaluation of 18F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

PubMed Central

Daumar, Pierre; Zeglis, Brian M.; Ramos, Nicholas; Divilov, Vadim; Sevak, Kuntal Kumar; Pillarsetty, NagaVaraKishore; Lewis, Jason S.

2015-01-01

Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, 18F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [18F]-18 and [18F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression. PMID:25240701

Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB.

PubMed

Chowdhury, Somenath Roy; Kumar, Ashish; Godinho, Joseane Lima Prado; De Macedo Silva, Sara Teixeira; Zuma, Aline Araujo; Saha, Sourav; Kumari, Neha; Rodrigues, Juliany Cola Fernandes; Sundar, Shyam; Dujardin, Jean-Claude; Roy, Syamal; De Souza, Wanderley; Mukhopadhyay, Sibabrata; Majumder, Hemanta K

2017-08-15

Indole alkaloids possess a large spectrum of biological activities including anti-protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B-specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200μM concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections. Copyright © 2017 Elsevier Inc. All rights reserved.

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling

PubMed Central

de la Campa, Adela G.; Ferrándiz, María J.; Martín-Galiano, Antonio J.; García, María T.; Tirado-Vélez, Jose M.

2017-01-01

The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (∼2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase subunit B inhibitor), or by seconeolitsine (a topoisomerase I inhibitor), has revealed the existence of topological domains that specifically respond to such changes. The control of DNA-supercoiling in S. pneumoniae occurs mainly via the regulation of topoisomerase gene transcription: relaxation triggers the up-regulation of gyrase and the down-regulation of topoisomerases I and IV, while hypernegative supercoiling down-regulates the expression of topoisomerase I. Relaxation affects 13% of the genome, with the majority of the genes affected located in 15 domains. Hypernegative supercoiling affects 10% of the genome, with one quarter of the genes affected located in 12 domains. However, all the above domains overlap, suggesting that the chromosome is organized into topological domains with fixed locations. Based on its response to relaxation, the pneumococcal chromosome can be said to be organized into five types of domain: up-regulated, down-regulated, position-conserved non-regulated, position-variable non-regulated, and AT-rich. The AT content is higher in the up-regulated than in the down-regulated domains. Genes within the different domains share structural and functional characteristics. It would seem that a topology-driven selection pressure has defined the chromosomal location of the metabolism, virulence and competence genes, which suggests the existence of topological rules that aim to improve bacterial fitness. PMID:28824578

Structure-activity relationships of aminocoumarin-type gyrase and topoisomerase IV inhibitors obtained by combinatorial biosynthesis.

PubMed

Flatman, Ruth H; Eustaquio, Alessandra; Li, Shu-Ming; Heide, Lutz; Maxwell, Anthony

2006-04-01

Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH3 versus Cl at position 8' of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3"-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH3, or Cl at 8' and H, carbamoyl, or MePC at 3"-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3"-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8' further enhanced activity, but the effect of CH3 or Cl depended on the nature of the acyl group at 3": in the presence of carbamoyl at 3", CH3 resulted in higher activity; in the presence of MePC at 3", Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4"-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

PubMed Central

Flatman, Ruth H.; Eustaquio, Alessandra; Li, Shu-Ming; Heide, Lutz; Maxwell, Anthony

2006-01-01

Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH3 versus Cl at position 8′ of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3"-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH3, or Cl at 8′ and H, carbamoyl, or MePC at 3"-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3"-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8′ further enhanced activity, but the effect of CH3 or Cl depended on the nature of the acyl group at 3": in the presence of carbamoyl at 3", CH3 resulted in higher activity; in the presence of MePC at 3", Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4"-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics. PMID:16569821

Topoisomerase poisoning activity of novel disaccharide anthracyclines.

PubMed

Guano, F; Pourquier, P; Tinelli, S; Binaschi, M; Bigioni, M; Animati, F; Manzini, S; Zunino, F; Kohlhagen, G; Pommier, Y; Capranico, G

1999-07-01

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.

The Ubiquitin-Conjugating Enzyme E2-EPF Is Overexpressed in Primary Breast Cancer and Modulates Sensitivity to Topoisomerase II Inhibition1

PubMed Central

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-01-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER- MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G2/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G2 checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIα protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness. PMID:17710163

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

PubMed

Tedesco, Donato; Zhang, Jianhuan; Trinh, Lan; Lalehzadeh, Guita; Meisner, Rene; Yamaguchi, Ken D; Ruderman, Daniel L; Dinter, Harald; Zajchowski, Deborah A

2007-07-01

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.

A glass fiber/diethylaminoethyl double filter binding assay that measures apoptotic internucleosomal DNA fragmentation.

PubMed

Erusalimsky, J D; John, J; Hong, Y; Moore, M

1996-11-15

A filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis is described. The assay is based on a novel principle that consists of using simultaneously two kinds of glass fiber filters to harvest [3H]thymidine-prelabeled cells following their incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filter, which is positively charged with DEAE active groups, traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the radioactivity retained by each of the filters. The assay was evaluated with the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filter and a parallel increase of radioactivity in the DEAE filter. Irradiation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detection of internucleosomal DNA fragmentation, the effects measured by this assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the assay in a mock screen demonstrated that the introduction of the DEAE filter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches to discover novel modulators of apoptosis.

[Poisons of DNA topoisomerases I and II].

PubMed

Charcosset, J Y; Soues, S; Laval, F

1993-11-01

Over the past decade, DNA topoisomerase I and II appeared to be the targets of some antitumor agents: CPT-11 and Topotecan derived from Camptothecin which interact with topoisomerase I; Actinomycin D, Adriamycin and Daunorubicin, Elliptinium Acetate, Mitoxantrone, Etoposide and Teniposide, Amsacrine which interact with topoisomerase II. The multiple functions of these enzymes are important as they play a role during replication, transcription, recombination, repair and chromatine organisation. Particularly, they relax torsional constraints which appear when intertwined DNA strands are separated while replication fork or RNA polymerases are moving. To some extent, topoisomerase I and II are structurally and functionally different. Moreover, topoisomerase I is not indispensable for a living cell whereas topoisomerase II is. Drug-topoisomerase interaction which probably leads to antitumoral effect of the compounds studied in this review is not a trivial inhibition of the enzyme but rather a poisoning due to stabilization of cleavable complexes between the enzyme and DNA. These stabilized complexes are likely to induce apoptosis-like programmed cell death, which is characterised by DNA fragmentation. However, it appears that it is the collision of the replication fork with the drug-stabilized cleavable complex that is responsible for the cytotoxicity of the drug: poisoning of topoisomerases by antitumor agents leads to a new concept of "dynamic toxicity". Although they interact with a common target, topoisomerase II poisons have differential effects on macromolecules syntheses, cell cycle and chromosome fragmentation; a few compounds may produce free radicals. Because of these differential effects in addition to quantitative and qualitative variations of stabilized cleavable complexes, in particular DNA sequences on which topoisomerase II is stabilized, these antitumor agents do not resemble each other. Cellular resistance to topoisomerases poisons results of two principal types of alteration: target and/or drug transport modification. Decreased ability to form the cleavable complex in resistant cells may be the consequence of both decreased amount of topoisomerase or altered enzyme. On the other hand, overexpression of membrane P-glycoprotein, which pumps drugs out of the cell by an energy dependent process provokes a decreased accumulation of these drugs. Cross resistances to other drugs are mainly under control of these two different mechanisms of resistance. A complete knowledge of their individual effects and mechanisms of resistance would allow a better clinical use of topoisomerases poisons, especially when administered in combination chemotherapy.

Topotecan Injection

MedlinePlus

... also used together with other medications to treat cervical cancer (cancer that begins in the opening of the uterus [womb]) that has not improved or has come back after other treatments. Topotecan is in a class of medications called topoisomerase type I inhibitors. It works by killing cancer cells.

A survey of the sequence-specific interaction of damaging agents with DNA: emphasis on antitumor agents.

PubMed

Murray, V

1999-01-01

This article reviews the literature concerning the sequence specificity of DNA-damaging agents. DNA-damaging agents are widely used in cancer chemotherapy. It is important to understand fully the determinants of DNA sequence specificity so that more effective DNA-damaging agents can be developed as antitumor drugs. There are five main methods of DNA sequence specificity analysis: cleavage of end-labeled fragments, linear amplification with Taq DNA polymerase, ligation-mediated polymerase chain reaction (PCR), single-strand ligation PCR, and footprinting. The DNA sequence specificity in purified DNA and in intact mammalian cells is reviewed for several classes of DNA-damaging agent. These include agents that form covalent adducts with DNA, free radical generators, topoisomerase inhibitors, intercalators and minor groove binders, enzymes, and electromagnetic radiation. The main sites of adduct formation are at the N-7 of guanine in the major groove of DNA and the N-3 of adenine in the minor groove, whereas free radical generators abstract hydrogen from the deoxyribose sugar and topoisomerase inhibitors cause enzyme-DNA cross-links to form. Several issues involved in the determination of the DNA sequence specificity are discussed. The future directions of the field, with respect to cancer chemotherapy, are also examined.

Genetic abnormalities in leukemia secondary to treatment in patients with Hodgkin's disease.

PubMed

Salas, Consuelo; Pérez-Vera, Patricia; Frías, Sara

2011-01-01

Hodgkin's disease has been treated mainly with two chemotherapy schedules, MOPP (nitrogen mustard, Oncovin, procarbazine and prednisone), which includes alkylating agents, and ABVD (adriamycin, bleomycin, vinblastine and dacarbazine), which includes topoisomerase II inhibitors, either with or without radiation therapy. Due to the types of agents used, patients with Hodgkin's disease often develop secondary leukemias. The alkylating agents included in the MOPP scheme were the first drugs associated with the development of therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML); both entities are the result of the clonal selection of cells with accumulated genomic lesions induced by antineoplastic therapy. In patients who developed t-MDS and t-AML, eight alternative routes with specific cytogenetic and molecular changes have been identified, and the routes are related to the type of therapy, alkylating agents or DNA topoisomerase II inhibitors. At the cytogenetic level, patients treated with alkylating agents show deletion 5q/monosomy 5 and deletion 7q/monosomy 7; in contrast, those who were treated with topoisomerase II inhibitors show 11q23 translocations involving the MLL gene. At the molecular level, there are two types of mutations: Class I, which alter the RAS-BRAF signal transduction pathways and increase cell proliferation; Class II, which disrupt genes that encode transcription factors and NPM1 that are involved in cell differentiation, and the inactivation of p53 tumor suppressor gene. Knowledge of the genetic alterations in these conditions is important for the classification, treatment and prognosis of patients as well as essential for increasing the knowledge of the biology of these diseases, which leads to identifying potential therapeutic targets.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

PubMed

Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

2016-10-25

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

Ruthenium(II) polypyridyl complexes as dual inhibitors of telomerase and topoisomerase.

PubMed

Liao, Guoliang; Chen, Xiang; Wu, Jingheng; Qian, Chen; Wang, Yi; Ji, Liangnian; Chao, Hui

2015-09-14

One novel ruthenium polypyridyl complex, [Ru(bpy)2(icip)](2+) (1), and two previously reported ruthenium polypyridyl complexes, [Ru(bpy)2(pdppz)](2+) ()2 and [Ru(bpy)2(tactp)](2+) (3) (bpy = 2,2'-bipyridine, icip = 2-(indeno[2,1-b]chromen-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, pdppz = phenanthro[4,5-abc]dipyrido[3,2-h:2',3'-j]phenazine, tactp = 4,5,9,18-tetraazachryseno[9,10-b]-triphenylene), have been synthesised. As expected, these complexes show inhibition towards telomerase by inducing and stabilising the G-quadruplex structure, and behave as topoisomerase I/II poisons at the same time. Additionally, the acute and chronic cytotoxicities of the complexes are considered. Furthermore, cell apoptosis experiments are used to briefly study the mechanism. Because studies involving multi-target inhibition towards topoisomerase and telomerase of Ru(II) complexes have not been reported previously, the present research may help to develop innovative chemical strategies and therapies.

Mass spectrometry reveals that the antibiotic simocyclinone D8 binds to DNA gyrase in a "bent-over" conformation: evidence of positive cooperativity in binding.

PubMed

Edwards, Marcus J; Williams, Mark A; Maxwell, Anthony; McKay, Adam R

2011-05-03

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.

In vitro characterization of the antibacterial spectrum of novel bacterial type II topoisomerase inhibitors of the aminobenzimidazole class.

PubMed

Mani, Nagraj; Gross, Christian H; Parsons, Jonathan D; Hanzelka, Brian; Müh, Ute; Mullin, Steve; Liao, Yusheng; Grillot, Anne-Laure; Stamos, Dean; Charifson, Paul S; Grossman, Trudy H

2006-04-01

Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class

PubMed Central

Mani, Nagraj; Gross, Christian H.; Parsons, Jonathan D.; Hanzelka, Brian; Müh, Ute; Mullin, Steve; Liao, Yusheng; Grillot, Anne-Laure; Stamos, Dean; Charifson, Paul S.; Grossman, Trudy H.

2006-01-01

Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities. PMID:16569833

DUPA conjugation of a cytotoxic indenoisoquinoline topoisomerase I inhibitor for selective prostate cancer cell targeting.

PubMed

Roy, Jyoti; Nguyen, Trung Xuan; Kanduluru, Ananda Kumar; Venkatesh, Chelvam; Lv, Wei; Reddy, P V Narasimha; Low, Philip S; Cushman, Mark

2015-04-09

Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate cancer cells while being present at low or undetectable levels in normal cells. This difference provides an opportunity to selectively deliver cytotoxic drugs to prostate cancer cells while sparing normal cells that lack PSMA, thus improving potencies and reducing toxicities. PSMA has high affinity for 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) (Ki = 8 nM). After binding to a DUPA-drug conjugate, PSMA internalizes, unloads the conjugate, and returns to the surface. In the present studies, an indenoisoquinoline topoisomerase I inhibitor was conjugated to DUPA via a peptide linker and a drug-release segment that facilitates intracellular cleavage to liberate the drug cargo. The DUPA-indenoisoquinoline conjugate exhibited an IC50 in the low nanomolar range in 22RV1 cell cultures and induced a complete cessation of tumor growth with no toxicity, as determined by loss of body weight and death of treated mice.

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I inhibitors by disabling DNA replication initiation and fork elongation responses

PubMed Central

Jossé, Rozenn; Martin, Scott E.; Guha, Rajarshi; Ormanoglu, Pinar; Pfister, Thomas D.; Reaper, Philip M.; Barnes, Christopher S.; Jones, Julie; Charlton, Peter; Pollard, John R.; Morris, Joel; Doroshow, James H.; Pommier, Yves

2014-01-01

Camptothecin and its derivatives, topotecan and irinotecan are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821, confirmed marked antiproliferative synergy with camptothecin, and even greater synergy with LMP-400. Single cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing check point induced by camptothecin and LMP-400. As expected, the combination ofTop1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed overtime from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970 enhanced the in vivo tumor response to irinotecan without additional toxicity. Akey implication of our work is the mechanistic rationale and proof-of-principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials. PMID:25269479

[Dexrazoxane (ICRF-187)--a cardioprotectant and modulator of action of some anticancer drugs].

PubMed

Kik, Krzysztof; Szmigiero, Leszek

2006-01-01

The nthracycline antibiotics are among the most widely used and effective anticancer drugs. The therapeutic efficacy of this class of drugs is limited by cumulative cardiac toxicity. Dexrazoxane is the only clinically approved cardioprotective agent used in anthracycline-containing anticancer therapy. Its cardioprotective action allows the use of a much higher cumulative dose of anthracyclines and improvement in the effectiveness of treatment. Anthracyclines form complexes with iron ions, which are very active in the production of reactive oxygen species responsible for the lipid peroxidation of mitochondrial and endoplasmatic reticulum membranes. This process seems to be the major cause of anthracycline-induced cardiotoxicity. Dexrazoxane exerts its protective effects by rapid and complete binding of ferric and ferrous ions, even by displacing the metal ions from complexes with anthracyclines. Besides its cardioprotective effect, dexrazoxane also exhibits anticancer properties. Like other derivatives of bisdioxopiperazine, dexrazoxane is a catalytic inhibitor of eukaryotic DNA topoisomerase II, the key enzyme controlling DNA topology and contributing to the replication and transcription processes. Dexrazoxane is able to lock topoisomerase II at the stage of the enzyme reaction cycle where the enzyme forms a closed clamp around the DNA. This phenomenon seems to be the main reason for the generation of DNA double-strand breaks by dexrazoxane as well as its cytotoxicity against quickly proliferating cancer cells. Other effects of its topoisomerase II catalytic inhibition is the induction of cell differentiation and apoptosis. Dexrazoxane may be used not only as a cardioprotective agent, but also as a modulator of action of some anticancer drugs by enhancing their selectivity or by delaying the development of multidrug resistance.

How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisomerases.

PubMed

Stuchinskaya, Tanya; Mitchenall, Lesley A; Schoeffler, Allyn J; Corbett, Kevin D; Berger, James M; Bates, Andrew D; Maxwell, Anthony

2009-02-06

DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range approximately 2-9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.

The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

PubMed Central

Chowdhury, Sayan; Mukherjee, Tulika; Mukhopadhyay, Rupkatha; Mukherjee, Budhaditya; Sengupta, Souvik; Chattopadhyay, Sharmila; Jaisankar, Parasuraman; Roy, Syamal; Majumder, Hemanta K

2012-01-01

Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topoisomerase of L. donovani and acts as a non-competitive inhibitor interacting with both subunits of the enzyme. Niranthin interacts with DNA–protein binary complexes and thus stabilizes the ‘cleavable complex’ formation and subsequently inhibits the religation of cleaved strand. The compound inhibits the proliferation of Leishmania amastigotes in infected cultured murine macrophages with limited cytotoxicity to the host cells and is effective against antimony-resistant Leishmania parasites by modulating upregulated P-glycoprotein on host macrophages. Importantly, besides its in vitro efficacy, niranthin treatment leads to a switch from a Th2- to a Th1-type immune response in infected BALB/c mice. The immune response causes production of nitric oxide, which results in almost complete clearance of the liver and splenic parasite burden after intraperitoneal or intramuscular administration of the drug. These findings can be exploited to develop niranthin as a new drug candidate against drug-resistant leishmaniasis. PMID:23027614

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

PubMed

Thougaard, Annemette V; Langer, Seppo W; Hainau, Bo; Grauslund, Morten; Juhl, Birgitte Ravn; Jensen, Peter Buhl; Sehested, Maxwell

2010-02-28

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 to be similar to findings in humans: massive necrosis in the subcutis, dermis and epidermis followed by sequestration and healing with granulation tissue, and a graft-versus-host-like reaction with hyperkeratotic and acanthotic keratinocytes, occasional apoptoses, epidermal invasion by lymphocytes and healing 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 tested two major hypotheses: (1) interaction with topoisomerase II alpha and (2) the formation of tissue damaging reactive oxygen species following redox cycling of an anthracycline Fe(2+) complex. Dexrazoxane could minimise skin damage via both mechanisms, as it stops the catalytic activity 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 in the topoisomerase II alpha gene (Top2a(Y165S/+)), we found that dexrazoxane provided a protection against anthracycline-induced skin wounds that was indistinguishable from that found in wildtype mice. Thus, interaction with topoisomerase II alpha is not central in the pathogenesis of anthracycline-induced skin damage. In contrast to dexrazoxane, the iron-chelating bisdioxopiperazine ICRF-161 do not inhibit the catalytic cycle of topoisomerase II alpha. This compound was used to isolate and test the importance of iron in the wound pathogenesis. ICRF-161 was found ineffective in the treatment of anthracycline-induced skin damage, suggesting that iron does not play a dominant role in the genesis of wounds. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Cell-cycle-dependent three-dimensional redistribution of nuclear proteins, P 120, pKi-67, and SC 35 splicing factor, in the presence of the topoisomerase I inhibitor camptothecin.

PubMed

Elias, Emmanuel; Lalun, Nathalie; Lorenzato, Marianne; Blache, Laurent; Chelidze, Pavel; O'Donohue, Marie-Françoise; Ploton, Dominique; Bobichon, Hélène

2003-11-15

Topoisomerase I (Topo I) is mostly known for its role in DNA relaxation, which is required for duplication and transcription. Topo I acts as a protein kinase mainly directed to the mRNA splicing factor SC35. Camptothecin is one of the specific Topo I inhibitors and is effective on the two functions of the enzyme. In this study we demonstrated that treatment of KB cells with camptothecin for only 30 min induced the 3D reorganization and redistribution of three proteins involved in the nucleus machinery, P 120, pKi-67, and SC 35, and this occurred in a cell cycle-dependent manner. Our data were obtained from confocal microscopic studies after immunolabeling, 3D reconstruction, and measurement of the nuclear components volumes. In the presence of camptothecin, P 120, which occupied the nucleolar volume, lost its reticulation and pKi-67 was redistributed within the nucleoplasm and even into the cytoplasm. Finally, for SC 35 the fusion of its dots into bigger volumes was observed specifically during the G1 phase. Variations of volumes were also observed for the nucleolus and for the nucleus. These results pointed out that, depending on the cell cycle phase, Topo I functions were selective toward the three different proteins.

B-ring-aryl substituted luotonin A analogues with a new binding mode to the topoisomerase 1-DNA complex show enhanced cytotoxic activity.

PubMed

González-Ruiz, Víctor; Pascua, Irene; Fernández-Marcelo, Tamara; Ribelles, Pascual; Bianchini, Giulia; Sridharan, Vellaisamy; Iniesta, Pilar; Ramos, M Teresa; Olives, Ana I; Martín, M Antonia; Menéndez, J Carlos

2014-01-01

Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.

DNA Topoisomerases of Leishmania parasites; druggable targets for drug discovery.

PubMed

Reguera, Rosa Mª; Elmahallawy, Ehab Kotb; Garcia-Estrada, Carlos; Carbajo-Andres, Ruben; Balana-Fouce, Rafael

2018-05-17

DNA topoisomerases (Top) are a group of isomerase enzymes responsible for controlling the topological problems caused by DNA double helix in the cell during the processes of replication, transcription and recombination. Interestingly, these enzymes have been known since long to be key molecular machines in several cellular processes through overwinding or underwinding of DNA in all-living organisms. Leishmania, a trypanosomatid parasite responsible for causing fatal diseases mostly in impoverished populations of low-income countries, have a set of six classes of Top enzymes. These are placed in the nucleus and the single mitochondrion and can be deadly targets of suitable drugs. Given the fact that there are clear differences in structure and expression between parasite and host enzymes, numerous studies have reported the therapeutic potential of Top inhibitors as antileishmanial drugs. In this regard, numerous compounds have been described as Top type IB and Top type II inhibitors in Leishmania parasites, such as camptothecin derivatives, indenoisoquinolines, indeno-1,5-naphthyridines, fluoroquinolones, antracyclines and podophyllotoxins. The aim of this review is to highlight several facts about Top and Top inhibitors as potential antileishmanial drugs, which may represent a promising strategy for the control of this disease of public health importance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A kinetic clutch governs uncoiling by type IB topoisomerases

NASA Astrophysics Data System (ADS)

Neuman, Keir

2013-03-01

Type IB topoisomerases (Top1B) are essential enzymes that relax excessive DNA supercoiling associated with replication and transcription and are important drug targets for cancer chemotherapy. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Type IB topoisomerase (nTop1). We employed a magnetic-tweezers based single-molecule DNA supercoil relaxation assay to measure the torque dependence of human nuclear Top1 relaxation (nTop1) and inhibition by CPT. For comparison, we examined the human mitochondrial (Top1mt) topoisomerase and an N-terminal deletion mutant of nTop1 (Top68). Despite substantial sequence homology in their core domains, nTop1 and Top1mt exhibit dramatic differences in sensitivity to torque and CPT, with Top68 betraying intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation, and the efficiency of CPT binding. This ``kinetic clutch'' mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics.

PubMed

Afzal, Mohd; Al-Lohedan, Hamad A; Usman, Mohammad; Tabassum, Sartaj

2018-04-18

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu II -Sn IV (1) and Ni II -Sn IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI 50 values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases

PubMed Central

2016-01-01

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10–100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption. PMID:26132160

How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of non-supercoiling type II topoisomerases

PubMed Central

Stuchinskaya, Tanya; Mitchenall, Lesley A.; Schoeffler, Allyn J.; Corbett, Kevin D.; Berger, James M.; Bates, Andrew D.; Maxwell, Anthony

2015-01-01

DNA topoisomerases control the topology of DNA (e.g. the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e. more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower than equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analysed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topo VI (which is only distantly related to the type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range ~2–9 kbp, and is not altered by reducing the free energy available from ATP hydrolysis by varying the ATP:ADP ratio. A direct test of one model (DNA tracking, i.e. sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect but that it is possible that other factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance. PMID:19094994

Flavonoids in Helichrysum pamphylicum inhibit mammalian type I DNA topoisomerase.

PubMed

Topcu, Zeki; Ozturk, Bintug; Kucukoglu, Ozlem; Kilinc, Emrah

2008-01-01

DNA topoisomerases are important targets for cancer chemotherapy. We investigated the effects of a methanolic extract of Helichrysum pamphylicum on mammalian DNA topoisomerase I via in vitro plasmid supercoil relaxation assays. The extracts manifested a considerable inhibition of the enzyme's activity in a dose-dependent manner. We also performed a HPLC analysis to identify the flavonoid content of the H. pamphylicum extract and tested the identified flavonoids; luteolin, luteolin-4-glucoside, naringenin, helichrysinA and isoquercitrin, on DNA topoisomerase I activity. The measurement of the total antioxidant capacity of the flavonoid standards suggested that the topoisomerase inhibition might be correlated with the antioxidant capacity of the plant.

Resveratrol-3-O-glucuronide and resveratrol-4′-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin

PubMed Central

Zunino, Susan J.; Storms, David H.

2017-01-01

Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4′-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4′-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with topoisomerase I inhibitors. PMID:28781690

Resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin.

PubMed

Zunino, Susan J; Storms, David H

2017-08-01

Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with topoisomerase I inhibitors.

Cell cycle arrest induced by inhibitors of epigenetic modifications in maize (Zea mays) seedling leaves: characterization of the process and possible mechanisms involved.

PubMed

Wang, Pu; Zhang, Hao; Hou, Haoli; Wang, Qing; Li, Yingnan; Huang, Yan; Xie, Liangfu; Gao, Fei; He, Shibin; Li, Lijia

2016-07-01

Epigenetic modifications play crucial roles in the regulation of chromatin architecture and are involved in cell cycle progression, including mitosis and meiosis. To explore the relationship between epigenetic modifications and the cell cycle, we treated maize (Zea mays) seedlings with six different epigenetic modification-related inhibitors and identified the postsynthetic phase (G2 ) arrest via flow cytometry analysis. Total H4K5ac levels were significantly increased and the distribution of H3S10ph signalling was obviously changed in mitosis under various treatments. Further statistics of the cells in different periods of mitosis confirmed that the cell cycle was arrested at preprophase. Concentrations of hydrogen peroxide were relatively higher in the treated plants and the antioxidant thiourea could negate the influence of the inhibitors. Moreover, all of the treated plants displayed negative results in the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and γ-H2AX immunostaining assays after exposure for 3 d. Additionally, the expression level of topoisomerase genes in the treated plants was relatively lower than that in the untreated plants. These results suggest that these inhibitors of epigenetic modifications could cause preprophase arrest via reactive oxygen species formation inhibiting the expression of DNA topoisomerase genes, accompanied by changes in the H4K5ac and H3S10ph histone modifications. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Spectroscopic detection of etoposide binding to chromatin components: The role of histone proteins

NASA Astrophysics Data System (ADS)

Chamani, Elham; Rabbani-Chadegani, Azra; Zahraei, Zohreh

2014-12-01

Chromatin has been introduced as a main target for most anticancer drugs. Etoposide is known as a topoisomerase II inhibitor, but its effect on chromatin components is unknown. This report, for the first time, describes the effect of etoposide on DNA, histones and DNA-histones complex in the structure of nucleosomes employing thermal denaturation, fluorescence, UV absorbance and circular dichroism spectroscopy techniques. The results showed that the binding of etoposide decreased UV absorbance and fluorescence emission intensity, altered secondary structure of chromatin and hypochromicity was occurred in thermal denaturation profiles. The drug exhibited higher affinity to chromatin compared to DNA. Quenching of drug chromophores with tyrosine residues of histones indicated that globular domain of histones is the site of etoposide binding. Moreover, the binding of etoposide to histones altered their secondary structure accompanied with hypochromicity revealing compaction of histones in the presence of the drug. From the results it is concludes that apart from topoisomerase II, chromatin components especially its protein moiety can be introduced as a new site of etoposide binding and histone proteins especially H1 play a fundamental role in this process and anticancer activity of etoposide.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

PubMed

Singh, Sheo B; Kaelin, David E; Meinke, Peter T; Wu, Jin; Miesel, Lynn; Tan, Christopher M; Olsen, David B; Lagrutta, Armando; Fukuda, Hideyuki; Kishii, Ryuta; Takei, Masaya; Takeuchi, Tomoko; Takano, Hisashi; Ohata, Kohei; Kurasaki, Haruaki; Nishimura, Akinori; Shibata, Takeshi; Fukuda, Yasumichi

2015-09-01

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human autoantibody to topoisomerase II.

PubMed

Hoffmann, A; Heck, M M; Bordwell, B J; Rothfield, N F; Earnshaw, W C

1989-02-01

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

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

PubMed Central

Alam, Sarfaraz; Khan, Feroz

2014-01-01

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. PMID:24516330

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

PubMed

Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-01-01

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells.

PubMed

Lee, W C; Lin, K Y; Chen, C M; Chen, Z T; Liu, H J; Lai, Y K

1991-10-01

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat-shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat-shock protein 70 (HSP70) since both proteins migrated to the same position on two-dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 65 kDa protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.

Ubiquitous Nature of Fluoroquinolones: The Oscillation between Antibacterial and Anticancer Activities

PubMed Central

Schweizer, Frank

2017-01-01

Fluoroquinolones are synthetic antibacterial agents that stabilize the ternary complex of prokaryotic topoisomerase II enzymes (gyrase and Topo IV), leading to extensive DNA fragmentation and bacteria death. Despite the similar structural folds within the critical regions of prokaryotic and eukaryotic topoisomerases, clinically relevant fluoroquinolones display a remarkable selectivity for prokaryotic topoisomerase II, with excellent safety records in humans. Typical agents that target human topoisomerases (such as etoposide, doxorubicin and mitoxantrone) are associated with significant toxicities and secondary malignancies, whereas clinically relevant fluoroquinolones are not known to exhibit such propensities. Although many fluoroquinolones have been shown to display topoisomerase-independent antiproliferative effects against various human cancer cells, those that are significantly active against eukaryotic topoisomerase show the same DNA damaging properties as other topoisomerase poisons. Empirical models also show that fluoroquinolones mediate some unique immunomodulatory activities of suppressing pro-inflammatory cytokines and super-inducing interleukin-2. This article reviews the extended roles of fluoroquinolones and their prospects as lead for the unmet needs of “small and safe” multimodal-targeting drug scaffolds. PMID:29112154

Tipin functions in the protection against topoisomerase I inhibitor.

PubMed

Hosono, Yoshifumi; Abe, Takuya; Higuchi, Masato; Kajii, Kosa; Sakuraba, Shuichi; Tada, Shusuke; Enomoto, Takemi; Seki, Masayuki

2014-04-18

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

PubMed

Singh, Badri Nath; Mudgil, Yashwanti; John, Riffat; Achary, V Mohan Murali; Tripathy, Manas Kumar; Sopory, Sudhir K; Reddy, Malireddy K; Kaul, Tanushri

2015-11-01

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Development and Validation of an Immunoassay for Quantification of Topoisomerase I in Solid Tumor Tissues

PubMed Central

Pfister, Thomas D.; Hollingshead, Melinda; Kinders, Robert J.; Zhang, Yiping; Evrard, Yvonne A.; Ji, Jiuping; Khin, Sonny A.; Borgel, Suzanne; Stotler, Howard; Carter, John; Divelbiss, Raymond; Kummar, Shivaani; Pommier, Yves; Parchment, Ralph E.; Tomaszewski, Joseph E.; Doroshow, James H.

2012-01-01

Background Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. Methodology/Principal Findings We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. Conclusions/Significance Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors. PMID:23284638

The Effect of Dimethyl Sulfoxide on Supercoiled DNA Relaxation Catalyzed by Type I Topoisomerases

PubMed Central

Lv, Bei; Dai, Yunjia; Liu, Ju; Zhuge, Qiang; Li, Dawei

2015-01-01

The effects of dimethyl sulfoxide (DMSO) on supercoiled plasmid DNA relaxation catalyzed by two typical type I topoisomerases were investigated in our studies. It is shown that DMSO in a low concentration (less than 20%, v/v) can induce a dose-related enhancement of the relaxation efficiency of Escherichia coli topoisomerase I (type IA). Conversely, obvious inhibitory effect on the activity of calf thymus topoisomerase I (type IB) was observed when the same concentration of DMSO is used. In addition, our studies demonstrate that 20% DMSO has an ability to reduce the inhibitory effect on EcTopo I, which was induced by double-stranded oligodeoxyribonucleotides while the same effect cannot be found in the case of CtTopo I. Moreover, our AFM examinations suggested that DMSO can change the conformation of negatively supercoiled plasmid by creating some locally loose regions in DNA molecules. Combining all the lines of evidence, we proposed that DMSO enhanced EcTopo I relaxation activity by (1) increasing the single-stranded DNA regions for the activities of EcTopo I in the early and middle stages of the reaction and (2) preventing the formation of double-stranded DNA-enzyme complex in the later stage, which can elevate the effective concentration of the topoisomerase in the reaction solution. PMID:26682217

Improvement of the Pharmacokinetics and In Vivo Antibacterial Efficacy of a Novel Type IIa Topoisomerase Inhibitor by Formulation in Liposomes

PubMed Central

Newman, Joseph; Goteti, Kosalaram; Beaudoin, Marie-Eve; Harrison, Rane; Hopkins, Sussie; Agrawal, Nikunj; Rivin, Olga

2013-01-01

Several useful properties of liposome-based formulations of various existing antibacterial drugs have been reported. These properties include lower MICs, improved pharmacokinetics, lower toxicity, selective distribution to infected tissues, and enhanced in vivo efficacy. Here we report in vivo studies of a liposomal formulation of a member of a novel class of antibacterial type II topoisomerase inhibitors, others of which have progressed to early phases of clinical trials. The free (i.e., nonliposomal) compound has broad-spectrum MICs but suboptimal pharmacokinetics in rats and mice, characterized by a high volume of distribution and rapid clearance. The liposomal formulation of the compound had essentially unchanged MICs but greatly reduced volume of distribution and clearance in rats and mice. In an in vivo mouse model of Staphylococcus aureus infection of one thigh, the liposomal compound localized preferentially to the infected thigh, whereas the free compound showed no preference for the infected versus the uninfected thigh. Most importantly, the liposomal compound had enhanced efficacy at clearing the infection compared with the free compound. Delivery of this class of compounds as liposomal formulations may offer clinical advantages compared with free compounds. PMID:23877679

A new compound, withangulatin A, promotes type II DNA topoisomerase-mediated DNA damage.

PubMed

Juang, J K; Huang, H W; Chen, C M; Liu, H J

1989-03-31

Withangulatin A, a new compound with a known chemical structure and from the antitumor Chinese herb Physalis angulata L, was found to act on topoisomerase II to induce topoisomerase II-mediated DNA damage in vitro. It has two effective dosage ranges of approximate 0.5 and 20 microM, with about one-third the activity of 20 microM VM-26.

Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

PubMed

Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

2004-06-01

Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

GS-2, a pyrazolo[1,5-a]indole derivative with inhibitory activity of topoisomerases, exerts its potent cytotoxic activity by ROS generation.

PubMed

Ji, Yuan Yuan; Zhu, Yong Ming; Wang, Jian Wen

2013-11-01

Pyrazolo[1,5-a]indole derivatives, a new type of topoisomerase (topo) inhibitor, demonstrate a broad spectrum of antitumor activities. However, the mechanism underlying the induced cytotoxicity remains unclear. In this study, we investigated whether GS-2, one of the derivatives, altered the levels of ROS in breast cancer MDA-231 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that GS-2 caused a time- and dose-dependent elevation of intracellular ROS level in MDA-231 cells. GS-2 subsequently elicited notable inhibition on the expression of topos, DNA damage, activation of caspase-3, -9. The loss of mitochondrial membrane potential (MMP) was observed during the induction. The addition of N-acetyl cysteine (NAC, a well-known antioxidant) could effectively attenuate the GS-2-induced ROS enhancement and subsequent apoptosis. NAC attenuated the induced inhibition on expression of topos, indicating that topos might be the target of GS-2-induced ROS. The finding of the induced ROS provides new evidence for the molecular mechanisms of antitumor activity of pyrazolo[1,5-a]indole derivatives. Copyright © 2013 Elsevier B.V. All rights reserved.

Topoisomerase IIα maintains genomic stability through decatenation G2 checkpoint signaling

PubMed Central

Bower, Jacquelyn J.; Karaca, Gamze F.; Zhou, Yingchun; Simpson, Dennis A.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability. PMID:20562910

Machine learning and docking models for Mycobacterium tuberculosis topoisomerase I.

PubMed

Ekins, Sean; Godbole, Adwait Anand; Kéri, György; Orfi, Lászlo; Pato, János; Bhat, Rajeshwari Subray; Verma, Rinkee; Bradley, Erin K; Nagaraja, Valakunja

2017-03-01

There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pharmacology of irinotecan.

PubMed

Kuhn, J G

1998-08-01

Irinotecan (CPT-11 [Camptosar]), a semisynthetic derivative of the plant alkaloid camptothecin, is bioactivated by carboxylesterases (EC3.1.1-) to the topoisomerase I inhibitor SN-38, a minor metabolite. Bioactivation of intravenously administered irinotecan by carboxylesterases occurs predominantly in the liver. Two human carboxylesterase isoforms responsible for SN-38 formation have been characterized. At relevant hepatic irinotecan concentrations up to 12 micrograms/mL, a low-Km isoform is responsible for irinotecan bioactivation. High concentrations of drugs commonly coadministered with irinotecan do not inhibit carboxylesterase activity. Intestinal carboxylesterases can also generate SN-38, followed by subsequent oral absorption. A second major polar metabolite of irinotecan, aminopentanecarboxylic acid (APC), is the product of CYP3A4-mediated oxidation of the terminal piperidine ring. APC is 100-fold less active than SN-38 as a topoisomerase I inhibitor and is a relatively weak inhibitor of acetylcholinesterase. SN-38 is eliminated mainly through conjugation by hepatic uridine glucuronosyltransferase (UGT*1.1), the same isoezyme responsible for glucuronidation of bilirubin. Grade 4 irinotecan-related toxicity (ie, neutropenia, diarrhea) has recently been reported in two patients with deficient UGT*1.1 activity. SN-38 glucuronide (SN-38G), which has only 1/100th the antitumor activity of SN-38, is actively secreted into the bile by a canalicular multispecific organic anion transporter. Deconjugation of SN-38G to SN-38 by beta-glucuronidase produced by the intestinal flora may contribute to enterohepatic recirculation of SN-38 and delayed intestinal toxicity.

Identification of Bisindolylmaleimide IX as a potential agent to treat drug-resistant BCR-ABL positive leukemia

PubMed Central

Liu, Huijuan; Zang, Yi; Azam, Mohammad; Habib, Samy L.; Li, Jia; Ruan, Xinsen; Jia, Hao; Wang, Xueying; Li, Baojie

2016-01-01

Chronic myeloid leukemia (CML) treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. In a screen for novel chemotherapeutic drug candidates with genotoxic activity, we identified a bisindolylmaleimide derivative, IX, as a small molecule compound with therapeutic potential against CML including drug-resistant CML. We show that Bisindolylmaleimide IX inhibits DNA topoisomerase, generates DNA breaks, activates the Atm-p53 and Atm-Chk2 pathways, and induces cell cycle arrest and cell death. Interestingly, Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL. BCR-ABL positive cells display enhanced DNA damage and increased cell cycle arrest in response to Bisindolylmaleimide IX due to decreased expression of topoisomerases. Cells positive for BCR-ABL or drug-resistant T315I BCR-ABL also display increased cytotoxicity since Bisindolylmaleimide IX inhibits B-Raf and the downstream oncogene addiction pathway. Mouse cancer model experiments showed that Bisindolylmaleimide IX, at doses that show little side effect, was effective in treating leukemia-like disorders induced by BCR-ABL or T315I BCR-ABL, and prolonged the lifespan of these model mice. Thus, Bisindolylmaleimide IX presents a novel drug candidate to treat drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway activated by BCR-ABL. PMID:27564101

Synthesis of 6-substituted 6H-indolo[2,3-b]quinolines as novel cytotoxic agents and topoisomerase II inhibitors.

PubMed

Kaczmarek, Lukasz; Luniewski, Wojciech; Zagrodzki, Bogdan; Godlewska, Joanna; Osiadacz, Jarosław; Wietrzyk, Joanna; Opolski, Adam; Peczyńska-Czoch, Wanda

2002-01-01

A systematic investigation into the impact of the substituents introduced into the indolo[2,3-b]quinoline system is described. The findings clearly demonstrate that the compounds bearing a methyl group or a longer aliphatic chain at the N-6 position are inactive against prokaryotic and eukaryotic cells. The introduction of alkyl-amino-alkyl substituent at the N-6 position of indolo[2,3-b]quinoline accounts for the appearance of the antimicrobial and.cytotoxic properties. The cytotoxicity against oral epidermoid carcinoma KB (ID50) is in the range from 2.0 to 9.0 microM, and the antimicrobial activity (MIC) falls between 0.03 and 0.50 mM. The structural relation within 6H-indolo[2,3-b]quinolines, concerning their antimicrobial and cytotoxic activity, corresponds well with their ability to bind DNA and to inhibit topoisomerase II activity.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

PubMed

Cotman, Andrej E; Trampuž, Marko; Brvar, Matjaž; Kikelj, Danijel; Ilaš, Janez; Peterlin-Mašič, Lucija; Montalvão, Sofia; Tammela, Päivi; Frlan, Rok

2017-08-01

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC 50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC 90 ) of 14 and 28 µg/mL, respectively. © 2017 Deutsche Pharmazeutische Gesellschaft.

The application of click chemistry in the synthesis of agents with anticancer activity

PubMed Central

Ma, Nan; Wang, Ying; Zhao, Bing-Xin; Ye, Wen-Cai; Jiang, Sheng

2015-01-01

The copper(I)-catalyzed 1,3-dipolar cycloaddition between alkynes and azides (click chemistry) to form 1,2,3-triazoles is the most popular reaction due to its reliability, specificity, and biocompatibility. This reaction has the potential to shorten procedures, and render more efficient lead identification and optimization procedures in medicinal chemistry, which is a powerful modular synthetic approach toward the assembly of new molecular entities and has been applied in anticancer drugs discovery increasingly. The present review focuses mainly on the applications of this reaction in the field of synthesis of agents with anticancer activity, which are divided into four groups: topoisomerase II inhibitors, histone deacetylase inhibitors, protein tyrosine kinase inhibitors, and antimicrotubule agents. PMID:25792812

Immunodetection of human topoisomerase I-DNA covalent complexes

PubMed Central

Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

2016-01-01

A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo. PMID:26917015

Biological evaluation of omega-(dialkylamino)alkyl derivatives of 6H-indolo[2,3-b]quinoline--novel cytotoxic DNA topoisomerase II inhibitors.

PubMed

Godlewska, Joanna; Luniewski, Wojciech; Zagrodzki, Bogdan; Kaczmarek, Lukasz; Bielawska-Pohl, Aleksandra; Dus, Danuta; Wietrzyk, Joanna; Opolski, Adam; Siwko, Magdalena; Jaromin, Anna; Jakubiak, Anna; Kozubek, Arkadiusz; Peczyñska-Czoch, Wanda

2005-01-01

A series of novel 6H-indolo[2,3-b]quinoline derivatives, substituted at C-2, C-9 or N-6 position with dialkyl(alkylamino)alkyl chains differing in the number of methylene groups, was prepared. These compounds were evaluated in vitro for their antimicrobial and cytotoxic activity against several cell lines of different origin and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. Liphophilic and calf thymus DNA-binding properties of these compounds were also investigated. All the compounds tested inhibited the growth of Gram-positive bacteria and fungi at MIC values ranging between 0.25 and 1 mM. They also showed cytotoxic activity against KB (human cervix carcinoma) cells (ID50 varied from 2.1 to 9.0 microM) and were able to overcome multidrug resistance in colorectal adenocarcinoma LoVo/DX, uterine sarcoma MES-SA/DX5 and promyelocytic leukemia HL-60/MX2 cells (the values of the resistance index RI fell between 0.54 and 2.4). The compounds induced G2M-phase cell cycle arrest in Jurkat T-cell leukemia cells, revealed DNA-binding properties and inhibited topoisomerase II activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Yan; Chen, Wang; Zhao, Baobing

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,more » 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.« less

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

PubMed

Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan

2015-11-24

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

Cancer.gov

SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

Regulation of Hepatic Cholesteryl Ester Transfer Protein Expression and Reverse Cholesterol Transport by Inhibition of DNA Topoisomerase II.

PubMed

Liu, Mengyang; Chen, Yuanli; Zhang, Ling; Wang, Qixue; Ma, Xingzhe; Li, Xiaoju; Xiang, Rong; Zhu, Yan; Qin, Shucun; Yu, Yang; Jiang, Xian-cheng; Duan, Yajun; Han, Jihong

2015-06-05

Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high density lipoprotein to triglyceride-rich lipoproteins. CETP expression can be transcriptionally activated by liver X receptor (LXR). Etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors. Etoposide has been reported to inhibit atherosclerosis in rabbits with un-fully elucidated mechanisms. In this study we determined if Topo II activity can influence cholesterol metabolism by regulating hepatic CETP expression. Inhibition of Topo II by etoposide, teniposide, or Topo II siRNA increased CETP expression in human hepatic cell line, HepG2 cells, which was associated with increased CETP secretion and mRNA expression. Meanwhile, inhibition of LXR expression by LXR siRNA attenuated induction of CETP expression by etoposide and teniposide. Etoposide and teniposide induced LXRα expression and LXRα/β nuclear translocation while inhibiting expression of receptor interacting protein 140 (RIP140), an LXR co-repressor. In vivo, administration of teniposide moderately reduced serum lipid profiles, induced CETP expression in the liver, and activated reverse cholesterol transport in CETP transgenic mice. Our study demonstrates a novel function of Topo II inhibitors in cholesterol metabolism by activating hepatic CETP expression and reverse cholesterol transport. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent pathway.

PubMed

Nayak, V Lakshma; Nagesh, Narayana; Ravikumar, A; Bagul, Chandrakant; Vishnuvardhan, M V P S; Srinivasulu, Vunnam; Kamal, Ahmed

2017-01-01

Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.

DS-8201a, a new HER2-targeting antibody-drug conjugate incorporating a novel DNA topoisomerase I inhibitor, overcomes HER2-positive gastric cancer T-DM1 resistance.

PubMed

Takegawa, Naoki; Nonagase, Yoshikane; Yonesaka, Kimio; Sakai, Kazuko; Maenishi, Osamu; Ogitani, Yusuke; Tamura, Takao; Nishio, Kazuto; Nakagawa, Kazuhiko; Tsurutani, Junji

2017-10-15

Anti-HER2 therapies are beneficial for patients with HER2-positive breast or gastric cancer. T-DM1 is a HER2-targeting antibody-drug conjugate (ADC) comprising the antibody trastuzumab, a linker, and the tubulin inhibitor DM1. Although effective in treating advanced breast cancer, all patients eventually develop T-DM1 resistance. DS-8201a is a new ADC incorporating an anti-HER2 antibody, a newly developed, enzymatically cleavable peptide linker, and a novel, potent, exatecan-derivative topoisomerase I inhibitor (DXd). DS-8201a has a drug-to-antibody-ratio (DAR) of 8, which is higher than that of T-DM1 (3.5). Owing to these unique characteristics and unlike T-DM1, DS-8201a is effective against cancers with low-HER2 expression. In the present work, T-DM1-resistant cells (N87-TDMR), established using the HER2-positive gastric cancer line NCI-N87 and continuous T-DM1 exposure, were shown to be susceptible to DS-8201a. The ATP-binding cassette (ABC) transporters ABCC2 and ABCG2 were upregulated in N87-TDMR cells, but HER2 overexpression was retained. Furthermore, inhibition of ABCC2 and ABCG2 by MK571 restored T-DM1 sensitivity. Therefore, resistance to T-DM1 is caused by efflux of its payload DM1, due to aberrant expression of ABC transporters. In contrast to DM1, DXd payload of DS-8201a inhibited the growth of N87-TDMR cells in vitro. This suggests that either DXd may be a poor substrate of ABCC2 and ABCG2 in comparison to DM1, or the high DAR of DS-8201a relative to T-DM1 compensates for increased efflux. Notably, N87-TDMR xenograft tumor growth was prevented by DS-8201a. In conclusion, the efficacy of DS-8201a as a treatment for patients with T-DM1-resistant breast or gastric cancer merits investigation. © 2017 UICC.

Mechanism of Growth Inhibition of Human Cancer Cells by Conjugated Eicosapentaenoic Acid, an Inhibitor of DNA Polymerase and Topoisomerase

PubMed Central

Yonezawa, Yuko; Yoshida, Hiromi; Mizushina, Yoshiyuki

2007-01-01

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We found that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) inhibited the activities of eukaryotic pols and topos in vitro, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC50 values for mammalian pols and human topos of 11.0 – 31.8 and 0.5 – 2.5 μM, respectively. cEPA inhibited the proliferation of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, and the inhibitory effect was stronger than that of normal EPA. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins, such as RPA70, ATR and phosphorylated-Chk1/2, were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of conjugated PUFA, such as cEPA, as a leading anti-cancer compound that inhibited pols and topos activities.

Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

PubMed Central

Khan, Shabana I.; Nimrod, Alison C.; Mehrpooya, Mohammed; Nitiss, John L.; Walker, Larry A.; Clark, Alice M.

2002-01-01

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets. PMID:12019091

Antifungal activity of eupolauridine and its action on DNA topoisomerases.

PubMed

Khan, Shabana I; Nimrod, Alison C; Mehrpooya, Mohammed; Nitiss, John L; Walker, Larry A; Clark, Alice M

2002-06-01

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 microg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.

Oxidative Metabolites of Curcumin Poison Human Type II Topoisomerases†

PubMed Central

Ketron, Adam C.; Gordon, Odaine N.; Schneider, Claus; Osheroff, Neil

2013-01-01

The polyphenol curcumin is the principal flavor and color component of the spice turmeric. Beyond its culinary uses, curcumin is believed to positively impact human health and displays antioxidant, anti-inflammatory, antibacterial, and chemopreventive properties. It also is in clinical trials as an anticancer agent. In aqueous solution at physiological pH, curcumin undergoes spontaneous autoxidation that is enhanced by oxidizing agents. The reaction proceeds through a series of quinone methide and other reactive intermediates to form a final dioxygenated bicyclopentadione product. Several naturally occurring polyphenols that can form quinones have been shown to act as topoisomerase II poisons (i.e., increase levels of topoisomerase II-mediated DNA cleavage). Because several of these compounds have chemopreventive properties, we determined the effects of curcumin, its oxidative metabolites, and structurally related degradation products (vanillin, ferulic acid, and feruloylmethane), on the DNA cleavage activities of human topoisomerase IIα and IIβ. Intermediates in the curcumin oxidation pathway increased DNA scission mediated by both enzymes ~4-5–fold. In contrast, curcumin and the bicyclopentadione, as well as vanillin, ferulic acid, and feruloylmethane, had no effect on DNA cleavage. As found for other quinone-based compounds, curcumin oxidation intermediates acted as redox-dependent (as opposed to interfacial) topoisomerase II poisons. Finally, under conditions that promote oxidation, the dietary spice turmeric enhanced topoisomerase II-mediated DNA cleavage. Thus, even within the more complex spice formulation, oxidized curcumin intermediates appear to function as topoisomerase II poisons. PMID:23253398

Screening for Natural Inhibitors of Topoisomerases I from Rhamnus davurica by Affinity Ultrafiltration and High-Performance Liquid Chromatography–Mass Spectrometry

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Topoisomerase I (Topo I) catalyzes topological interconversion of duplex DNA during DNA replication and transcription, and has been deemed as important antineoplastic targets. In this study, the fraction R.d-60 from ethyl acetate extracts of Rhamnus davurica showed higher inhibitory rates against SGC-7901 and HT-29 compared with the R.d-30 fraction in vitro. However, the specific active components of R.d-60 fraction remain elusive. To this end, a method based on bio-affinity ultrafiltration and high performance liquid chromatography/electrospray mass spectrometry (HPLC- ESI-MS/MS) was developed to rapidly screen and identify the Topo I inhibitors in this fraction. The enrichment factors (EFs) were calculated to evaluate the binding affinities between the bioactive constituents and Topo I. As a result, eight ligands were identified and six of which with higher EFs showed more potential antitumor activity. Furthermore, antiproliferative assays in vitro (IC50 values) with two representative candidates (apigenin, quercetin) against SGC-7901, HT-29 and Hep G2 cells were conducted and further validated. Finally, the structure-activity relationships revealed that flavones contain a C2-C3 double bond of C ring exhibited higher bio-affinities to Topo I than those without it. This integrated method combining Topo I ultrafiltration with HPLC-MS/MS proved to be very efficient in rapid screening and identification of potential Topo I inhibitors from the complex extracts of medicinal plants, and could be further explored as a valuable high-throughput screening platform in the early drug discovery stage. PMID:28919906

Quinone-induced Enhancement of DNA Cleavage by Human Topoisomerase IIα: Adduction of Cysteine Residues 392 and 405†

PubMed Central

Bender, Ryan P.; Ham, Amy-Joan L.; Osheroff, Neil

2010-01-01

Several quinone-based metabolites of drugs and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein, and appear to increase levels of enzyme-DNA cleavage complexes by at least two potentially independent mechanisms. Treatment of topoisomerase IIα with quinones inhibits DNA religation, and blocks the N-terminal gate of the protein by crosslinking its two protomer subunits. It is not known whether these two effects result from quinone adduction to the same amino acid residue(s) in topoisomerase IIα or whether they are mediated by modification of separate residues. Therefore, the present study identified amino acid residues in human topoisomerase IIα that are modified by quinones and determined their role in the actions of these compounds as topoisomerase II poisons. Four cysteine residues were identified by mass spectrometry as sites of quinone adduction: cys170, cys392, cys405, and cys455. Mutations (cys–>ala) were individually generated at each position. Only mutations at cys392 or cys405 reduced sensitivity (~50% resistance) to benzoquinone. Top2αC392A and top2αC405A displayed faster rates (~2–fold) of DNA religation than wild-type topoisomerase IIα in the presence of the quinone. In contrast, as determined by DNA binding, protein clamp closing, and protomer crosslinking experiments, mutations at cys392 and cys405 did not affect the ability of benzoquinone to block the N-terminal gate of topoisomerase IIα. These findings indicate that adduction of cys392 and cys405 is important for the actions of quinones against the enzyme, and increases levels of cleavage complexes primarily by inhibiting DNA religation. PMID:17298034

Dose escalation study to evaluate safety, tolerability and efficacy of intravenous etoposide phosphate administration in 27 dogs with multicentric lymphoma

PubMed Central

Hordeaux, Juliette; Bouchaert, Emmanuel; Gomes, Bruno

2017-01-01

Comparative oncology has shown that naturally occurring canine cancers are of valuable and translatable interest for the understanding of human cancer biology and the characterization of new therapies. This work was part of a comparative oncology project assessing a new, clinical-stage topoisomerase II inhibitor and comparing it with etoposide in dogs with spontaneous lymphoma with the objective to translate findings from dogs to humans. Etoposide is a topoisomerase II inhibitor widely used in various humans’ solid and hematopoietic cancer, but little data is available concerning its potential antitumor efficacy in dogs. Etoposide phosphate is a water-soluble prodrug of etoposide which is expected to be better tolerated in dogs. The objectives of this study were to assess the safety, the tolerability and the efficacy of intravenous etoposide phosphate in dogs with multicentric lymphoma. Seven dose levels were evaluated in a traditional 3+3 phase I design. Twenty-seven owned-dogs with high-grade multicentric lymphoma were enrolled and treated with three cycles of etoposide phosphate IV injections every 2 weeks. Adverse effects were graded according to the Veterinary Cooperative Oncology Group criteria. A complete end-staging was realized 45 days after inclusion. The maximal tolerated dose was 300 mg/m2. At this dose level, the overall response rate was 83.3% (n = 6, 3 PR and 2 CR). Only a moderate reversible gastrointestinal toxicity, no severe myelotoxicity and no hypersensitivity reaction were reported at this dose level. Beyond the characterization of etoposide clinical efficacy in dogs, this study underlined the clinical and therapeutic homologies between dog and human lymphomas. PMID:28505195

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

PubMed Central

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

2010-01-01

Summary 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 shows how topoisomerase V may interact with DNA. PMID:20637419

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taneja, Bhupesh; Patel, Asmita; Slesarev, Alexei

Topoisomerases are involved in controlling and maintaining the topology of DNA and are present in all kingdoms of life. Unlike all other types of topoisomerases, similar type IB enzymes have only been identified in bacteria and eukarya. The only putative type IB topoisomerase in archaea is represented by Methanopyrus kandleri topoisomerase V. Despite several common functional characteristics, topoisomerase V shows no sequence similarity to other members of the same type. The structure of the 61 kDa N-terminal fragment of topoisomerase V reveals no structural similarity to other topoisomerases. Furthermore, the structure of the active site region is different, suggesting nomore » conservation in the cleavage and religation mechanism. Additionally, the active site is buried, indicating the need of a conformational change for activity. The presence of a topoisomerase in archaea with a unique structure suggests the evolution of a separate mechanism to alter DNA.« less

Antibacterial mechanism of fraxetin against Staphylococcus aureus

PubMed Central

WANG, HAITING; ZOU, DAN; XIE, KUNPEING; XIE, MINGJIE

2014-01-01

Fraxetin is one of the main constituents of the traditional medicinal plant Fraxinus rhynchophylla. The inhibitory effect of fraxetin on various bacterial strains has been extensively reported, however, its mechanism of action on bacterial cells remains to be elucidated. In the present study, the antibacterial mechanism of fraxetin on Staphylococcus aureus was systematically investigated by examining its effect on cell membranes, protein synthesis, nucleic acid content and topoisomerase activity. The results indicated that fraxetin increased the permeability of the cell membrane but did not render it permeable to macromolecules, such as DNA and RNA. Additionally, the quantity of protein, DNA and RNA decreased to 55.74, 33.86 and 48.96%, respectively following treatment with fraxetin for 16 h. The activity of topoisomerase I and topoisomerase II were also markedly inhibited as fraxetin concentration increased. The result of the ultraviolet-visible spectrophotometry demonstrated that the DNA characteristics exhibited a blue shift and hypochromic effect following treatment with fraxetin. These results indicated that fraxetin had a marked inhibitory effect on S.aureus proliferation. Further mechanistic studies showed that fraxetin could disrupt nucleic acid and protein synthesis by preventing topoisomerase from binding to DNA. PMID:25189268

Solvent Dependency of the UV-Vis Spectrum of Indenoisoquinolines: Role of Keto-Oxygens as Polarity Interaction Probes

PubMed Central

Coletta, Andrea; Castelli, Silvia; Chillemi, Giovanni; Sanna, Nico; Cushman, Mark; Pommier, Yves; Desideri, Alessandro

2013-01-01

Indenoisoquinolines are the most promising non-campthotecins topoisomerase IB inhibitors. We present an integrated experimental/computational investigation of the UV-Vis spectra of the IQNs parental compound (NSC314622) and two of its derivatives (NSC724998 and NSC725776) currently undergoing Phase I clinical trials. In all the three compounds a similar dependence of the relative absorption intensities at 270 nm and 290 nm on solvent polarity is found. The keto-oxygens in positions 5 and 11 of the molecular scaffold of the molecule are the principal chromophores involved in this dependence. Protic interactions on these sites are also found to give rise to absorptions at wavelength <250 nm observed in water solution, due to the stabilization of highly polarized tautomers of the molecule. These results suggest that the keto-oxygens are important polarizable groups that can act as useful interactors with the molecular receptor, providing at the same time an useful fingerprint for the monitoring of the drug binding to topoisomerase IB. PMID:24086299

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals

PubMed Central

Ahmad, Muzammil; Xue, Yutong; Lee, Seung Kyu; Martindale, Jennifer L.; Shen, Weiping; Li, Wen; Zou, Sige; Ciaramella, Maria; Debat, Hélène; Nadal, Marc; Leng, Fenfei; Zhang, Hongliang; Wang, Quan; Siaw, Grace Ee-Lu; Niu, Hengyao; Pommier, Yves; Gorospe, Myriam; Hsieh, Tao-Shih; Tse-Dinh, Yuk-Ching; Xu, Dongyi; Wang, Weidong

2016-01-01

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals. PMID:27257063

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

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 themore » 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.« less

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

PubMed

Esselen, Melanie; Boettler, Ute; Teller, Nicole; Bachler, Simone; Hutter, Melanie; Rufer, Corinna E; Skrbek, Susanne; Marko, Doris

2011-07-13

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

PubMed

Karkare, Shantanu; Chung, Terence T H; Collin, Frederic; Mitchenall, Lesley A; McKay, Adam R; Greive, Sandra J; Meyer, Jacobus J M; Lall, Namrita; Maxwell, Anthony

2013-02-15

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action*

PubMed Central

Karkare, Shantanu; Chung, Terence T. H.; Collin, Frederic; Mitchenall, Lesley A.; McKay, Adam R.; Greive, Sandra J.; Meyer, Jacobus J. M.; Lall, Namrita; Maxwell, Anthony

2013-01-01

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents. PMID:23275348

Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation, and anaphase entry

PubMed Central

Fasulo, Barbara; Koyama, Carol; Yu, Kristina R.; Homola, Ellen M.; Hsieh, Tao S.; Campbell, Shelagh D.; Sullivan, William

2012-01-01

Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events. PMID:22262459

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

PubMed

Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

PubMed Central

Barker, Catherine R.; McNamara, Anne V.; Rackstraw, Stephen A.; Nelson, David E.; White, Mike R.; Watson, Alastair J. M.; Jenkins, John R.

2006-01-01

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death. PMID:16504968

A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase I and pBAD/Thio supercoiled plasmid DNA

PubMed Central

Tiwari, Purushottam Babu; Annamalai, Thirunavukkarasu; Cheng, Bokun; Narula, Gagandeep; Wang, Xuewen; Tse-Dinh, Yuk-Ching; He, Jin; Darici, Yesim

2014-01-01

To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg2+. In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoI-pBAD/Thio interactions is determined to be about 8 nM. We then studied the effect of Mg2+ on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg2+ coordinated EctopoI (Mg2+EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg2+EctopoI-pBAD/Thio interactions (~0.043 s−1), compared to EctopoI-pBAD/Thio interactions (~0.017 s−1). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg2+ and furthers the understanding of importance of the Mg2+ ion for bacterial topoisomerase I catalytic activity. PMID:24530905

Natural variation in a single amino acid substitution underlies physiological responses to topoisomerase II poisons.

PubMed

Zdraljevic, Stefan; Strand, Christine; Seidel, Hannah S; Cook, Daniel E; Doench, John G; Andersen, Erik C

2017-07-01

Many chemotherapeutic drugs are differentially effective from one patient to the next. Understanding the causes of this variability is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate sensitivity to a group of anti-neoplastic drugs that target topoisomerase II using the model organism Caenorhabditis elegans. We show that wild strains of C. elegans vary in their sensitivity to these drugs, and we use an unbiased genetic approach to demonstrate that this natural variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and its poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this amino acid difference between the two human topoisomerase isoforms influences cytotoxicity of topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility of C. elegans in understanding the genetics of drug responses.

BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.

PubMed

Yang, Jay; Bachrati, Csanad Z; Hickson, Ian D; Brown, Grant W

2012-01-01

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA.

A natural anticancer agent thaspine targets human topoisomerase IB.

PubMed

Castelli, Silvia; Katkar, Prafulla; Vassallo, Oscar; Falconi, Mattia; Linder, Stig; Desideri, Alessandro

2013-02-01

The different steps of the topoisomerase I catalytic cycle have been analyzed in the presence of the plant alkaloid thaspine (1- (2-(Dimethylamino)ethyl)-3,8-dimethoxychromeno[5,4,3-cde]chromene-5,10-dione), known to induce apoptosis in colon carcinoma cells. The experiments indicate that thaspine inhibits both the cleavage and the religation steps of the enzyme reaction. The inhibition is reversible and the effect is enhanced upon pre-incubation. Molecular docking simulations of thaspine over topoisomerase I, in the presence or absence of the DNA substrate, show that thaspine, when interacting with the enzyme alone in the closed or in the open state, can bind in proximity of the active residues preventing the cleavage reaction, whilst when docked with the enzyme-DNA cleavable complex intercalates between the DNA bases in a way similar to that found for camptothecin, explaining its religation inhibition. These results unequivocally demonstrate that thaspine targets human topoisomerase I .

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

PubMed

Ahmed, Wareed; Sala, Claudia; Hegde, Shubhada R; Jha, Rajiv Kumar; Cole, Stewart T; Nagaraja, Valakunja

2017-05-01

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase

PubMed Central

Ahmed, Wareed; Sala, Claudia; Hegde, Shubhada R.; Jha, Rajiv Kumar

2017-01-01

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase. PMID:28463980

A high-throughput fluorescence polarization assay for inhibitors of gyrase B.

PubMed

Glaser, Bryan T; Malerich, Jeremiah P; Duellman, Sarah J; Fong, Julie; Hutson, Christopher; Fine, Richard M; Keblansky, Boris; Tang, Mary J; Madrid, Peter B

2011-02-01

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.

PubMed

Ahmad, Muzammil; Shen, Weiping; Li, Wen; Xue, Yutong; Zou, Sige; Xu, Dongyi; Wang, Weidong

2017-03-17

Human cells contain five topoisomerases in the nucleus and cytoplasm, but which one is the major topoisomerase for mRNAs is unclear. To date, Top3β is the only known topoisomerase that possesses RNA topoisomerase activity, binds mRNA translation machinery and interacts with an RNA-binding protein, FMRP, to promote synapse formation; and Top3β gene deletion has been linked to schizophrenia. Here, we show that Top3β is also the most abundant mRNA-binding topoisomerase in cells. Top3β, but not other topoisomerases, contains a distinctive RNA-binding domain; and deletion of this domain diminishes the amount of Top3β that associates with mRNAs, indicating that Top3β is specifically targeted to mRNAs by its RNA binding domain. Moreover, Top3β mutants lacking either its RNA-binding domain or catalytic residue fail to promote synapse formation, suggesting that Top3β requires both its mRNA-binding and catalytic activity to facilitate neurodevelopment. Notably, Top3β proteins bearing point mutations from schizophrenia and autism individuals are defective in association with FMRP; whereas one of the mutants is also deficient in binding mRNAs, catalyzing RNA topoisomerase reaction, and promoting synapse formation. Our data suggest that Top3β is the major topoisomerase for mRNAs, and requires both RNA binding and catalytic activity to promote neurodevelopment and prevent mental dysfunction. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaiserman, H.B.; Ingebritsen, T.S.; Benbow, R.M.

1988-05-03

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purifiedmore » fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, (..gamma..-/sup 32/P)ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.« less

In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.

PubMed

Freitas, Ferdinando B; Frouco, Gonçalo; Martins, Carlos; Leitão, Alexandre; Ferreira, Fernando

2016-10-01

African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes.

PubMed

Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

2017-04-21

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes

PubMed Central

Guha, Mithu; Saare, Mario; Maslovskaja, Julia; Kisand, Kai; Liiv, Ingrid; Haljasorg, Uku; Tasa, Tõnis; Metspalu, Andres; Milani, Lili; Peterson, Pärt

2017-01-01

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes. PMID:28242760

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

PubMed

Meersma, G J; Bakker, M; Groen, H J; Van der Zee, A G; Jensen, P B; Giaccone, G; De Vries, E G; Smit, E F

1999-01-01

Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

Role of the protein in the DNA sequence specificity of the cleavage site stabilized by the camptothecin topoisomerase IB inhibitor: a metadynamics study

PubMed Central

Coletta, Andrea; Desideri, Alessandro

2013-01-01

Camptothecin (CPT) is a topoisomerase IB (TopIB) selective inhibitor whose derivatives are currently used in cancer therapy. TopIB cleaves DNA at any sequence, but in the presence of CPT the only stabilized protein–DNA covalent complex is the one having a thymine in position −1 with respect to the cleavage site. A metadynamics simulation of two TopIB–DNA–CPT ternary complexes differing for the presence of a thymine or a cytosine in position −1 indicates the occurrence of two different drug’s unbinding pathways. The free-energy difference between the bound state and the transition state is large when a thymine is present in position −1 and is strongly reduced in presence of a cytosine, in line with the different drug stabilization properties of the two systems. Such a difference is strictly related to the changes in the hydrogen bond network between the protein, the DNA and the drug in the two systems, indicating a direct role of the protein in determining the specificity of the cleavage site sequence stabilized by the CPT. Calculations carried out in presence of one compound of the indenoisoquinoline family (NSC314622) indicate a comparable energy difference between the bound and the transition state independently of the presence of a thymine or a cytosine in position −1, in line with the experimental results. PMID:24003027

3′-Phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1) knockdown sensitizes non-small cell lung cancer cells to DNA damaging agents

PubMed Central

Leung, Ada W. Y.; Dragowska, Wieslawa H.; Ricaurte, Daniel; Kwok, Brian; Mathew, Veena; Roosendaal, Jeroen; Ahluwalia, Amith; Warburton, Corinna; Laskin, Janessa J.; Stirling, Peter C.; Qadir, Mohammed A.; Bally, Marcel B.

2015-01-01

Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30–33%. Through an siRNA screen, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC. PMID:26220590

On the binding of indeno[1,2-c]isoquinolines in the DNA-topoisomerase I cleavage complex.

PubMed

Xiao, Xiangshu; Antony, Smitha; Pommier, Yves; Cushman, Mark

2005-05-05

An ab initio quantum mechanics calculation is reported which predicts the orientation of indenoisoquinoline 4 in the ternary cleavage complex formed from DNA and topoisomerase I (top1). The results of this calculation are consistent with the hypothetical structures previously proposed for the indenoisoquinoline-DNA-top1 ternary complexes based on molecular modeling, the crystal structure of a recently reported ternary complex, and the biological results obtained with a pair of diaminoalkyl-substituted indenoisoquinoline enantiomers. The results of these studies indicate that the pi-pi stacking interactions between the indenoisoquinolines and the neighboring DNA base pairs play a major role in determining binding orientation. The calculation of the electrostatic potential surface maps of the indenoisoquinolines and the adjacent DNA base pairs shows electrostatic complementarity in the observed binding orientation, leading to the conclusion that electrostatic attraction between the intercalators and the base pairs in the cleavage complex plays a major stabilizing role. On the other hand, the calculation of LUMO and HOMO energies of indenoisoquinoline 13b and neighboring DNA base pairs in conjunction with NBO analysis indicates that charge transfer complex formation plays a relatively minor role in stabilizing the ternary complexes derived from indenoisoquinolines, DNA, and top1. The results of these studies are important in understanding the existing structure-activity relationships for the indenoisoquinolines as top1 inhibitors and as anticancer agents, and they will be important in the future design of indenoisoquinoline-based top1 inhibitors.

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

PubMed Central

Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

2016-01-01

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. PMID:26908655

Variola virus topoisomerase: DNA cleavage specificity and distribution of sites in Poxvirus genomes.

PubMed

Minkah, Nana; Hwang, Young; Perry, Kay; Van Duyne, Gregory D; Hendrickson, Robert; Lefkowitz, Elliot J; Hannenhalli, Sridhar; Bushman, Frederic D

2007-08-15

Topoisomerase enzymes regulate superhelical tension in DNA resulting from transcription, replication, repair, and other molecular transactions. Poxviruses encode an unusual type IB topoisomerase that acts only at conserved DNA sequences containing the core pentanucleotide 5'-(T/C)CCTT-3'. In X-ray structures of the variola virus topoisomerase bound to DNA, protein-DNA contacts were found to extend beyond the core pentanucleotide, indicating that the full recognition site has not yet been fully defined in functional studies. Here we report quantitation of DNA cleavage rates for an optimized 13 bp site and for all possible single base substitutions (40 total sites), with the goals of understanding the molecular mechanism of recognition and mapping topoisomerase sites in poxvirus genome sequences. The data allow a precise definition of enzyme-DNA interactions and the energetic contributions of each. We then used the resulting "action matrix" to show that favorable topoisomerase sites are distributed all along the length of poxvirus DNA sequences, consistent with a requirement for local release of superhelical tension in constrained topological domains. In orthopox genomes, an additional central cluster of sites was also evident. A negative correlation of predicted topoisomerase sites was seen relative to early terminators, but no correlation was seen with early or late promoters. These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo.

Effect of short peptides containing lysine and epsilon-aminocaproic acid on fibrinolytic activity of plasmin and topoisomerase II action on supercoiled DNA.

PubMed

Midura-Nowaczek, Krystyna; Purwin, Maciej; Markowska, Agnieszka; Drozdowska, Danuta; Bruzgo, Magdalena

2013-01-01

Effects of eight short peptides containing lysine and epsilon-aminocaproic acid (EACA) on prolongation of the clot lysis time, as well as hemolytic and antibacterial activities were investigated. Interaction with plasmids pBR322 and pUC19 with the use of ethidium bromide assay and determination of influence on the activity of topoisomerase I and II were also tested. Examined compounds inhibited fibrinolytic activity of plasmin and five of them were more active than EACA. Amides of dipeptides were most active antifibrinolytics (IC50 < 0.2 mM). According to the obtained data, the significant inhibition of fibrinolytic activity of plasmin was not associated with hemolytic effects. Examined compounds did not show antibacterial activity (MIC > 512 mg/L). DNA binding effects determined with the use of ethidium bromide were weak for all peptides and similar to those observed with EACA. Six compounds inhibited topoisomerase II action on supercoiled DNA.

Topoisomerases interlink genetic network underlying autism.

PubMed

Vokálová, Lenka; Durdiaková, Jaroslava; Ostatníková, Daniela

2015-12-01

DNA topoisomerases belong to the group of proteins that play an important role in the organizational dynamics of the human genome. Their enzymatic activity solves topological strain rising from DNA supercoiling occurring during transcription. DNA topoisomerases are especially important for transcription of genes involved in neurodevelopment. Disruption of topoisomerase activity in animal models resulted in impaired neurodevelopment and changed brain architecture. Recent research revealed that topoisomerases induced expression of the same group of genes as those associated with autism. Transcriptional inhibition of neuronal genes during critical stages of brain development may be responsible for pathology of neurodevelopmental disorders such as autism. In this review we aim to outline the role of topoisomerase in neurodevelopment and its possible linkage to neuropathology of autism. Copyright © 2015 Elsevier Ltd. All rights reserved.

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

PubMed

Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

2017-05-30

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

Renal medullary carcinoma: molecular, pathological and clinical evidence for treatment with topoisomerase-inhibiting therapy.

PubMed

Schaeffer, Edward M; Guzzo, Thomas J; Furge, Kyle A; Netto, George; Westphal, Michael; Dykema, Karl; Yang, Ximing; Zhou, Ming; Teh, Bin Tean; Pavlovich, Christian P

2010-07-01

Aetiology (case series) Level of Evidence 4. To present the molecular rationale and potential clinical benefit of topoisomerase II (TopoII)-inhibiting therapy for renal medullary carcinoma (RMC), a rare but extremely lethal form of kidney cancer that classically afflicts young men with sickle-cell trait. The current therapeutic approach with these aggressive tumours is radical nephrectomy followed by systemic chemotherapy, but the prognosis remains dismal. The whole-genome expression was analysed in four RMC tumours. We also report a case of metastatic RMC in which a complete response was achieved for 9 months using a TopoII-inhibiting therapy. Expanded whole-genome expression analysis showed increases of TopoII in all cases. There was also overall deregulation of DNA remodelling and repair, and an ontological association between RMC and urothelial carcinoma. Using a TopoII-inhibiting agent, there was a complete response for 9 months in a patient with metastatic RMC. This report provides molecular evidence for the rational use of TopoII inhibitors in the treatment of RMC.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

PubMed

Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

2009-12-29

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

PubMed Central

2017-01-01

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process. PMID:29290109

Dual targeting DNA gyrase B (GyrB) and topoisomerse IV (ParE) inhibitors: A review.

PubMed

Azam, Mohammed Afzal; Thathan, Janarthanan; Jubie, Selvaraj

2015-10-01

GyrB and ParE are type IIA topoisomerases and found in most bacteria. Its function is vital for DNA replication, repair and decatenation. The highly conserved ATP-binding subunits of DNA GyrB and ParE are structurally related and have been recognized as prime candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, no natural product or small molecule inhibitors targeting ATPase catalytic domain of both GyrB and ParE enzymes have succeeded in the clinic. Moreover, no inhibitors of these enzymes with broad-spectrum antibacterial activity against Gram-negative pathogens have been reported. Availability of high resolution crystal structures of GyrB and ParE made it possible for the design of many different classes of inhibitors with dual mechanism of action. Among them benzimidazoles, benzothiazoles, thiazolopyridines, imidiazopyridazoles, pyridines, indazoles, pyrazoles, imidazopyridines, triazolopyridines, pyrrolopyrimidines, pyrimidoindoles as well as related structures are disclosed in literatures. Unfortunately most of these inhibitors are found to be active against Gram-positive pathogens. In the present review we discuss about studies on novel dual targeting ATPase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

PubMed

Shcherbakov, Victor P; Kudryashova, Elena

2014-09-01

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification

PubMed Central

Seol, Yeonee; Hardin, Ashley H.; Strub, Marie-Paule; Charvin, Gilles; Neuman, Keir C.

2013-01-01

Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed. The kinetic proofreading (KPR) model postulates that strand passage requires a DNA-bound topoisomerase to collide twice in rapid succession with a second DNA segment, implying a quadratic relationship between DNA collision frequency and relaxation rate. To test this model, we used a single-molecule assay to measure the unlinking rate as a function of DNA collision frequency for Escherichia coli topoisomerase IV (topo IV) that displays efficient non-equilibrium topology simplification activity, and for E. coli topoisomerase III (topo III), a type IA topoisomerase that unlinks and unknots DNA to equilibrium levels. Contrary to the predictions of the KPR model, topo IV and topo III unlinking rates were linearly related to the DNA collision frequency. Furthermore, topo III exhibited decatenation activity comparable with that of topo IV, supporting proposed roles for topo III in DNA segregation. This study enables us to rule out the KPR model for non-equilibrium topology simplification. More generally, we establish an experimental approach to systematically control DNA collision frequency. PMID:23460205

In vitro antibacterial potency and spectrum of ABT-492, a new fluoroquinolone.

PubMed

Nilius, Angela M; Shen, Linus L; Hensey-Rudloff, Dena; Almer, Laurel S; Beyer, Jill M; Balli, Darlene J; Cai, Yingna; Flamm, Robert K

2003-10-01

ABT-492 demonstrated potent antibacterial activity against most quinolone-susceptible pathogens. The rank order of potency was ABT-492 > trovafloxacin > levofloxacin > ciprofloxacin against quinolone-susceptible staphylococci, streptococci, and enterococci. ABT-492 had activity comparable to those of trovafloxacin, levofloxacin, and ciprofloxacin against seven species of quinolone-susceptible members of the family Enterobacteriaceae, although it was less active than the comparators against Citrobacter freundii and Serratia marcescens. The activity of ABT-492 was greater than those of the comparators against fastidious gram-negative species, including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella spp. and against Pseudomonas aeruginosa and Helicobacter pylori. ABT-492 was as active as trovafloxacin against Chlamydia trachomatis, indicating good intracellular penetration and antibacterial activity. In particular, ABT-492 was more active than trovafloxacin and levofloxacin against multidrug-resistant Streptococcus pneumoniae, including strains resistant to penicillin and macrolides, and H. influenzae, including beta-lactam-resistant strains. It retained greater in vitro activity than the comparators against S. pneumoniae and H. influenzae strains resistant to other quinolones due to amino acid alterations in the quinolone resistance-determining regions of the target topoisomerases. ABT-492 was a potent inhibitor of bacterial topoisomerases, and unlike the comparators, DNA gyrase and topoisomerase IV from either Staphylococcus aureus or Escherichia coli were almost equally sensitive to ABT-492. The profile of ABT-492 suggested that it may be a useful agent for the treatment of community-acquired respiratory tract infections, as well as infections of the urinary tract, bloodstream, and skin and skin structure and nosocomial lung infections.

In Vitro Antibacterial Potency and Spectrum of ABT-492, a New Fluoroquinolone

PubMed Central

Nilius, Angela M.; Shen, Linus L.; Hensey-Rudloff, Dena; Almer, Laurel S.; Beyer, Jill M.; Balli, Darlene J.; Cai, Yingna; Flamm, Robert K.

2003-01-01

ABT-492 demonstrated potent antibacterial activity against most quinolone-susceptible pathogens. The rank order of potency was ABT-492 > trovafloxacin > levofloxacin > ciprofloxacin against quinolone-susceptible staphylococci, streptococci, and enterococci. ABT-492 had activity comparable to those of trovafloxacin, levofloxacin, and ciprofloxacin against seven species of quinolone-susceptible members of the family Enterobacteriaceae, although it was less active than the comparators against Citrobacter freundii and Serratia marcescens. The activity of ABT-492 was greater than those of the comparators against fastidious gram-negative species, including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella spp. and against Pseudomonas aeruginosa and Helicobacter pylori. ABT-492 was as active as trovafloxacin against Chlamydia trachomatis, indicating good intracellular penetration and antibacterial activity. In particular, ABT-492 was more active than trovafloxacin and levofloxacin against multidrug-resistant Streptococcus pneumoniae, including strains resistant to penicillin and macrolides, and H. influenzae, including β-lactam-resistant strains. It retained greater in vitro activity than the comparators against S. pneumoniae and H. influenzae strains resistant to other quinolones due to amino acid alterations in the quinolone resistance-determining regions of the target topoisomerases. ABT-492 was a potent inhibitor of bacterial topoisomerases, and unlike the comparators, DNA gyrase and topoisomerase IV from either Staphylococcus aureus or Escherichia coli were almost equally sensitive to ABT-492. The profile of ABT-492 suggested that it may be a useful agent for the treatment of community-acquired respiratory tract infections, as well as infections of the urinary tract, bloodstream, and skin and skin structure and nosocomial lung infections. PMID:14506039

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

PubMed

Infante Lara, Lorena; Sledge, Alexis; Laradji, Amine; Okoro, Cosmas O; Osheroff, Neil

2017-02-01

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons. Published by Elsevier Ltd.

Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism

PubMed Central

Farnet, C. M.; Wang, B.; Hansen, M.; Lipford, J. R.; Zalkow, L.; Robinson, W. E.; Siegel, J.; Bushman, F.

1998-01-01

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8,12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development. PMID:9736543

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

PubMed

Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

2003-07-01

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

DNA topoisomerase I and DNA gyrase as targets for TB therapy.

PubMed

Nagaraja, Valakunja; Godbole, Adwait A; Henderson, Sara R; Maxwell, Anthony

2017-03-01

Tuberculosis (TB) is the deadliest bacterial disease in the world. New therapeutic agents are urgently needed to replace existing drugs for which resistance is a significant problem. DNA topoisomerases are well-validated targets for antimicrobial and anticancer chemotherapies. Although bacterial topoisomerase I has yet to be exploited as a target for clinical antibiotics, DNA gyrase has been extensively targeted, including the highly clinically successful fluoroquinolones, which have been utilized in TB therapy. Here, we review the exploitation of topoisomerases as antibacterial targets and summarize progress in developing new agents to target DNA topoisomerase I and DNA gyrase from Mycobacterium tuberculosis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

All tangled up: how cells direct, manage and exploit topoisomerase function

PubMed Central

Vos, Seychelle M.; Tretter, Elsa M.; Schmidt, Bryan H.; Berger, James M.

2015-01-01

Preface Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases frequently are linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication, and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation, and utility as therapeutic targets. PMID:22108601

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

PubMed

Patel, S; Sprung, A U; Keller, B A; Heaton, V J; Fisher, L M

1997-10-01

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Camptothecin Attenuates Cytochrome P450 3A4 Induction by Blocking the Activation of Human Pregnane X ReceptorS⃞

PubMed Central

Chen, Yakun; Tang, Yong; Robbins, Gregory T.

2010-01-01

Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs. PMID:20504912

Selective inhibition of histone deacetylase 6 (HDAC6) induces DNA damage and sensitizes transformed cells to anticancer agents.

PubMed

Namdar, Mandana; Perez, Gisela; Ngo, Lang; Marks, Paul A

2010-11-16

Histone deacetylase 6 (HDAC6) is structurally and functionally unique among the 11 human zinc-dependent histone deacetylases. Here we show that chemical inhibition with the HDAC6-selective inhibitor tubacin significantly enhances cell death induced by the topoisomerase II inhibitors etoposide and doxorubicin and the pan-HDAC inhibitor SAHA (vorinostat) in transformed cells (LNCaP, MCF-7), an effect not observed in normal cells (human foreskin fibroblast cells). The inactive analogue of tubacin, nil-tubacin, does not sensitize transformed cells to these anticancer agents. Further, we show that down-regulation of HDAC6 expression by shRNA in LNCaP cells enhances cell death induced by etoposide, doxorubicin, and SAHA. Tubacin in combination with SAHA or etoposide is more potent than either drug alone in activating the intrinsic apoptotic pathway in transformed cells, as evidenced by an increase in PARP cleavage and partial inhibition of this effect by the pan-caspase inhibitor Z-VAD-fmk. HDAC6 inhibition with tubacin induces the accumulation of γH2AX, an early marker of DNA double-strand breaks. Tubacin enhances DNA damage induced by etoposide or SAHA as indicated by increased accumulation of γH2AX and activation of the checkpoint kinase Chk2. Tubacin induces the expression of DDIT3 (CHOP/GADD153), a transcription factor up-regulated in response to cellular stress. DDIT3 induction is further increased when tubacin is combined with SAHA. These findings point to mechanisms by which HDAC6-selective inhibition can enhance the efficacy of certain anti-cancer agents in transformed cells.

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

PubMed Central

Bradford, Patricia A.; Otterson, Linda G.; Basarab, Gregory S.; Kutschke, Amy C.; Giacobbe, Robert A.; Patey, Sara A.; Alm, Richard A.; Johnstone, Michele R.; Potter, Marie E.; Miller, Paul F.; Mueller, John P.

2014-01-01

AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development. PMID:25385112

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Cao, Nan; Cheng, Bokun

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) andmore » 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.« less

Switch in Site of Inhibition: A Strategy for Structure-Based Discovery of Human Topoisomerase IIα Catalytic Inhibitors

PubMed Central

2015-01-01

A study of structure-based modulation of known ligands of hTopoIIα, an important enzyme involved in DNA processes, coupled with synthesis and in vitro assays led to the establishment of a strategy of rational switch in mode of inhibition of the enzyme’s catalytic cycle. 6-Arylated derivatives of known imidazopyridine ligands were found to be selective inhibitors of hTopoIIα, while not showing TopoI inhibition and DNA binding. Interestingly, while the parent imidazopyridines acted as ATP-competitive inhibitors, arylated derivatives inhibited DNA cleavage similar to merbarone, indicating a switch in mode of inhibition from ATP-hydrolysis to the DNA-cleavage stage of catalytic cycle of the enzyme. The 6-aryl-imidazopyridines were relatively more cytotoxic than etoposide in cancer cells and less toxic to normal cells. Such unprecedented strategy will encourage research on “choice-based change” in target-specific mode of action for rapid drug discovery. PMID:25941559

The Dynamic Interplay Between DNA Topoisomerases and DNA Topology.

PubMed

Seol, Yeonee; Neuman, Keir C

2016-09-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell DNA topology is constantly in flux. Transcription and other essential processes including DNA replication and repair, alter the topology of the genome, while introducing additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases, is a pervasive factor that influences DNA metabolism in vivo . Building on the extensive structural and biochemical characterization over the past four decades that established the fundamental mechanistic basis of topoisomerase activity, the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases have begun to be explored. In this review we survey established and emerging DNA topology dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

The dynamic interplay between DNA topoisomerases and DNA topology.

PubMed

Seol, Yeonee; Neuman, Keir C

2016-11-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

3D visualization software to analyze topological outcomes of topoisomerase reactions

PubMed Central

Darcy, I. K.; Scharein, R. G.; Stasiak, A.

2008-01-01

The action of various DNA topoisomerases frequently results in characteristic changes in DNA topology. Important information for understanding mechanistic details of action of these topoisomerases can be provided by investigating the knot types resulting from topoisomerase action on circular DNA forming a particular knot type. Depending on the topological bias of a given topoisomerase reaction, one observes different subsets of knotted products. To establish the character of topological bias, one needs to be aware of all possible topological outcomes of intersegmental passages occurring within a given knot type. However, it is not trivial to systematically enumerate topological outcomes of strand passage from a given knot type. We present here a 3D visualization software (TopoICE-X in KnotPlot) that incorporates topological analysis methods in order to visualize, for example, knots that can be obtained from a given knot by one intersegmental passage. The software has several other options for the topological analysis of mechanisms of action of various topoisomerases. PMID:18440983

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies

PubMed Central

Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H.; Mondragón, Alfonso

2013-01-01

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix–hairpin–helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase

PubMed Central

Yang, Xi; Débat, Hélène; Fogg, Jonathan M; Zechiedrich, Lynn; Strick, Terence R; Garnier, Florence

2018-01-01

Abstract DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus. PMID:29253195

Structural Studies of E. coli Topoisomerase III-DNA Complexes Reveal A Novel Type IA Topoisomerase-DNA Conformational Intermediate

PubMed Central

Changela, Anita; DiGate, Russell J.; Mondragón, Alfonso

2007-01-01

Summary E. coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5′ phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an 8-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding. PMID:17331537

Interaction of cholinesterase modulators with DNA and their cytotoxic activity.

PubMed

Janockova, Jana; Gulasova, Zuzana; Plsikova, Jana; Musilek, Kamil; Kuca, Kamil; Mikes, Jaromir; Culka, Lubomir; Fedorocko, Peter; Kozurkova, Maria

2014-03-01

This research was focused on a study of the binding properties of a series of cholinesterase reactivators compounds K075 (1), K027 (2) and inhibitors compounds K524, K009 and 7-MEOTA (3-5) with calf thymus DNA. The nature of the interactions between compounds 1-5 and DNA were studied using spectroscopic techniques (UV-vis, fluorescence spectroscopy and circular dichroism). The binding constants for complexes of cholinesterase modulators with DNA were determined from UV-vis spectroscopic titrations (K=0.5 × 10(4)-8.9 × 10(5)M(-1)). The ability of the prepared analogues to relax topoisomerase I was studied with electrophoretic techniques and it was proved that ligands 4 and 5 inhibited this enzyme at a concentration of 30 μM. The biological activity of the novel compounds was assessed through an examination of changes in cell cycle distribution, mitochondrial membrane potential and cellular viability. Inhibitors 3-5 exhibited a cytotoxic effect on HL-60 (human acute promyelocytic leukaemia) cell culture, demonstrated a tendency to affect mitochondrial physiology and viability, and also forced cells to accumulate in the G1/G0-phase of the cell cycle. The cholinesterase reactivators 1 and 2 were found relatively save from the point of view of DNA binding, whereas cholinesterase inhibitors 3-5 resulted as strong DNA binding agents that limit their plausible use. Copyright © 2013 Elsevier B.V. All rights reserved.

Amplification and overexpression of topoisomerase IIalpha predict response to anthracycline-based therapy in locally advanced breast cancer.

PubMed

Coon, John S; Marcus, Elizabeth; Gupta-Burt, Shalina; Seelig, Steven; Jacobson, Kris; Chen, Shande; Renta, Vivian; Fronda, Geraldo; Preisler, Harvey D

2002-04-01

The putative association between erbB-2 overexpression and favorable response to anthracyline-based therapy in breast cancer is controversial, and the mechanism unclear. We sought to determine whether coamplification and overexpression of the topoisomerase IIalpha gene, near erbB-2 on chromosome 17, and a known anthracycline target, may underlie the association. Thirty-five patients who had locally advanced breast cancer (LABC) and who had received neoadjuvant, anthracycline-based therapy were studied. Copy number of topoisomerase IIalpha and erbB-2 was determined by fluorescence in situ hybridization, and expression by immunohistochemistry. Of 8 patients with erbB-2 amplification, 5 had a complete response (CR) or minimal residual disease (MRD), 3 had a partial response (PR), and none had stable (StD) or progressive disease (PD) at the time of mastectomy, versus 3 CR or MRD, 16 PR, and 8 StD or PD for patients without amplification (P = 0.008). In contrast, erbB-2 overexpression was not significantly associated with response (P = 0.114). Of 6 patients with topoisomerase IIalpha amplification, 4 had CR or MRD, 2 PR, and none StD or PD, versus 4 CR or MRD, 17 PR, and 8 StD or PD for patients without amplification (P = 0.034). All of the tumors with topoisomerase IIalpha amplification also had erbB-2 amplification, but not vice versa. Overexpression of topoisomerase IIalpha (9 patients) was also associated with favorable response (P = 0.021). Coamplification of erbB-2 and topoisomerase IIalpha is significantly associated with favorable local response to anthracycline-based therapy in LABC. The expression data favor a plausible mechanism based on topoisomerase IIalpha biology.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

PubMed

Rybak, Paulina; Hoang, Agnieszka; Bujnowicz, Lukasz; Bernas, Tytus; Berniak, Krzysztof; Zarębski, Mirosław; Darzynkiewicz, Zbigniew; Dobrucki, Jerzy

2016-08-02

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

PubMed

Gros, Laurent; Renodon-Cornière, Axelle; de Saint Vincent, Bruno Robert; Feder, Marcin; Bujnicki, Janusz M; Jacquemin-Sablon, Alain

2006-11-01

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II.

PubMed

Barker, Catherine R; Mouchel, Nathalie A P; Jenkins, John R

2006-04-07

Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.

DNA end-processing enzyme polynucleotide kinase as a potential target in the treatment of cancer.

PubMed

Allinson, Sarah L

2010-06-01

Pharmacological inhibition of DNA-repair pathways as an approach for the potentiation of chemo- and radio-therapeutic cancer treatments has attracted increasing levels of interest in recent years. Inhibitors of several enzymes involved in the repair of DNA strand breaks are currently at various stages of the drug development process. Polynucleotide kinase (PNK), a bifunctional DNA-repair enzyme that possesses both 3'-phosphatase and 5'-kinase activities, plays an important role in the repair of both single strand and double strand breaks and as a result, RNAi-mediated knockdown of PNK sensitizes cells to a range of DNA-damaging agents. Recently, a small molecule inhibitor of PNK has been developed that is able to sensitize cells to ionizing radiation and the topoisomerase I poison, camptothecin. Although still in the early stages of development, PNK inhibition represents a promising means of enhancing the efficacy of existing cancer treatments.

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

Dynamic investigation of DNA bending and wrapping by type II topoisomerases

NASA Astrophysics Data System (ADS)

Shao, Qing; Finzi, Laura; Dunlap, David

2009-11-01

Type II topoisomerases catalyze DNA decatenation and unwinding which is crucial for cell division, and therefore type II topoisomerases are some of the main targets of anti-cancer drugs. A recent crystal structure shows that, during the catalytic cycle, a yeast type II topoimerase can bend a 10 base pair DNA segment by up to 150 degrees. Bacterial gyrase, another type II topoisomerase, can wrap DNA into a tight 180 degree turn. Bending a stiff polymer like DNA requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. Using modified deoxyribonucleotides in PCR reactions, stiffer DNA fragments have been produced and used as substrates for topoisomerase II-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. The wrapping ability of gyrase decreases for diamino-purine-substituted DNA in which every base pair has three hydrogen-bonds. The overall rate of relaxation of plectonemes by recombinant human topoisomerase II alpha also decreases. These results reveal the dynamic properties of DNA bending and wrapping by type II topisomerases and suggest that A:T base pair melting is a rate determining step for bending and wrapping.

DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1.

PubMed

Ogitani, Yusuke; Aida, Tetsuo; Hagihara, Katsunobu; Yamaguchi, Junko; Ishii, Chiaki; Harada, Naoya; Soma, Masako; Okamoto, Hiromi; Oitate, Masataka; Arakawa, Shingo; Hirai, Takehiro; Atsumi, Ryo; Nakada, Takashi; Hayakawa, Ichiro; Abe, Yuki; Agatsuma, Toshinori

2016-10-15

An anti-HER2 antibody-drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. DS-8201a exhibited a HER2 expression-dependent cell growth-inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1-insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1-insensitive HER2-positive cancers and low HER2-expressing cancers. Clin Cancer Res; 22(20); 5097-108. ©2016 AACR. ©2016 American Association for Cancer Research.

A “Double-Edged” Scaffold: Antitumor Power within the 
Antibacterial Quinolone

PubMed Central

Bisacchi, Gregory S.; Hale, Michael R.

2016-01-01

In the late 1980s, reports emerged describing experimental antibacterial quinolones having significant potency against eukaryotic Type II topoisomerases (topo II) and showing cytotoxic activity against tumor cell lines. As a result, several pharmaceutical companies initiated quinolone anticancer programs to explore the potential of this class in comparison to conventional human topo II inhibiting antitumor drugs such as doxorubicin and etoposide. In this review, we present a modern re-evaluation of the anticancer potential of the quinolone class in the context of today’s predominantly pathway-based (rather than cytotoxicity-based) oncology drug R&D environment. The quinolone eukaryotic SAR is comprehensively discussed, contrasted with the corresponding prokaryotic data, and merged with recent structural biology information which is now beginning to help explain the basis for that SAR. Quinolone topo II inhibitors appear to be much less susceptible to efflux-mediated resistance, a current limitation of therapy with conventional agents. Recent advances in the biological understanding of human topo II isoforms suggest that significant progress might now be made in overcoming two other treatment-limiting disadvantages of conventional topo II inhibitors, namely cardiotoxicity and drug-induced secondary leukemias. We propose that quinolone class topo II inhibitors could have a useful future therapeutic role due to the continued need for effective topo II drugs in many cancer treatment settings, and due to the recent biological and structural advances which can now provide, for the first time, specific guidance for the design of a new class of inhibitors potentially superior to existing agents. PMID:26695512

PARP inhibition causes premature loss of cohesion in cancer cells

PubMed Central

Kukolj, Eva; Kaufmann, Tanja; Dick, Amalie E.; Zeillinger, Robert; Gerlich, Daniel W.; Slade, Dea

2017-01-01

Poly(ADP-ribose) polymerases (PARPs) regulate various aspects of cellular function including mitotic progression. Although PARP inhibitors have been undergoing various clinical trials and the PARP1/2 inhibitor olaparib was approved as monotherapy for BRCA-mutated ovarian cancer, their mode of action in killing tumour cells is not fully understood. We investigated the effect of PARP inhibition on mitosis in cancerous (cervical, ovary, breast and osteosarcoma) and non-cancerous cells by live-cell imaging. The clinically relevant inhibitor olaparib induced strong perturbations in mitosis, including problems with chromosome alignment at the metaphase plate, anaphase delay, and premature loss of cohesion (cohesion fatigue) after a prolonged metaphase arrest, resulting in sister chromatid scattering. PARP1 and PARP2 depletion suppressed the phenotype while PARP2 overexpression enhanced it, suggesting that olaparib-bound PARP1 and PARP2 rather than the lack of catalytic activity causes this phenotype. Olaparib-induced mitotic chromatid scattering was observed in various cancer cell lines with increased protein levels of PARP1 and PARP2, but not in non-cancer or cancer cell lines that expressed lower levels of PARP1 or PARP2. Interestingly, the sister chromatid scattering phenotype occurred only when olaparib was added during the S-phase preceding mitosis, suggesting that PARP1 and PARP2 entrapment at replication forks impairs sister chromatid cohesion. Clinically relevant DNA-damaging agents that impair replication progression such as topoisomerase inhibitors and cisplatin were also found to induce sister chromatid scattering and metaphase plate alignment problems, suggesting that these mitotic phenotypes are a common outcome of replication perturbation. PMID:29262611

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

PubMed

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

PubMed Central

Wendorff, Timothy J

2018-01-01

Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to engage two DNA segments and link ATP turnover to DNA transport is poorly understood. Using multiple biochemical approaches, we show that Top6B, which harbors the ATPase activity of topo VI, recognizes and exploits the DNA crossings present in supercoiled DNA to stimulate subunit dimerization by ATP. Top6B self-association in turn induces extensive DNA bending, which is needed to support duplex cleavage by Top6A. Our observations explain how topo VI tightly coordinates DNA crossover recognition and ATP binding with strand scission, providing useful insights into the operation of type IIB topoisomerases and related meiotic recombination and GHKL ATPase machineries. PMID:29595473

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors

PubMed Central

Sankar, Savita; Patterson, Ethan; Lewis, Emily M.; Waller, Laura E.; Tong, Caili; Dearborn, Joshua; Wozniak, David; Rubin, Joshua B.; Kroll, Kristen L.

2017-01-01

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies. PMID:29234490

Sarcosine influences apoptosis and growth of prostate cells via cell-type specific regulation of distinct sets of genes.

PubMed

Rodrigo, Miguel A Merlos; Strmiska, Vladislav; Horackova, Eva; Buchtelova, Hana; Michalek, Petr; Stiborova, Marie; Eckschlager, Tomas; Adam, Vojtech; Heger, Zbynek

2018-02-01

Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets. © 2017 Wiley Periodicals, Inc.

ERBB-Receptors and Drug Response in Breast Cancer

DTIC Science & Technology

1997-09-01

in topoisomerase II a (topo II a ) levels seen may be understood by global changes in DNA repair enzymes, brought about by signaling through the EGFR...ErbB-mediated signaling and topoisomerase II a levels specifically as they relate to drug sensitivity/resistance. This information will allow the use...topoisomerase II a levels and increased sensitivity to doxorubicin. In order to determine which, if any, of the specific receptors involved are

Feasibility of Screening for Antibiotic Resistance - Part I

DTIC Science & Technology

2005-09-01

Klebsiella pneumonia (AF052258), Providencia stuartii (AF052259), Serratia marcescens (AF052260). After alignment of the sequences of the various...intracellular targets of fluoroquinolones , the type II DNA topoisomerases gyrase and topoisomerase IV [Balas et al., 1998; Weigel et al., 1998]. Mutations in...the quinolones resistant determining region (QRDR) of the parC gene, which encodes the A subunit of topoisomerase , and the gyrA gene, which encodes

Promising new developments in cancer chemotherapy.

PubMed

Ferrante, K; Winograd, B; Canetta, R

1999-01-01

The positive impact on survival of traditional chemotherapeutic agents has renewed interest in developing newer cytotoxic agents and orally active compounds with improved therapeutic indices. In addition, new insights into the pathways of human tumorigenesis have led to novel approaches aimed at specific mechanism-based targets. The taxane class, of which paclitaxel was the first member, has the unique ability to promote and stabilize microtubule function directly, thereby inhibiting mitotic progression and inducing apoptotic cell death. Paclitaxel provides treatment benefit in a broad range of solid tumors including breast, ovarian, and lung cancer. The success with paclitaxel stimulated interest in the microtubule as a new therapeutic target. Taxane analogues with improved preclinical efficacy have been identified and are entering clinical trials. The enthusiasm for oral anticancer agents and the therapeutic importance of platinum compounds has led to the development of JM216 (satraplatin), a novel platinum IV coordination complex with oral activity in cisplatin-resistant cell lines, which is now in phase III trials in prostate cancer. Another compound in late development is DPPE, a chemopotentiator that enhances the in vivo antitumor effects of cytotoxic agents such as doxorubicin, cyclophosphamide, and cisplatin. Agents that inhibit topoisomerase I and II have also been of interest. TAS-103 is a dual topoisomerase I and II inhibitor with preclinical efficacy in a broad spectrum of tumors and in multidrug-resistant tumor cell lines. Vaccination strategies represent a rational therapeutic approach in the minimal residual disease or high-risk adjuvant therapy setting. The GMK and MGV vaccines utilizing ganglioside antigens overexpressed on human tumors such as melanoma and small cell lung cancer appear to induce antibody production reliably at tolerable doses and are under further clinical investigation. Inhibition of matrix metalloproteinases (MMPs) is another attractive target for intervention in several aspects of tumor progression. Local production of MMPs with subsequent degradation of the extracellular matrix is implicated in supporting tumor growth, invasion, and angiogenesis. The development of orally active, nontoxic MMP inhibitors is critical since these compounds will likely require chronic administration in conjunction with other therapies. Oncogenes and tumor suppressor genes are appealing targets for therapy since they are thought to be responsible for a significant number of cancers. Mutations in the Ras oncogene occur with great frequency in a number of human cancers including lung, pancreas, and colon cancer. Clinical development of potent and selective inhibitors of farnesyltransferase, the Ras-processing enzyme, is ongoing. These compounds uncouple Ras activity, affect tumor growth, and have demonstrated significant antitumor activity against experimental models of human cancer. The exciting compounds and novel therapeutic approaches currently under investigation by Bristol-Myers Squibb Pharmaceutical Research Institute offer great potential as effective cancer chemotherapy agents for the near future.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

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)more » 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.« less

Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid.

PubMed

Mudgil, Y; Singh, B N; Upadhyaya, K C; Sopory, S K; Reddy, M K

2002-05-01

We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons*

PubMed Central

Huelsenbeck, Stefanie C.; Schorr, Anne; Roos, Wynand P.; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-01-01

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons. PMID:23012366

Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

PubMed

Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-11-09

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

Structural basis for suppression of hypernegative DNA supercoiling by E. coli topoisomerase I

DOE PAGES

Tan, Kemin; Zhou, Qingxuan; Cheng, Bokun; ...

2015-10-20

Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain andmore » zinc ribbon-like domain bind ssDNA with primarily π -stacking interactions. Finally, this novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.« less

Polyclonal antibodies against a structure mimicking the covalent linkage unit between picornavirus RNA and VPg: an immunochemical study.

PubMed

Ivanova, O A; Venyaminova, A G; Repkova, M N; Drygin, Yu F

2005-09-01

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

NASA Astrophysics Data System (ADS)

Strick, Terence R.; Croquette, Vincent; Bensimon, David

2000-04-01

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

An RNA Topoisomerase

NASA Astrophysics Data System (ADS)

Wang, Hui; di Gate, Russell J.; Seeman, Nadrian C.

1996-09-01

A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule. The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments. This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be interconverted only by a strand passage event. We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase. The conversion of circles to knots is accompanied by a small amount of RNA catenane generation. These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.

Camptothecin (CPT) and its derivatives are known to target topoisomerase I (Top1) as their mechanism of action: did we miss something in CPT analogue molecular targets for treating human disease such as cancer?

PubMed Central

Li, Fengzhi; Jiang, Tao; Li, Qingyong; Ling, Xiang

2017-01-01

Camptothecin (CPT) was discovered from plant extracts more than 60 years ago. Since then, only two CPT analogues (irinotecan and topotecan) have been approved for cancer treatment, although several thousand CPT derivatives have been synthesized and many of them were actively studied in our research community over the past 6+ decades. In this review article, we briefly summarize: (1) the discovery and early development of CPTs, (2) the recognized CPT mechanism of action (MOA), (3) the synthesis of CPT and CPT analogues, and (4) the structure-activity relationship (SAR) of CPT and its analogues. Next, we provide evidence that certain CPT analogues can exert improved efficacy with low toxicity independently of topoisomerase I (Top1) inhibition; instead, these CPT analogues use novel MOAs by targeting important cancer survival-associated oncogenic proteins and/or by bypassing various treatment-resistant mechanisms. We then present a comprehensive review of the most advanced CPT analogues in clinical development, with the goal of resolving why no new CPTs have been FDA approved for cancer treatment, beyond irinotecan and topotecan. We argue that new CPT Top1 inhibitor drugs are unlikely being found to be significantly better than irinotecan and/or topotecan in terms of the overall antitumor activity and toxicity. The significance of CPT analogues that possess novel MOAs has not been sufficiently recognized so far. In our opinion, this is a research area with great potential to make a breakthrough for development of the next generation of CPT analogues that possess high efficacy (due to novel targets) and low toxicity (due to low inhibition of Top1 activity/function) for effective treatment of human disease, including cancer. PMID:29312794

Ru/Fe bimetallic complexes: Synthesis, characterization, cytotoxicity and study of their interactions with DNA/HSA and human topoisomerase IB.

PubMed

Takarada, Jessica E; Guedes, Adriana P M; Correa, Rodrigo S; Silveira-Lacerda, Elisângela de P; Castelli, Silvia; Iacovelli, Federico; Deflon, Victor Marcelo; Batista, Alzir Azevedo; Desideri, Alessandro

2017-12-15

Three ruthenium/iron-based compounds, 1: [Ru(MIm)(bipy)(dppf)]PF 6 (MIm = 2-mercapto-1-methylimidazole anion), 2: [RuCl(Im)(bipy)(dppf)]PF 6 (Im = imidazole), and 3: [Ru(tzdt)(bipy)(dppf)]PF 6 (tzdt = 1,3-thiazolidine-2-thione anion) (dppf = 1,1'-bis(diphenylphosphine)ferrocene and bipy = 2,2'-bipyridine), were synthesized, and characterized by elemental analyses, conductivity, UV/Vis, IR, 1 H, 13 C and 31 P{1H} NMR spectroscopies, and by electrochemical technique. The complex 3 was also characterized by single-crystal X-ray. The three ruthenium(II) complexes show cytotoxicity against DU-145 (prostate carcinoma cells) and A549 (lung carcinoma cells) tumor cells. The free ligands do not exhibit any cytotoxic activity, such as evident by the IC 50 values higher than 200 μM. UV/Vis and viscosity experiments showed that the complexes interact weakly with the DNA molecule, via electrostatic forces. The interaction of the complexes 1-3 with the HSA is moderate, with K b values in range of 10 5 -10 7  M -1 , presenting a static mechanism of interaction stabilized by hydrophobic. Complexes 2 and 3 showed high affinity for the FA7 HSA site as evidenced by fluorescence spectroscopy and molecular docking. Complexes 1-3 were tested as potential human Topoisomerase IB inhibitors by analysing the different steps of the enzyme catalytic cycle. The results indicate that all compounds efficiently inhibit the DNA relaxation and the cleavage reaction, in which the effect increases upon pre-incubation. Complexes 1 and 2 are also able to slow down the religation reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes*S⃞

PubMed Central

Lin, Chao-Po; Ban, Yi; Lyu, Yi Lisa; Desai, Shyamal D.; Liu, Leroy F.

2008-01-01

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process. PMID:18515798

Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer.

PubMed

Chen, Ina; Mathews-Greiner, Lesley; Li, Dandan; Abisoye-Ogunniyan, Abisola; Ray, Satyajit; Bian, Yansong; Shukla, Vivek; Zhang, Xiaohu; Guha, Raj; Thomas, Craig; Gryder, Berkley; Zacharia, Athina; Beane, Joal D; Ravichandran, Sarangan; Ferrer, Marc; Rudloff, Udo

2017-05-01

Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function. Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor

PubMed Central

Gongerowska, Martyna; Gutkowski, Paweł; Zakrzewska-Czerwińska, Jolanta; Jakimowicz, Dagmara

2016-01-01

ABSTRACT Maintaining an optimal level of chromosomal supercoiling is critical for the progression of DNA replication and transcription. Moreover, changes in global supercoiling affect the expression of a large number of genes and play a fundamental role in adapting to stress. Topoisomerase I (TopA) and gyrase are key players in the regulation of bacterial chromosomal topology through their respective abilities to relax and compact DNA. Soil bacteria such as Streptomyces species, which grow as branched, multigenomic hyphae, are subject to environmental stresses that are associated with changes in chromosomal topology. The topological fluctuations modulate the transcriptional activity of a large number of genes and in Streptomyces are related to the production of antibiotics. To better understand the regulation of topological homeostasis in Streptomyces coelicolor, we investigated the interplay between the activities of the topoisomerase-encoding genes topA and gyrBA. We show that the expression of both genes is supercoiling sensitive. Remarkably, increased chromosomal supercoiling induces the topA promoter but only slightly influences gyrBA transcription, while DNA relaxation affects the topA promoter only marginally but strongly activates the gyrBA operon. Moreover, we showed that exposure to elevated temperatures induces rapid relaxation, which results in changes in the levels of both topoisomerases. We therefore propose a unique mechanism of S. coelicolor chromosomal topology maintenance based on the supercoiling-dependent stimulation, rather than repression, of the transcription of both topoisomerase genes. These findings provide important insight into the maintenance of topological homeostasis in an industrially important antibiotic producer. IMPORTANCE We describe the unique regulation of genes encoding two topoisomerases, topoisomerase I (TopA) and gyrase, in a model Streptomyces species. Our studies demonstrate the coordination of topoisomerase gene regulation, which is crucial for maintenance of topological homeostasis. Streptomyces species are producers of a plethora of biologically active secondary metabolites, including antibiotics, antitumor agents, and immunosuppressants. The significant regulatory factor controlling the secondary metabolism is the global chromosomal topology. Thus, the investigation of chromosomal topology homeostasis in Streptomyces strains is crucial for their use in industrial applications as producers of secondary metabolites. PMID:27551021

Isolation of bioactive and other oxoaporphine alkaloids from two annonaceous plants, Xylopia aethiopica and Miliusa cf. banacea.

PubMed

Harrigan, G G; Gunatilaka, A A; Kingston, D G; Chan, G W; Johnson, R K

1994-01-01

The oxoaporphine alkaloids oxophoebine [1] and liriodenine [2] have been isolated from Xylopia aethiopica (Annonaceae). Both showed selective toxicity against DNA repair and recombination deficient mutants of the yeast Saccharomyces cerevisae. Three related but inactive compounds, oxoglaucine [3], O-methylmoschatoline [4], and lysicamine [5], were also isolated from this plant. Selective toxicity was also observed for 10-methoxyliriodenine (lauterine) [6] and 10-hydroxyliriodenine [7], two oxoaporphine alkaloids isolated from Miliusa cf. banacea (Annonaceae). The structure of 10-hydroxyliriodenine [7], a novel oxoaporphine, was determined by spectroscopic methods and chemical conversion to compound 6. The role of the bioactive oxoaporphine alkaloids as DNA topoisomerase inhibitors is discussed.

S-1 induced secondary acute erythroid leukemia with a chromosome inv(12)(p13;q13)

PubMed Central

Matsumoto, Kensuke; Kitanaka, Akira; Uemura, Makiko; Waki, Fusako; Fukumoto, Tetsuya; Ohnishi, Hiroaki; Kubota, Yoshitsugu; Ishida, Toshihiko

2011-01-01

Adjuvant chemotherapy by S-1 following gastrectomy is considered standard treatment in Japan. Analysis of follow-up data have proved the efficacy of S-1 administration, and that hematological adverse events were relatively rare. Pyrimidine anti-metabolites, including S-1, have shown relatively lower risks for secondary hematological malignancies in comparison to alkylating agents and topoisomerase-II inhibitors. We here report a case of therapy-related leukemia after S-1 administration. A patient who had received S-1as the sole adjuvant chemotherapy was diagnosed with acute erythroid leukemia. To the best of our knowledge, our patient represents the first report of S-1 induced acute leukemia. PMID:22147971

Comparative analysis of topoisomerase IB inhibition and DNA intercalation by flavonoids and similar compounds: structural determinates of activity

PubMed Central

2004-01-01

Flavonoids and other polyphenolic compounds have been shown to inhibit human topoisomerase IB (topo I) through both inhibition of relaxation activity and through stabilization of the cleavable complex (poisoning). Some flavonoids have also been shown to intercalate DNA, and an association of topoisomerase inhibition with intercalation has been noted. We surveyed 34 polyphenolic compounds, primarily flavonoid glycones and aglycones, for their ability to inhibit topo I and to intercalate DNA using an in vitro gel electrophoresis method. We show that the most potent topo I poisons are the flavones and flavonols, and that these generally, but not always, are found to be DNA intercalators. There was no clear correlation, however, of topo-I-poisoning activity with the degree of DNA unwinding. Surprisingly, both DNA intercalation and topo I poisoning were shown to occur with some flavone glycones, including the C-glycosylflavone orientin. Inhibition of relaxation activity by flavonoids was found to be difficult to quantify and was most likely to be due to non-specific inhibition through flavonoid aggregation. As part of a structure–activity analysis, we also investigated the acid–base chemistry of flavonoids and determined that many flavonoids show acid–base activity with a pKa in the physiological pH region. For this reason, subtle pH changes can have significant effects on solution activity of flavonoids and their concomitant biological activity. In addition, these effects may be complicated by pH-dependent aggregation and oxidative degradation. Finally, we develop a simple model for the intercalation of flavonoids into DNA and discuss possible consequences of intercalation and topoisomerase inhibition on a number of cellular processes. PMID:15312049

Exploring the gyrase ATPase domain for tailoring newer anti-tubercular drugs: hit to lead optimization of a novel class of thiazole inhibitors.

PubMed

Jeankumar, Variam Ullas; Kotagiri, Sonali; Janupally, Renuka; Suryadevara, Priyanka; Sridevi, Jonnalagadda Padma; Medishetti, Raghavender; Kulkarni, Pushkar; Yogeeswari, Perumal; Sriram, Dharmarajan

2015-02-01

Gyrase ATPase domain, the pharmaceutical underexploited segment of DNA gyrase, the sole Type II topoisomerase present in Mycobacterium tuberculosis represents an attractive target for anti-tubercular drug discovery. Here we report, the development of a novel series of MTB DNA gyraseB inhibitor identified through a medium throughput screening (MTS) of BITS in-house chemical library (3000 compounds). The MTS hit was further remodeled by chemical synthesis to identify the most potent analogue 27 exhibiting an in vitro gyrB inhibitory IC50 of 0.15 μM. The series also demonstrated well correlating gyrase super coiling activity and in vitro anti-mycobacterial potency against MTB H37Rv strain. Furthermore the compounds displayed good safety profile in their subsequent cytotoxicity and hERG toxicity evaluations, to be worked out from a pharmaceutical point of view as potential anti-tubercular agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.

PubMed

Maxwell, Anthony; Lawson, David M

2003-01-01

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

The Escherichia coli supX locus is topA, the structural gene for DNA topoisomerase I.

PubMed Central

Margolin, P; Zumstein, L; Sternglanz, R; Wang, J C

1985-01-01

Mutations in the supX locus, which result in the absence of DNA topoisomerase I enzyme activity in both Salmonella typhimurium and Escherichia coli, are all selected as suppressors of the leu-500 promoter mutation in S. typhimurium. To determine whether the supX locus is the structural gene topA for the DNA topoisomerase I enzyme or is a positive-acting regulator/activator gene for a nearby topA structural gene, nonsense mutations were selected in the E. coli supX gene carried on an F' episome in S. typhimurium cells. The cysB-topA region of the episomes with nonsense-mutant supX alleles were then cloned onto plasmid pBR322 and transformed into E. coli cells lacking a chromosomal supX gene. Three such E. coli strains, each carrying cloned DNA from episomes with different nonsense-mutant supX alleles, all lacked DNA topoisomerase I activity but expressed antigenic determinants specific to the enzyme; control cells lacked both enzyme activity and antigenic determinants. Maxicell studies of plasmid-coded proteins demonstrated the absence of the DNA topoisomerase I protein (100 kDa) in the three strains but the appearance of a new smaller peptide in each (36, 47, and 64 kDa). These new peptides must represent fragments of the enzyme resulting from translation termination at the supX nonsense codons and confirm the interpretation that the supX gene is topA, the structural gene for DNA topoisomerase I. Images PMID:2991925

The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity

PubMed Central

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A.; Kim, Sungjoon; Wilson, Christopher J.; Lehár, Joseph; Kryukov, Gregory V.; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F.; Monahan, John E.; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A.; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H.; Cheng, Jill; Yu, Guoying K.; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D.; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C.; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P.; Gabriel, Stacey B.; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E.; Weber, Barbara L.; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L.; Meyerson, Matthew; Golub, Todd R.; Morrissey, Michael P.; Sellers, William R.; Schlegel, Robert; Garraway, Levi A.

2012-01-01

The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic avenues remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacologic annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacologic profiles for 24 anticancer drugs across 479 of the lines, this collection allowed identification of genetic, lineage, and gene expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Altogether, our results suggest that large, annotated cell line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of “personalized” therapeutic regimens2. PMID:22460905

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

PubMed

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A; Kim, Sungjoon; Wilson, Christopher J; Lehár, Joseph; Kryukov, Gregory V; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F; Monahan, John E; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H; Cheng, Jill; Yu, Guoying K; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P; Gabriel, Stacey B; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E; Weber, Barbara L; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L; Meyerson, Matthew; Golub, Todd R; Morrissey, Michael P; Sellers, William R; Schlegel, Robert; Garraway, Levi A

2012-03-28

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ze-xin; Li, Jian; Li, Yan

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 themore » 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.« less

Sodium phenylbutyrate abrogates African swine fever virus replication by disrupting the virus-induced hypoacetylation status of histone H3K9/K14.

PubMed

Frouco, Gonçalo; Freitas, Ferdinando B; Martins, Carlos; Ferreira, Fernando

2017-10-15

African swine fever virus (ASFV) causes a highly lethal disease in swine for which neither a vaccine nor treatment are available. Recently, a new class of drugs that inhibit histone deacetylases enzymes (HDACs) has received an increasing interest as antiviral agents. Considering studies by others showing that valproic acid, an HDAC inhibitor (HDACi), blocks the replication of enveloped viruses and that ASFV regulates the epigenetic status of the host cell by promoting heterochromatinization and recruitment of class I HDACs to viral cytoplasmic factories, the antiviral activity of four HDACi against ASFV was evaluated in this study. Results showed that the sodium phenylbutyrate fully abrogates the ASFV replication, whereas the valproic acid leads to a significant reduction of viral progeny at 48h post-infection (-73.9%, p=0.046), as the two pan-HDAC inhibitors tested (Trichostatin A: -82.2%, p=0.043; Vorinostat: 73.9%, p=0.043). Further evaluation showed that protective effects of NaPB are dose-dependent, interfering with the expression of late viral genes and reversing the ASFV-induced histone H3 lysine 9 and 14 (H3K9K14) hypoacetylation status, compatible to an open chromatin state and possibly enabling the expression of host genes non-beneficial to infection progression. Additionally, a synergic antiviral effect was detected when NaPB is combined with an ASFV-topoisomerase II poison (Enrofloxacin). Altogether, our results strongly suggest that cellular HDACs are involved in the establishment of ASFV infection and emphasize that further in vivo studies are needed to better understand the antiviral activity of HDAC inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

When Maxwellian demon meets action at a distance. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

NASA Astrophysics Data System (ADS)

Rybenkov, Valentin V.

2016-09-01

The ability of living systems to defy thermodynamics without explicitly violating it is a continued source of inspiration to many biophysicists. The story of type-2 DNA topoisomerases is a beautiful example from that book. DNA topoisomerases catalyze a concerted DNA cleavage-religation reaction, which is interjected by a strand passage event. This sequence of events results in a seemingly unhindered transfer of one piece of DNA through another upon their random collision. An obvious consequence of such transfer is a change in the topological state of the colliding DNAs; hence the name of the enzymes, topoisomerases. There are several classes of topoisomerases, which differ in how they capture the cleaved and transported DNA segments (which are often referred to as the gate and transfer segments; or the G- and T-segments, to be short). Type-2 topoisomerases have two cleavage-religation centers. They open a gate in double stranded DNA and transfer another piece of double stranded DNA through it [1]. And in doing so, they manage to collect information about the rest of the DNA and perform strand passage in a directional manner so as to take the molecule away from the thermodynamic equilibrium [2].

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors

PubMed Central

Kalra, Sean; Cleaver, Timothy G.; Merrick, Daniel; Wang, Xiao-Jing; Malkoski, Stephen P.

2015-01-01

Non-small cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer, however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC datasets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms including mutation, homozygous deletion, and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4−/− mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage while Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4 deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4 deficient tumors can be exploited by specific therapeutic strategies. PMID:25893305

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells.

PubMed

Kumar, Ashutosh; Ehrenshaft, Marilyn; Tokar, Erik J; Mason, Ronald P; Sinha, Birandra K

2016-07-01

Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions. Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin. Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines. Inhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells. As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic. Published by Elsevier B.V.

Evaluation of in vitro inhibitory effect of enoxacin on Babesia and Theileria parasites.

PubMed

Omar, Mosaab A; Salama, Akram; Elsify, Ahmed; Rizk, Mohamed Abdo; Al-Aboody, Mohammad Saleh; AbouLaila, Mahmoud; El-Sayed, Shimaa Abd El-Salam; Igarashi, Ikuo

2016-02-01

Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by micro molar concentrations of enoxacin (IC50 values = 33.5, 15.2, 7.5 and 23.2 μM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Enoxacin IC50 values for Babesia and Theileria parasites were satisfactory as the drug is potent antibacterial drug with minimum side effects. Therefore, enoxacin might be used for treatment of Babesiosis and Theileriosis especially in case of mixed infections with bacterial diseases or incase of animal sensitivity against diminazin toxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

Common TDP1 Polymorphisms in Relation to Survival among Small Cell Lung Cancer Patients: A Multicenter Study from the International Lung Cancer Consortium.

PubMed

Lohavanichbutr, Pawadee; Sakoda, Lori C; Amos, Christopher I; Arnold, Susanne M; Christiani, David C; Davies, Michael P A; Field, John K; Haura, Eric B; Hung, Rayjean J; Kohno, Takashi; Landi, Maria Teresa; Liu, Geoffrey; Liu, Yi; Marcus, Michael W; O'Kane, Grainne M; Schabath, Matthew B; Shiraishi, Kouya; Slone, Stacey A; Tardón, Adonina; Yang, Ping; Yoshida, Kazushi; Zhang, Ruyang; Zong, Xuchen; Goodman, Gary E; Weiss, Noel S; Chen, Chu

2017-12-15

Purpose: DNA topoisomerase inhibitors are commonly used for treating small-cell lung cancer (SCLC). Tyrosyl-DNA phosphodiesterase (TDP1) repairs DNA damage caused by this class of drugs and may therefore influence treatment outcome. In this study, we investigated whether common TDP1 single-nucleotide polymorphisms (SNP) are associated with overall survival among SCLC patients. Experimental Design: Two TDP1 SNPs (rs942190 and rs2401863) were analyzed in 890 patients from 10 studies in the International Lung Cancer Consortium (ILCCO). The Kaplan-Meier method and Cox regression analyses were used to evaluate genotype associations with overall mortality at 36 months postdiagnosis, adjusting for age, sex, race, and tumor stage. Results: Patients homozygous for the minor allele (GG) of rs942190 had poorer survival compared with those carrying AA alleles, with a HR of 1.36 [95% confidence interval (CI): 1.08-1.72, P = 0.01), but no association with survival was observed for patients carrying the AG genotype (HR = 1.04, 95% CI, 0.84-1.29, P = 0.72). For rs2401863, patients homozygous for the minor allele (CC) tended to have better survival than patients carrying AA alleles (HR = 0.79; 95% CI, 0.61-1.02, P = 0.07). Results from the Genotype Tissue Expression (GTEx) Project, the Encyclopedia of DNA Elements (ENCODE), and the ePOSSUM web application support the potential function of rs942190. Conclusions: We found the rs942190 GG genotype to be associated with relatively poor survival among SCLC patients. Further investigation is needed to confirm the result and to determine whether this genotype may be a predictive marker for treatment efficacy of DNA topoisomerase inhibitors. Clin Cancer Res; 23(24); 7550-7. ©2017 AACR . ©2017 American Association for Cancer Research.

Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex

PubMed Central

Veselkov, Dennis A.; Laponogov, Ivan; Pan, Xiao-Su; Selvarajah, Jogitha; Skamrova, Galyna B.; Branstrom, Arthur; Narasimhan, Jana; Prasad, Josyula V. N. Vara; Fisher, L. Mark; Sanderson, Mark R.

2016-01-01

Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved. PMID:27050128

Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors

PubMed Central

Kumala, Slawomir; Fujarewicz, Krzysztof; Jayaraju, Dheekollu; Rzeszowska-Wolny, Joanna; Hancock, Ronald

2013-01-01

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. PMID:23382828

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site.

PubMed

Cao, Nan; Tan, Kemin; Annamalai, Thirunavukkarasu; Joachimiak, Andrzej; Tse-Dinh, Yuk-Ching

2018-06-14

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changela, Anita; DiGate, Russell J.; Mondragon, Alfonso

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5{prime} phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is boundmore » along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.« less

Mechanisms of chiral discrimination by topoisomerase IV

PubMed Central

Neuman, K. C.; Charvin, G.; Bensimon, D.; Croquette, V.

2009-01-01

Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results. PMID:19359479

Bioactive Pentacyclic Triterpene Ester Derivatives from Alnus viridis ssp. viridis Bark.

PubMed

Novakovic, Miroslav; Nikodinovic-Runic, Jasmina; Veselinovic, Jovana; Ilic-Tomic, Tatjana; Vidakovic, Vera; Tesevic, Vele; Milosavljevic, Slobodan

2017-05-26

Seven derivatives of pentacyclic triterpene acids (1-7) were isolated from the bark of Alnus viridis ssp. viridis using a combination of column chromatography and semipreparative HPLC. Compounds 1-3, 6, and 7 were determined to be new after spectroscopic data interpretation and were assigned as 27-hydroxyalphitolic acid derivatives (1-3), a 27-hydroxybetulinic acid derivative (6), and a 3-epi-maslinic acid derivative (7), respectively. Pentacyclic triterpenoids with a C-27 hydroxymethyl group have been found in species of the genus Alnus for the first time. These compounds were subjected to cytotoxicity testing against a number of cancer cell lines. Also, selected pentacyclic triterpenoids were selected as potential inhibitors of topoisomerases I and IIα for an in silico investigation.

MC70 potentiates doxorubicin efficacy in colon and breast cancer in vitro treatment.

PubMed

Azzariti, Amalia; Quatrale, Anna E; Porcelli, Letizia; Colabufo, Nicola A; Cantore, Mariangela; Cassano, Giuseppe; Gasparre, Giuseppe; Iannelli, Giuseppina; Tommasi, Stefania; Panaro, Maria A; Paradiso, Angelo

2011-11-16

A major limitation of cancer treatment is the ability of cancer cells to develop resistance to chemotherapeutic drugs, by the establishment of multidrug resistance. Here, we characterize MC70 as ABC transporters inhibitor and anticancer agent, alone or with chemotherapy. MC70 was analyzed for its interaction with ABCB1, ABCG2 and ABCC1 by specific transport assays. In breast and colon cancer cell lines, cell growth and apoptosis were measured by MTT assay and DNA laddering Elisa kit, respectively. Cell cycle perturbation and cellular targets modulation were analyzed by Flow-cytometry and Western blotting, respectively. MC70 interacted with ABC transporters. In breast cancer cells, MC70 slightly inhibited cell proliferation strongly enhancing doxorubicin effectiveness. By contrast, MC70 was found to inhibit cell growth in colon cancer cells without affecting doxorubicin efficacy and in combination with topoisomerase I inhibitors it could be a promising therapeutic approach. What is more, it was also observed that MC70 induced apoptosis, canceled in favor of necrosis when given in combination with high doses of doxorubicin. MC70 inhibited cell migration probably through its interaction with sigma-1 receptor. Modulations of i) cell cycle, ii) pAkt and the phosphorylation of the three MAPKs were highlighted, while any activity was excluded at transcription level, thus accounting for the phenotypic effects observed. MC70 might be considered as a new potential anticancer agent capable to i) enhance chemotherapy effectiveness and ii) to play a contributory role in the treatment of chemotherapy resistant tumors. Copyright © 2011 Elsevier B.V. All rights reserved.

Amsacrine as a Topoisomerase II Poison: Importance of Drug-DNA Interactions†

PubMed Central

Ketron, Adam C.; Denny, William A.; Graves, David E.; Osheroff, Neil

2012-01-01

Amsacrine (m-AMSA) is an anticancer agent that displays activity against refractory acute leukemias as well as Hodgkin’s and non-Hodgkin’s lymphomas. The drug is comprised of an intercalative acridine moiety coupled to a 4’-amino-methanesulfon-m-anisidide head group. m-AMSA is historically significant in that it was the first drug demonstrated to function as a topoisomerase II poison. Although m-AMSA was designed as a DNA binding agent, the ability to intercalate does not appear to be the sole determinant of drug activity. Therefore, to more fully analyze structure-function relationships and the role of DNA binding in the action of m-AMSA, we analyzed a series of derivatives for the ability to enhance DNA cleavage mediated by human topoisomerase IIα and topoisomerase IIβ and to intercalate DNA. Results indicate that the 3’-methoxy (m-AMSA) positively affects drug function, potentially by restricting the rotation of the head group in a favorable orientation. Shifting the methoxy to the 2’-position (o-AMSA), which abrogates drug function, appears to increase rotational freedom of the head group and may impair interactions of the 1’-substituent or other portions of the head group within the ternary complex. Finally, the non-intercalative m-AMSA head group enhanced enzyme-mediated DNA cleavage when it was detached from the acridine moiety, albeit with 100-fold lower affinity. Taken together, our results suggest that much of the activity and specificity of m-AMSA as a topoisomerase II poison is embodied in the head group, while DNA intercalation is used primarily to increase the affinity of m-AMSA for the topoisomerase II-DNA cleavage complex. PMID:22304499

High-throughput assays for DNA gyrase and other topoisomerases

PubMed Central

Maxwell, Anthony; Burton, Nicolas P.; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

High-throughput assays for DNA gyrase and other topoisomerases.

PubMed

Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum.

PubMed

Lubitz, Dorit; Wendisch, Volker F

2016-10-07

Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope. Ciprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin. Ciprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.

The Current Case of Quinolones: Synthetic Approaches and Antibacterial Activity.

PubMed

Naeem, Abdul; Badshah, Syed Lal; Muska, Mairman; Ahmad, Nasir; Khan, Khalid

2016-03-28

Quinolones are broad-spectrum synthetic antibacterial drugs first obtained during the synthesis of chloroquine. Nalidixic acid, the prototype of quinolones, first became available for clinical consumption in 1962 and was used mainly for urinary tract infections caused by Escherichia coli and other pathogenic Gram-negative bacteria. Recently, significant work has been carried out to synthesize novel quinolone analogues with enhanced activity and potential usage for the treatment of different bacterial diseases. These novel analogues are made by substitution at different sites--the variation at the C-6 and C-8 positions gives more effective drugs. Substitution of a fluorine atom at the C-6 position produces fluroquinolones, which account for a large proportion of the quinolones in clinical use. Among others, substitution of piperazine or methylpiperazine, pyrrolidinyl and piperidinyl rings also yields effective analogues. A total of twenty six analogues are reported in this review. The targets of quinolones are two bacterial enzymes of the class II topoisomerase family, namely gyrase and topoisomerase IV. Quinolones increase the concentration of drug-enzyme-DNA cleavage complexes and convert them into cellular toxins; as a result they are bactericidal. High bioavailability, relative low toxicity and favorable pharmacokinetics have resulted in the clinical success of fluoroquinolones and quinolones. Due to these superior properties, quinolones have been extensively utilized and this increased usage has resulted in some quinolone-resistant bacterial strains. Bacteria become resistant to quinolones by three mechanisms: (1) mutation in the target site (gyrase and/or topoisomerase IV) of quinolones; (2) plasmid-mediated resistance; and (3) chromosome-mediated quinolone resistance. In plasmid-mediated resistance, the efflux of quinolones is increased along with a decrease in the interaction of the drug with gyrase (topoisomerase IV). In the case of chromosome-mediated quinolone resistance, there is a decrease in the influx of the drug into the cell.

Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

NASA Astrophysics Data System (ADS)

Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R.

2015-05-01

Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01474c

Novel water soluble morpholine substituted Zn(II) phthalocyanine: Synthesis, characterization, DNA/BSA binding, DNA photocleavage and topoisomerase I inhibition.

PubMed

Barut, Burak; Demirbaş, Ümit; Özel, Arzu; Kantekin, Halit

2017-12-01

In this study, novel peripherally tetra 3-morpholinophenol substituted zinc(II) phthalocyanine (4) and its water soluble form quaternized zinc(II) phthalocyanine (ZnQ) were synthesized for the first time. These novel compounds were characterized by a combination of different spectroscopic techniques such as FT-IR, 1 H NMR, 13 C NMR, UV-vis and mass. The DNA binding of ZnQ was investigated using UV-vis absorption titration, competitive ethidium bromide, thermal denaturation and viscosity experiments that the ZnQ bound to CT-DNA via intercalation mode. ZnQ indicated photocleavage activity on supercoiled pBR322 plasmid DNA via formation of singlet oxygen under irradiation at 700nm. Besides, the topoisomerase I inhibitory effect experiments showed that ZnQ inhibited topoisomerase I enzyme in a concentration-dependent manner. The bovine serum albumin (BSA) binding experiments indicated that ZnQ bound to proteins through a static quenching mechanism. All of these results claim that ZnQ has potential agent for photodynamic therapy owing to its nucleic acid interactions and photobiological or photochemical properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing Photodynamyc Therapy Efficacy by Combination Therapy: Dated, Current and Oncoming Strategies

PubMed Central

Postiglione, Ilaria; Chiaviello, Angela; Palumbo, Giuseppe

2011-01-01

Combination therapy is a common practice in many medical disciplines. It is defined as the use of more than one drug to treat the same disease. Sometimes this expression describes the simultaneous use of therapeutic approaches that target different cellular/molecular pathways, increasing the chances of killing the diseased cell. This short review is concerned with therapeutic combinations in which PDT (Photodynamyc Therapy) is the core therapeutic partner. Besides the description of the principal methods used to assess the efficacy attained by combinations in respect to monotherapy, this review describes experimental results in which PDT was combined with conventional drugs in different experimental conditions. This inventory is far from exhaustive, as the number of photosensitizers used in combination with different drugs is very large. Reports cited in this work have been selected because considered representative. The combinations we have reviewed include the association of PDT with anti-oxidants, chemotherapeutics, drugs targeting topoisomerases I and II, antimetabolites and others. Some paragraphs are dedicated to PDT and immuno-modulation, others to associations of PDT with angiogenesis inhibitors, receptor inhibitors, radiotherapy and more. Finally, a look is dedicated to combinations involving the use of natural compounds and, as new entries, drugs that act as proteasome inhibitors. PMID:24212824

New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity.

PubMed

Durcik, Martina; Lovison, Denise; Skok, Žiga; Durante Cruz, Cristina; Tammela, Päivi; Tomašič, Tihomir; Benedetto Tiz, Davide; Draskovits, Gábor; Nyerges, Ákos; Pál, Csaba; Ilaš, Janez; Peterlin Mašič, Lucija; Kikelj, Danijel; Zidar, Nace

2018-06-25

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC 50 values against DNA gyrase, and submicromolar IC 50 values against topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compound in the series has an IC 50 value of 13 nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC 50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 μM against Enterococcus faecalis, and 3.13 μM against wild type S. aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) is 4.6 μM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Drug repurposing of novel quinoline acetohydrazide derivatives as potent COX-2 inhibitors and anti-cancer agents

NASA Astrophysics Data System (ADS)

Manohar, Chelli Sai; Manikandan, A.; Sridhar, P.; Sivakumar, A.; Siva Kumar, B.; Reddy, Sabbasani Rajasekhara

2018-02-01

Novel QuinolineAcetohydrazide (QAh) derivatives (9a-n) were firstly evaluated in silico to determine their anti-inflammatory and anti-cancer efficacy via the mechanisms of COX1 and COX2 inhibition, and NF-ĸB, HDAC and Human Topoisomerase I pathways respectively. In the studied set, the trifluoro substituted QAh derivatives: (E)-N'-(4-(trifluoro methyl) benzylidene)-2-(7-fluoro-2-methoxy quinolin-8-yl) acetohydrazid and (E)-N'-(3-(trifluoro methyl) benzylidene)-2-(7-fluoro-2-methoxy quinolin-8-yl) acetohydrazide are determined to be potential leads, indicated from their best docked scores, relative ligand efficiency, and significant structural attributes evaluated by ab initio simulations. The only setback being their partition co-efficient that retrieved a red flag in the evaluation of their Lipinski parameters. The experimental in vitro studies confirmed the significant enhancement as COX-2 inhibitors and appreciable enhancement in MTT assay of breast and skin cancer cell lines. Significantly, trifluoro substituent in the quinoline scaffold can be reasoned to note the excellent binding affinity to all the evaluated drug targets.

Structural and molecular docking studies of biologically active mercaptopyrimidine Schiff bases

NASA Astrophysics Data System (ADS)

Kirubavathy, S. Jone; Velmurugan, R.; Karvembu, R.; Bhuvanesh, N. S. P.; Enoch, Israel V. M. V.; Selvakumar, P. Mosae; Premnath, D.; Chitra, S.

2017-01-01

Novel Schiff bases derived from the treatment of mercapto-diamino pyrimidine with two different aldehydes are characterized using elemental analysis, single crystal X-ray diffraction and 1H NMR spectroscopy. The pharmacological action of the synthesized compounds viz., antimicrobial, anticancer and antitubercular activities is studied. The Schiff bases show a very good activity against various test pathogens. DNA and β-CD binding interactions of the compounds are studied using UV-Visible absorption and fluorescence spectral measurements. The binding constants of the compounds towards β-CD are in the order of 103 to 104. Molecular docking is done using MOE program on the 3D structure of the enzymes, viz., human thymidylate synthase complexed with dump and raltitrex, candida albicans N-myristoyltransferasepeptidic inhibitor, catalytic domain of protein kinase pKnb from mycobacterium tuberculosis in complex with mitoxantrone, pare, topoisomerase atpase inhibitor, E. coli and lactobacillus casdihydrofolatereductase. The MIC/IC50 values of the Schiff bases are compared with the glide scores from the molecular docking studies. The number of hydrogen bonding interactions between the Schiff bases and amino acid residues are also reported.

Emerging drug treatments for solid tumours.

PubMed

Schellens, J H; Pronk, L C; Verweij, J

1996-01-01

A number of novel anticancer agents have emerged during the past few decades, which show high activity in preclinical tumour models and promising activity in early trials in patients with solid tumours. Most of the agents have novel and unique mechanisms of action, and show activity against a variety of malignancies, including tumours which are notoriously resistant to systemic treatment. Recently, our understanding of the molecular basis of cancer has increased considerably. This is reflected in the development of agents that are directed at well defined molecular targets, such as the mitotic tubulin/microtubuli system (taxoids), nuclear enzymes (topoisomerase I inhibitors) and cell signal transduction pathways (protein kinase C inhibitors). In addition, significant advances have been made in our understanding of mechanisms of toxicity, especially of cisplatin. This has resulted in the development of agents modulating cisplatin toxicity, among which amifostine (WR-2721) is one of the most promising. The outlined emerging drug therapies with novel anticancer agents and treatment modalities will, it is hoped, result in increased response rates of advanced tumours, longer disease-free and total survival and better palliative care.

Targeting KRAS-mutant non-small cell lung cancer with the Hsp90 inhibitor ganetespib.

PubMed

Acquaviva, Jaime; Smith, Donald L; Sang, Jim; Friedland, Julie C; He, Suqin; Sequeira, Manuel; Zhang, Chaohua; Wada, Yumiko; Proia, David A

2012-12-01

Mutant KRAS is a feature of more than 25% of non-small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors.

Novel Hybrid Molecules on the Basis of Steroids and (5Z,9Z)-Tetradeca-5,9-dienoic Acid: Synthesis, Anti-Cancer Studies and Human Topoisomerase I Inhibitory Activity.

PubMed

D'yakonov, Vladimir A; Dzhemileva, Lilya U; Tuktarova, Regina A; Ishmukhametova, Svetlana R; Yunusbaeva, Milyausha M; Ramazanova, Ilfir R; Dzhemilev, Usein M

2017-01-01

Novel steroid derivatives of 5Z,9Z-dienoic acids were prepared by the DCC/DMAP-catalyzed esterification of (5Z,9Z)-tetradeca-5,9-dienoic acid with hydroxy steroids. High cytotoxicity towards the HEK293, Jurkat, K562 cancer cell lines and human topoisomerase I (hTop1) inhibitory activity in vitro were found for the synthesized acids. A probable mechanism of topoisomerase I inhibition was hypothesized on the basis of in silico studies resorting to docking. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

An improved synthesis for the (Z)-14-methyl-9-pentadecenoic acid and its topoisomerase I inhibitory activity

PubMed Central

Carballeira, Néstor M.; Sanabria, David; Oyola, Delise

2006-01-01

An improved synthesis for the (Z)-14-methyl-9-pentadecenoic acid was developed based on the appropriate use of (trimethylsilyl)acetylene as the key reagent in the synthesis. The reported synthesis started with commercially available 8-bromo-1-octanol and furnished the desired acid in seven steps and in a 16% overall yield, a significant improvement over the previous reported synthesis for this fatty acid. The synthesis reported herein afforded sufficient amounts to study the acid topoisomerase I inhibitory potential and it was found that the title acid inhibits the human placenta DNA topoisomerase I enzyme at concentrations of 500 μM. PMID:17680032

Discovery of a Novel Anti-Cancer Agent Targeting Both Topoisomerase I & II as Well as Telomerase Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo: Cinnamomum verum Component Cuminaldehyde.

PubMed

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

2016-01-01

Cinnamomum verum is used to make the spice cinnamon and has been used for more than 5000 years by both of the two most ancient forms of medicine in the words: Ayurveda and traditional Chinese herbal medicines for various applications such as adenopathy, rheumatism, dermatosis, dyspepsia, stroke, tumors, elephantiasis, trichomonas, yeast, and virus infections. We evaluated the anticancer effect of cuminaldehyde (CuA), 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 cuminaldehyde suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase 3 and 9, increase in annexin V+PI+ cells, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and comet with elevated tail intensity and moment. In addition, cuminaldehyde also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of both topoisomerase I & II as well as telomerase activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of cuminaldehyde was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of cuminaldehyde against A549 cells is accompanied by downregulations of proliferative control involving apoptosis, both topoisomerase I & II as well as telomerase activities, together with an upregulation of lysosomal vacuolation and VAC. Similar effects (including all of the above-mentioned effects) were found in other cell lines, including human lung squamous cell carcinoma NCI-H520 and colorectal adenocarcinoma COLO 205 (results not shown). Our data suggest that cuminaldehyde could be a potential agent for anticancer therapy.

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

PubMed

Okamura, Shinya; Nishiyama, Eri; Yamazaki, Tomohiro; Otsuka, Nao; Taniguchi, Shoko; Ogawa, Wakano; Hatano, Tsutomu; Tsuchiya, Tomofusa; Kuroda, Teruo

2015-06-01

Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 μM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug. Copyright © 2015 Elsevier B.V. All rights reserved.

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in cervical cancer and associates with tumor growth.

PubMed

Liang, Jing; Nishi, Hirotaka; Bian, Mei-Lu; Higuma, Chinatsu; Sasaki, Toru; Ito, Hiroe; Isaka, Keiichi

2012-10-01

We found that the ubiquitin-conjugating enzyme E2-EPF mRNA is highly expressed in cervical squamous cancer relative to normal tissues and its expression levels positively correlate with clinical stage. Reduction of E2-EPF protein levels by >80% using shRNA decreases the expression levels of HIF-1α, and the proliferation, invasion and tumorigenicity of SiHa, a cervical squamous cancer cell line. E2-EPF knockdown also increases the chemosensitivity to topoisomerase I inhibitor (topotecan) and II (etoposide and doxorubicin). Our results suggest that E2-EPF is associated with the growth and aggressivity of cervical tumor cells. Targeting the E2-EPF pathway may have potential clinical applications for the treatment of cervical cancer.

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid.

PubMed

Carballeira, Néstor M; Montano, Nashbly; Padilla, Luis F

2007-01-01

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.

Dietary polyphenols influence antimetabolite agents: methotrexate, 6-mercaptopurine and 5-fluorouracil in leukemia cell lines

PubMed Central

Mahbub, Amani; Le Maitre, Christine; Haywood-Small, Sarah; Cross, Neil; Jordan-Mahy, Nicola

2017-01-01

Polyphenols have been previously shown to sensitize leukemia cell lines to topoisomerase inhibitors. Here, we assess the effects of five polyphenols when used alone and in combination with antimetabolites: methotrexate, 6-mercaptopurine and 5-fluorouracil; in lymphoid and myeloid leukemia cells lines, and non-tumor control cells. The effects of combined treatments were investigated on ATP and glutathione levels, cell-cycle progression, DNA damage and apoptosis. Polyphenols antagonized methotrexate and 6-mercaptopurine induced cell-cycle arrest and apoptosis in most leukemia cell lines. This was associated with reduced DNA damage and increased glutathione levels, greater than that seen following individual treatments alone. In contrast, 5-fluorouracil when combined with quercetin, apigenin and rhein caused synergistic decrease in ATP levels, induction of cell-cycle arrest and apoptosis in some leukemia cell lines. However, antagonistic effects were observed when 5-fluorouracil was combined with rhein and cis-stilbene in myeloid cell lines. The effects were dependant on polyphenol type and chemotherapy agent investigated, and cell type treated. Interestingly treatment of non-tumor control cells with polyphenols protected cells from antimetabolite treatments. This suggests that polyphenols modulate the action of antimetabolite agents; more importantly they antagonized methotrexate and 6-mercaptopurine actions, thus suggesting the requirement of polyphenol-exclusion during their use. PMID:29285220

Pharmacophore Hybridization To Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

PubMed

Ortega, Jose Antonio; Riccardi, Laura; Minniti, Elirosa; Borgogno, Marco; Arencibia, Jose M; Greco, Maria L; Minarini, Anna; Sissi, Claudia; De Vivo, Marco

2018-02-08

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on human cancer cells, endorsing their further exploration for anticancer drug discovery.

Probing Conformational Changes in Human DNA Topoisomerase IIα by Pulsed Alkylation Mass Spectrometry*

PubMed Central

Chen, Yu-tsung; Collins, Tammy R. L.; Guan, Ziqiang; Chen, Vincent B.; Hsieh, Tao-Shih

2012-01-01

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5′-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193. PMID:22679013

Structure-activity studies of dicationically substituted bis-benzimidazoles against Giardia lamblia: correlation of antigiardial activity with DNA binding affinity and giardial topoisomerase II inhibition.

PubMed Central

Bell, C A; Dykstra, C C; Naiman, N A; Cory, M; Fairley, T A; Tidwell, R R

1993-01-01

Nine dicationically substituted bis-benzimidazoles were examined for their in vitro activities against Giardia lamblia WB (ATCC 30957). The potential mechanisms of action of these compounds were evaluated by investigating the relationship among in vitro antigiardial activity and the affinity of the molecules for DNA and their ability to inhibit the activity of giardial topoisomerase II. Each compound demonstrated antigiardial activity, as measured by assessing the incorporation of [methyl-3H]thymidine by giardial trophozoites exposed to the test agents. Three compounds exhibited excellent in vitro antigiardial activities, with 50% inhibitory concentrations which compared very favorably with those of two currently used drugs, quinacrine HCl and metronidazole. Putative mechanisms of action for these compounds were suggested by the strong correlation observed among in vitro antigiardial activity and the affinity of the molecules for natural and synthetic DNA and their ability to inhibit the relaxation activity of giardial topoisomerase II. A strong correlation between the DNA binding affinity of these compounds and their inhibition of giardial topoisomerase II activity was also observed. Images PMID:8109934

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Zhou, Qingxuan; Cheng, Bokun

Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain andmore » zinc ribbon-like domain bind ssDNA with primarily π -stacking interactions. Finally, this novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions.« less

African swine fever virus ORF P1192R codes for a functional type II DNA topoisomerase.

PubMed

Coelho, João; Martins, Carlos; Ferreira, Fernando; Leitão, Alexandre

2015-01-01

Topoisomerases modulate the topological state of DNA during processes, such as replication and transcription, that cause overwinding and/or underwinding of the DNA. African swine fever virus (ASFV) is a nucleo-cytoplasmic double-stranded DNA virus shown to contain an OFR (P1192R) with homology to type II topoisomerases. Here we observed that pP1192R is highly conserved among ASFV isolates but dissimilar from other viral, prokaryotic or eukaryotic type II topoisomerases. In both ASFV/Ba71V-infected Vero cells and ASFV/L60-infected pig macrophages we detected pP1192R at intermediate and late phases of infection, cytoplasmically localized and accumulating in the viral factories. Finally, we used a Saccharomyces cerevisiae temperature-sensitive strain in order to demonstrate, through complementation and in vitro decatenation assays, the functionality of P1192R, which we further confirmed by mutating its predicted catalytic residue. Overall, this work strengthens the idea that P1192R constitutes a target for studying, and possibly controlling, ASFV transcription and replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Multiple polysaccharide-drug complex-loaded liposomes: A unique strategy in drug loading and cancer targeting.

PubMed

Ruttala, Hima Bindu; Ramasamy, Thiruganesh; Gupta, Biki; Choi, Han-Gon; Yong, Chul Soon; Kim, Jong Oh

2017-10-01

In the present study, a unique strategy was developed to develop nanocarriers containing multiple therapeutics with controlled release characteristics. In this study, we demonstrated the synthesis of dextran sulfate-doxorubicin (DS-DOX) and alginate-cisplatin (AL-CIS) polymer-drug complexes to produce a transferrin ligand-conjugated liposome. The targeted nanoparticles (TL-DDAC) were nano-sized and spherical. The targeted liposome exhibited a specific receptor-mediated endocytic uptake in cancer cells. The enhanced cellular uptake of TL-DDAC resulted in a significantly better anticancer effect in resistant and sensitive breast cancer cells compared to that of the free drugs. Specifically, DOX and CIS at a molar ratio of 1:1 exhibited better therapeutic performance compared to that of other combinations. The combination of an anthracycline-based topoisomerase II inhibitor (DOX) and a platinum compound (CIS) resulted in significantly higher cell apoptosis (early and late) in both types of cancer cells. In conclusion, treatment with DS-DOX and AL-CIS based combination liposomes modified with transferrin (TL-DDAC) was an effective cancer treatment strategy. Further investigation in clinically relevant animal models is warranted to prove the therapeutic efficacy of this unique strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regression of recurrent malignant gliomas with convection-enhanced delivery of topotecan.

PubMed

Bruce, Jeffrey N; Fine, Robert L; Canoll, Peter; Yun, Jonathan; Kennedy, Benjamin C; Rosenfeld, Steven S; Sands, Stephen A; Surapaneni, Krishna; Lai, Rose; Yanes, Candix L; Bagiella, Emilia; DeLaPaz, Robert L

2011-12-01

Convection-enhanced delivery of chemotherapeutics for the treatment of malignant glioma is a technique that delivers drugs directly into a tumor and the surrounding interstitium through continuous, low-grade positive-pressure infusion. This allows high local concentrations of drug while overcoming the limitations imposed by toxicity and the blood-brain barrier in systemic therapies that prevent the use of many potentially effective drugs. To examine the safety profile of a conventional chemotherapeutic agent, topotecan, via convection-enhanced delivery in the treatment of recurrent malignant gliomas and secondarily to assess radiographic response and survival. We performed a prospective, dose-escalation phase Ib study of the topoisomerase-I inhibitor topotecan given by convection-enhanced delivery in patients with recurrent malignant gliomas. Significant antitumor activity as described by radiographic changes and prolonged overall survival with minimal drug-associated toxicity was demonstrated. A maximum tolerated dose was established for future phase II studies. Topotecan by convection-enhanced delivery has significant antitumor activity at concentrations that are nontoxic to normal brain. The potential for use of this therapy as a generally effective treatment option for malignant gliomas will be tested in subsequent phase II and III trials.

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

PubMed Central

2011-01-01

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. PMID:22111588

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

PubMed Central

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

2009-01-01

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 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 μM) 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

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

PubMed

Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

1999-09-01

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

Expression of p53 and selected proliferative markers (Ki-67, MCM3, PCNA, and topoisomerase IIα) in borderline ovarian tumors: Correlation with clinicopathological features.

PubMed

Ciepliński, Klaudiusz; Jóźwik, Maciej; Semczuk-Sikora, Anna; Gogacz, Marek; Lewkowicz, Dorota; Ignatov, Atanas; Semczuk, Andrzej

2018-02-01

The expression of p53 has been studied not only in primary human ovarian carcinomas, but also in borderline ovarian tumors, however, the results were discordant. Expression patterns of proteins involved in cell proliferation and apoptosis have been investigated in various human neoplasms, including female genital tract neoplasms. The aim of this investigation was to assess the staining pattern and immunolocalization of p53 and selected proliferative markers (Ki-67, MCM3, PCNA, and topoisomerase IIα) in borderline ovarian tumors (BOTs). The study group consisted of 42 women who underwent pelvic surgery between 2006-2015. The median patients' age was 46 years. The immunoperoxidase technique was employed using antibodies against p53, Ki-67, MCM3, PCNA, and topoisomerase IIα. For p53, nuclear expression was observed in BOTs, however, cytoplasmatic immunoreactivity was also detected. Altogether, 25 (60%) tumors demonstrated positive p53 immunostaining, including overexpression found in 6 (14%). There were no significant differences in p53 expression between subgroups of clinicopathological variables. Immunoexpression of Ki-67, MCM3, PCNA, and topoisomerase IIα was nuclear. Ki-67 expression was positive in 12 (29%) cases and there was a trend towards a relationship between patients' age and Ki-67 staining (P=0.08). Interestingly, a significantly higher Ki-67 expression was found in tumors of ≥10 cm in diameter compared to smaller tumors (P=0.008). MCM3 expression was detected in 38 (90%) tumors, and PCNA expression in 28 (67%), yet none of clinicopathological factors was related to them. Topoisomerase IIα expression was present in 14 (33%) cases and, interestingly, its significantly higher expression was observed in BOTs of ≥10 cm in diameter compared to smaller tumors (P=0.008). Moreover, Spearman's correlation revealed highly significant positive associations between Ki-67 and topoisomerase IIα (R=0.403, P=0.008) and Ki-67 and MCM3 (R=0.469, P=0.001). We report a high positive immunostaining rate for p53, suggesting a role of TP53 alterations in the development of BOTs in humans. The new finding of higher topoisomerase IIα immunostaining positivity in BOTs of ≥10 cm may be clinically relevant and requires further studies on larger patient groups.

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ*

PubMed Central

Xu, Yang; Wu, Xiling; Her, Chengtao

2015-01-01

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress. PMID:26055704

Application of Albumin-embedded Magnetic Nanoheaters for Release of Etoposide in Integrated Chemotherapy and Hyperthermia of U87-MG Glioma Cells.

PubMed

Babincová, Melánia; Vrbovská, Hana; Sourivong, Paul; Babinec, Peter; Durdík, Štefan

2018-05-01

Malignant gliomas remain refractory to several therapeutic approaches and the requirement for novel treatment modalities is critical to combat this disease. Etoposide is a topoisomerase-II inhibitor, which promotes DNA damage and apoptosis of cancer cells. In this study, we prepared albumin with embedded magnetic nanoparticles and etoposide for in vitro evaluation of combined hyperthermia and chemotherapy. Magnetic nanoparticles were prepared by a modified co-precipitation method in the presence of human serum albumin and etoposide. A cellular proliferation assay was used to determine the effects of these nanostructures on the viability of U87 glioma cells in an alternating magnetic field. The in vitro experiments showed that cell viability decreased to 59.4% after heat treatment alone and to 53.8% on that with free etoposide, while combined treatment resulted in 7.8% cell viability. Integrating hyperthermia and chemotherapy using albumin co-embedded magnetic nanoheaters and etoposide may represent a promising therapeutic option for glioblastoma. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

PubMed Central

Bases, Robert

2005-01-01

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

PubMed Central

Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

2015-01-01

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

Antitumor effects and mechanisms of Ganoderma extracts and spores oil

PubMed Central

Chen, Chun; Li, Peng; Li, Ye; Yao, Guan; Xu, Jian-Hua

2016-01-01

Ganoderma lucidum is a popular herbal medicine used in China to promote health. Modern studies have disclosed that the active ingredients of Ganoderma can exhibit several effects, including antitumor effects and immunomodulation. The present study evaluated the antitumor effects of self-prepared Ganoderma extracts and spores oil, and investigated the possible underlying mechanisms by observing the effects of the extracts and oil on topoisomerases and the cell cycle. The results showed that Ganoderma extracts and spores oil presented dose-dependent inhibitory effects on tumor cells. The half maximal inhibitory concentration (IC50) values of Ganoderma extracts on HL60, K562 and SGC-7901 cells for 24 h were 0.44, 0.39 and 0.90 mg/ml, respectively; for Ganoderma spores oil, the IC50 values were 1.13, 2.27 and 6.29 mg/ml, respectively. In the in vivo study, the inhibitory rates of Ganoderma extracts (4 g/kg/d, intragastrically) on S180 and H22 cells were 39.1 and 44.6%, respectively, and for Ganoderma spores oil (1.2 g/kg/d, intragastrically) the inhibitory rates were 30.9 and 44.9%, respectively. Ganoderma extracts and spores oil inhibited the activities of topoisomerase I and II. Ganoderma spores oil was shown block the cell cycle at the transition between the G1 and S phases and induce a marked decrease in cyclin D1 levels in K562 cells, with no significant change in cyclin E level. These results suggest that the Ganoderma extracts and spores oil possessed antitumor effects in the in vitro and in vivo studies. The antitumor mechanisms of the extracts and spores oil were associated with inhibitory effects on topoisomerase I and II activities, and for Ganoderma spores oil, the antitumor effects may also be associated with decreased cyclin D1 levels, thus inducing G1 arrest in the cell cycle. PMID:27900038

Therapeutic Applications of PARP Inhibitors: Anticancer Therapy and Beyond

PubMed Central

Curtin, Nicola; Szabo, Csaba

2013-01-01

The aim of this article is to describe the current and potential clinical translation of pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) for the therapy of various diseases. The first section of the present review summarizes the available preclinical and clinical data with PARP inhibitors in various forms of cancer. In this context, the role of PARP in single-strand DNA break repair is relevant, leading to replication-associated lesions that cannot be repaired if homologous recombination (HRR) repair is defective, and the synthetic lethality of PARP inhibitors in HRR-defective cancer. HRR defects are classically associated with BRCA1 and 2 mutations associated with familial breast and ovarian cancer, but there may be many other causes of HRR defects. Thus, PARP inhibitors may be the drugs of choice for BRCA mutant breast and ovarian cancers, and extend beyond these tumors if appropriate biomarkers can be developed to identify HRR defects. Multiple lines of preclinical data demonstrate that PARP inhibition increases cytotoxicity and tumor growth delay in combination with temozolomide, topoisomerase inhibitors and ionizing radiation. Both single agent and combination clinical trials are underway. The final part of the first section of the present review summarizes the current status of the various PARP inhibitors that are in various stages of clinical development. The second section of the present review summarizes the role of PARP in selected non-oncologic indications. In a number of severe, acute diseases (such as stroke, neurotrauma, circulatory shock and acute myocardial infarction) the clinical translatability of PARP inhibition is supported by multiple lines of preclinical data, as well as observational data demonstrating PARP activation in human tissue samples. In these disease indications, PARP overactivation due to oxidative and nitrative stress drives cell necrosis and pro-inflammatory gene expression, which contributes to disease pathology. Accordingly, multiple lines of preclinical data indicate the efficacy of PARP inhibitors to preserve viable tissue and to down-regulate inflammatory responses. As the clinical trials with PARP inhibitors in various forms of cancer progress, it is hoped that a second line of clinical investigations, aimed at testing of PARP inhibitors for various non-oncologic indications, will be initiated, as well. PMID:23370117

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Soo Kyung; Gwak, Jungsug; Song, Im-Sook

Highlights: {yields} TopIn activates p53-dependent transcription in colon cancer cells. {yields} TopIn induces apoptosis in colon cancer cells. {yields} TopIn selectively inhibits topoisomerase I activity. {yields} TopIn does not affect the activity of BCRP and MDR-1. -- Abstract: The tumor suppressor p53 plays an important role in cellular emergency mechanisms through regulating the genes involved in cell cycle arrest and apoptosis. To identify small molecules that can activate p53-responsive transcription, we performed chemical screening using genetically engineered HCT116 reporter cells. We found that TopIn (7-phenyl-6H-[1,2,5]oxadiazolo[3,4-e]indole 3-oxide) efficiently activated p53-mediated transcriptional activity and induced phosphorylation of p53 at Ser15, thereby stabilizingmore » the p53 protein. Furthermore, TopIn upregulated the expression of p21{sup WAF1/CIP1}, a downstream target of p53, and suppressed cellular proliferation in various colon cancer cells. Additionally, TopIn induced DNA fragmentation, caspase-3/7 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis, in p53 wild-type and mutated colon cancer cells. Finally, we found that TopIn inhibited topoisomerase I activity, but not topoisomerase II, in vitro and induced the formation of the topoisomerase I-DNA complex in HCT116 colon cancer cells. Unlike camptothecin (CPT) and its derivative SN38, TopIn did not affect the activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP) or multidrug-resistant protein-1 (MDR-1). These results suggest that TopIn may present a promising new topoisomerase I-targeting anti-tumor therapeutics.« less

Antitumor efficacy of PKI-587, a highly potent dual PI3K/mTOR kinase inhibitor.

PubMed

Mallon, Robert; Feldberg, Larry R; Lucas, Judy; Chaudhary, Inder; Dehnhardt, Christoph; Santos, Efren Delos; Chen, Zecheng; dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Venkatesan, Aranapakam; Hollander, Irwin

2011-05-15

The aim of this study was to show preclinical efficacy and clinical development potential of PKI-587, a dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor. In vitro class 1 PI3K enzyme and human tumor cell growth inhibition assays and in vivo five tumor xenograft models were used to show efficacy. In vitro, PKI-587 potently inhibited class I PI3Ks (IC(50) vs. PI3K-α = 0.4 nmol/L), PI3K-α mutants, and mTOR. PKI-587 inhibited growth of 50 diverse human tumor cell lines at IC(50) values of less than 100 nmol/L. PKI-587 suppressed phosphorylation of PI3K/mTOR effectors (e.g., Akt), and induced apoptosis in human tumor cell lines with elevated PI3K/mTOR signaling. MDA-MB-361 [breast; HER2(+), PIK3CA mutant (E545K)] was particularly sensitive to this effect, with cleaved PARP, an apoptosis marker, induced by 30 nmol/L PKI-587 at 4 hours. In vivo, PKI-587 inhibited tumor growth in breast (MDA-MB-361, BT474), colon (HCT116), lung (H1975), and glioma (U87MG) xenograft models. In MDA-MB-361 tumors, PKI-587 (25 mg/kg, single dose i.v.) suppressed Akt phosphorylation [at threonine(T)308 and serine(S)473] for up to 36 hours, with cleaved PARP (cPARP) evident up to 18 hours. PKI-587 at 25 mg/kg (once weekly) shrank large (∼1,000 mm(3)) MDA-MB-361 tumors and suppressed tumor regrowth. Tumor regression correlated with suppression of phosphorylated Akt in the MDA-MB-361 model. PKI-587 also caused regression in other tumor models, and efficacy was enhanced when given in combination with PD0325901 (MEK 1/2 inhibitor), irinotecan (topoisomerase I inhibitor), or HKI-272 (neratinib, HER2 inhibitor). Significant antitumor efficacy and a favorable pharmacokinetic/safety profile justified phase 1 clinical evaluation of PKI-587. ©2011 AACR.

The Reverse Gyrase from Pyrobaculum calidifontis, a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities*

PubMed Central

Jamroze, Anmbreen; Perugino, Giuseppe; Valenti, Anna; Rashid, Naeem; Rossi, Mosè; Akhtar, Muhammad; Ciaramella, Maria

2014-01-01

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability. PMID:24347172

Pharmacokinetically guided phase I trial of topotecan and etoposide phosphate in recurrent ovarian cancer.

PubMed

Levitt, N C; Propper, D J; Madhusudan, S; Braybrooke, J P; Echeta, C; Te Poele, R; Davies, S L; Flanagan, E; Hickson, I D; Joel, S; Ganesan, T S

2005-07-11

A pharmacokinetically guided phase I study of topotecan and etoposide phosphate was conducted in recurrent ovarian cancer. The scheduling of the topoisomerase I and II inhibitors was determined using in vitro activity data. All patients had recurrent disease following prior platinum-containing chemotherapy. Patients had a World Health Organisation performance status of 0-2 and adequate bone marrow, renal and hepatic function. Treatment was with topotecan intravenously for 5 days followed immediately by a 5-day intravenous infusion of etoposide phosphate (EP), with pharmacokinetically guided dose adjustment. Plasma etoposide levels were measured on days 2 and 4 of the infusion. A total of 21 patients entered the study. In all, 48% were platinum resistant and 71% had received prior paclitaxel. The main toxicities were haematological, short lived and reversible. A total of 29% of patients experienced grade 4 thrombocytopenia and 66% grade 4 neutropenia after the first cycle. Neutropenia and thrombocytopenia was dose limiting. The maximum-tolerated dose was topotecan 0.85 mg m(-2) day(-1) days 1-5 followed immediately by a 5-day infusion of EP at a plasma concentration of 1 mug ml(-1). The response rate (RR) was 28% in 18 evaluable patients. There was marked interpatient variability in topoisomerase IIalpha levels measured from peripheral lymphocytes, with no observed increase following topotecan. This regimen of topotecan followed by EP demonstrated good activity in recurrent ovarian cancer and was noncrossresistant with paclitaxel. Both the toxicity and RR was higher than would be expected from the single agent data, in keeping with synergy of action.

Design, synthesis, molecular modeling and anti-proliferative evaluation of novel quinoxaline derivatives as potential DNA intercalators and topoisomerase II inhibitors.

PubMed

Ibrahim, M K; Taghour, M S; Metwaly, A M; Belal, A; Mehany, A B M; Elhendawy, M A; Radwan, M M; Yassin, A M; El-Deeb, N M; Hafez, E E; ElSohly, M A; Eissa, I H

2018-06-04

New series of [1,2,4]triazolo [4,3-a]quinoxaline and bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives have been designed, synthesized and biologically evaluated for their cytotoxic activities against three tumor cell lines (HePG-2, Hep-2 and Caco-2). Compounds 16 e , 21, 25 a and 25 b exhibited the highest activities against the examined cell lines with IC 50 values ranging from 0.29 to 0.90 μM comparable to that of doxorubicin (IC 50 ranging from 0.51 to 0.73 μM). The most active members were further evaluated for their topoisomerase II (Topo II) inhibitory activities and DNA intercalating affinities as potential mechanisms for their anti-proliferative activities. Interestingly, the results of Topo II inhibition and DNA binding assays were consistent with that of the cytotoxicity data, where the most potent anti-proliferative derivatives exhibited good Topo II inhibitory activities and DNA binding affinities, comparable to that of doxorubicin. Moreover, the most active compound 25 a caused cell cycle arrest at G2/M phase and induced apoptosis in Caco-2 cells. In addition, Furthermore, molecular docking studies were performed for the novel compounds against DNA-Topo II complex to investigate their binding patterns. Based on these studies, it was concluded that DNA binding and/or Topo II inhibition may contribute to the observed cytotoxicity of the synthesized compounds. Copyright © 2018. Published by Elsevier Masson SAS.

Design, synthesis, anticancer screening, docking studies and in silico ADME prediction of some β-carboline derivatives.

PubMed

Abdelsalam, Mohamed A; AboulWafa, Omaima M; M Badawey, El-Sayed A; El-Shoukrofy, Mai S; El-Miligy, Mostafa M; Gouda, Noha; Elaasser, Mahmoud M

2018-05-22

Medicinal interest has focused on β-carbolines as anticancer agents. Several β-carbolines were designed, synthesized and evaluated for their cytotoxic activity against MCF-7 and A-549 cancer cell lines using MTT assay. Compounds 13a, 13c, 13d and 20a were the most promising showing high selectivity indices. Compounds 13c and 20a showed potent inhibition of topoisomerase (topo-I) and kinesin spindle protein (KSP/Eg5 ATPase) which was confirmed by their docking results into the active site of both enzymes. In silico physicochemical calculations predicted that compounds 13a, 13d and 20a obeyed Lipinski's rule of five. Compounds 13c and 20a are multitarget anticancer leads that act as potent inhibitors for both topo-I and/or KSP ATPase.

Synthesis, DNA-binding affinity and cytotoxicity of the dinuclear platinum(II) complexes with berenil and amines ligands.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Popławska, Bozena; Bołkun-Skórnicka, Urszula

2008-01-01

A series of platinium(II) complexes of formula [Pt2L4(berenil)2]Cl4.4HCl.2H2O where L is piperidine (1), 4-picoline (2), 3-picoline (3) or isopropylamine (4) was prepared and their cytotoxicity have been tested against the growth of human breast cancer cells. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than cisplatin. Data from the ethidium displacement assay indicated that these compounds show moderate specificity for AT base pairs of DNA. Compounds 1-4 were also potent topoisomerase II inhibitors, with 50% inhibitory concentrations (IC50) ranging from 5 to 50 microM.

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

PubMed Central

Carballeira, Néstor M.; Montano, Nashbly; Padilla, Luis F.

2006-01-01

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31–32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the ω end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors. PMID:17125759

The connection between BRG1, CTCF and topoisomerases at TAD boundaries.

PubMed

Barutcu, A Rasim; Lian, Jane B; Stein, Janet L; Stein, Gary S; Imbalzano, Anthony N

2017-03-04

The eukaryotic genome is partitioned into topologically associating domains (TADs). Despite recent advances characterizing TADs and TAD boundaries, the organization of these structures is an important dimension of genome architecture and function that is not well understood. Recently, we demonstrated that knockdown of BRG1, an ATPase driving the chromatin remodeling activity of mammalian SWI/SNF enzymes, globally alters long-range genomic interactions and results in a reduction of TAD boundary strength. We provided evidence suggesting that this effect may be due to BRG1 affecting nucleosome occupancy around CTCF sites present at TAD boundaries. In this review, we elaborate on our findings and speculate that BRG1 may contribute to the regulation of the structural and functional properties of chromatin at TAD boundaries by affecting the function or the recruitment of CTCF and DNA topoisomerase complexes.

Neutral and ionic platinum compounds containing a cyclometallated chiral primary amine: synthesis, antitumor activity, DNA interaction and topoisomerase I-cathepsin B inhibition.

PubMed

Albert, Joan; Bosque, Ramon; Crespo, Margarita; Granell, Jaume; López, Concepción; Martín, Raquel; González, Asensio; Jayaraman, Anusha; Quirante, Josefina; Calvis, Carme; Badía, Josefa; Baldomà, Laura; Font-Bardia, Mercè; Cascante, Marta; Messeguer, Ramon

2015-08-14

The synthesis and preliminary biological evaluation of neutral and cationic platinum derivatives of chiral 1-(1-naphthyl)ethylamine are reported, namely cycloplatinated neutral complexes [PtCl{(R or S)-NH(2)CH(CH(3))C(10)H(6)}(L)] [L = SOMe(2) ( 1-R or 1-S ), L = PPh(3) (2-R or 2-S), L = P(4-FC(6)H(4))(3) (3-R), L = P(CH(2))(3)N(3)(CH(2))(3) (4-R)], cycloplatinated cationic complexes [Pt{(R)-NH(2)CH(CH(3))C(10)H(6)}{L}]Cl [L = Ph(2)PCH(2)CH(2)PPh(2) (5-R), L = (C(6)F(5))(2)PCH(2)CH(2)P(C(6)F(5))(2) (6-R)] and the Pt(ii) coordination compound trans-[PtCl(2){(R)-NH(2)CH(CH(3))C(10)H(6)}(2)] (7-R). The X-ray molecular structure of 7-R is reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), cell cycle arrest and apoptosis, DNA interaction, topoisomerase I and cathepsin B inhibition, and Pt cell uptake of the studied compounds are presented. Remarkable cytotoxicity was observed for most of the synthesized Pt(ii) compounds regardless of (i) the absolute configuration R or S, and (ii) the coordinated/cyclometallated (neutral or cationic) nature of the complexes. The most potent compound 2-R (IC(50) = 270 nM) showed a 148-fold increase in potency with regard to cisplatin in HCT-116 colon cancer cells. Preliminary biological results point out to different biomolecular targets for the investigated compounds. Neutral cyclometallated complexes 1-R and 2-R, modify the DNA migration as cisplatin, cationic platinacycle 5-R was able to inhibit topoisomerase I-promoted DNA supercoiling, and Pt(ii) coordination compound 7-R turned out to be the most potent inhibitor of cathepsin B. Induction of G-1 phase ( 2-R and 5-R ), and S and G-2 phases (6-R) arrests are related to the antiproliferative activity of some representative compounds upon A-549 cells. Induction of apoptosis is also observed for 2-R and 6-R.

Expressions of topoisomerase IIα and BCRP in metastatic cells are associated with overall survival in small cell lung cancer patients.

PubMed

Rijavec, Matija; Silar, Mira; Triller, Nadja; Kern, Izidor; Cegovnik, Urška; Košnik, Mitja; Korošec, Peter

2011-09-01

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-naïve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-naïve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.

An atypical topoisomerase II sequence from the slime mold Physarum polycephalum.

PubMed

Hugodot, Yannick; Dutertre, Murielle; Duguet, Michel

2004-01-21

We have determined the complete nucleotide sequence of the cDNA encoding DNA topoisomerase II from Physarum polycephalum. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic enzymes, a 250-bp fragment was polymerase chain reaction (PCR) amplified. This fragment was used as a probe to screen a Physarum cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. Rapid amplification of cDNA ends (RACE)-PCR was employed to isolate the remaining portion of the gene. The complete sequence of 4613 bp contains an open reading frame of 4494 bp that codes for 1498 amino acid residues with a theoretical molecular weight of 167 kDa. The predicted amino acid sequence shares similarity with those of other eukaryotes and shows the highest degree of identity with the enzyme of Dictyostelium discoideum. However, the enzyme of P. polycephalum contains an atypical amino-terminal domain very rich in serine and proline, whose function is unknown. Remarkably, both a mitochondrial targeting sequence and a nuclear localization signal were predicted respectively in the amino and carboxy-terminus of the protein, as in the case of human topoisomerase III alpha. At the Physarum genomic level, the topoisomerase II gene encompasses a region of about 16 kbp suggesting a large proportion of intronic sequences, an unusual situation for a gene of a lower eukaryote, often free of introns. Finally, expression of topoisomerase II mRNA does not appear significantly dependent on the plasmodium cycle stage, possibly due to the lack of G1 phase or (and) to a mitochondrial localization of the enzyme.

Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA

PubMed Central

Rodríguez, A.Chapin; Stock, Daniela

2002-01-01

Reverse gyrase is the only topoisomerase known to positively supercoil DNA. The protein appears to be unique to hyperthermophiles, where its activity is believed to protect the genome from denaturation. The 120 kDa enzyme is the only member of the type I topoisomerase family that requires ATP, which is bound and hydrolysed by a helicase-like domain. We have determined the crystal structure of reverse gyrase from Archaeoglobus fulgidus in the presence and absence of nucleotide cofactor. The structure provides the first view of an intact supercoiling enzyme, explains mechanistic differences from other type I topoisomerases and suggests a model for how the two domains of the protein cooperate to positively supercoil DNA. Coordinates have been deposited in the Protein Data Bank under accession codes 1GKU and 1GL9. PMID:11823434

Variola Type IB DNA Topoisomerase: DNA Binding and Supercoil Unwinding Using Engineered DNA Minicircles

PubMed Central

2015-01-01

Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3′-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5′-CCCTT-3′) from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in regulating the steady-state superhelical density of DNA domains in the cell. PMID:24945825

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure.

PubMed

Cho, Min-Guk; Ahn, Ju-Hyun; Choi, Hee-Song; Lee, Jae-Ho

2017-07-01

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H 2 O 2 ) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H 2 O 2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H 2 O 2 -induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the "abscission checkpoint". Finally, we showed that KRAS-induced binucleated cell formation appeared to be also H 2 O 2 -dependent. In conclusion, we propose that a ROS, mainly H 2 O 2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases.

PubMed

Thomson, Neil H; Santos, Sergio; Mitchenall, Lesley A; Stuchinskaya, Tanya; Taylor, James A; Maxwell, Anthony

2014-08-21

DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the 'exit gate' may be an important determinant of this process.

Cytotoxicity and cellular uptake of doxorubicin and its formamidine derivatives in HL60 sensitive and HL60/MX2 resistant cells.

PubMed

Kik, Krzysztof; Wasowska-Lukawska, Malgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-04-01

In this work a comparison was made of the cytotoxicity and cellular uptake of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N=CH-N<) at the 3' position with morpholine (DOXM) or hexamethyleneimine (DOXH) ring. All tests were performed in doxorubicin-sensitive HL60 and -resistant HL60/MX2 cells which are known for the presence of altered topoisomerase II. Cytotoxic activity of DOX toward HL60/MX2 cells was about 195 times lower when compared with the sensitive HL60 cell line. DOXM and DOXH were approximately 20 times more active in resistant cells than DOX. It was found that the uptake of DOX was lower in resistant cells by about 16%, while that of DOXM and DOXH was lower by about 36% and 19%, respectively. Thus the changes in the cellular uptake of anthracyclines are not associated with the fact that cytotoxicity of DOXM and DOXH exceed the cytotoxicity of DOX. Experiments in cell-free system containing human topoisomerase II showed that topoisomerase II is not inhibited by DOXM and DOXH. Formamidinoanthracyclines may be more useful than parent drugs in therapy against tumor cells with altered topoisomerase II activity.

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases

NASA Astrophysics Data System (ADS)

Thomson, Neil H.; Santos, Sergio; Mitchenall, Lesley A.; Stuchinskaya, Tanya; Taylor, James A.; Maxwell, Anthony

2014-08-01

DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the `exit gate' may be an important determinant of this process.

Actin homolog MreB affects chromosome segregation by regulating topoisomerase IV in Escherichia coli.

PubMed

Madabhushi, Ram; Marians, Kenneth J

2009-01-30

In Escherichia coli, topoisomerase IV, a type II topoisomerase, mediates the resolution of topological linkages between replicated daughter chromosomes and is essential for chromosome segregation. Topo IV activity is restricted to only a short interval late in the cell cycle. However, the mechanism that confers this temporal regulation is unknown. Here we report that the bacterial actin homolog MreB participates in the temporal oscillation of Topo IV activity. We show that mreB mutant strains are deficient in Topo IV activity. In addition, we demonstrate that, depending upon whether it is in a monomeric or polymerized state, MreB affects Topo IV activity differentially. In addition, MreB physically interacts with the ParC subunit of Topo IV. Together, these results may explain how dynamics of the bacterial cytoskeleton are coordinated with the timing of chromosome segregation.

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

PubMed Central

Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel; Prendergast, James G.; Mati, Ioulia K.; Eijk, Paul P.; Cockroft, Scott L.; Bradley, Mark; Ylstra, Bauke; Gilbert, Nick

2013-01-01

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. PMID:23416946

Alteration of Escherichia coli topoisomerase IV to novobiocin resistance.

PubMed

Hardy, Christine D; Cozzarelli, Nicholas R

2003-03-01

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

Molecular modeling of NK-CT1, from Indian monocellate cobra (Naja kaouthia) and its docking interaction with human DNA topoisomerase II alpha

PubMed Central

Bandopadhyay, Pathikrit; Halder, Soma; Sarkar, Mrinmoy; Kumar Bhunia, Sujay; Dey, Sananda; Gomes, Antony; Giri, Biplab

2016-01-01

A 6.76 kDa molecular weight cardio and cytotoxic protein of 60 amino acids in length called NK-CT1, was purified from the venom of Indian monocellate cobra (Naja kaouthia) by ion-exchange chromatography and HPLC as described in our earlier report. Therefore it is of interest to utlize the sequence of NK-CT1 for further functional inference using molecular modeling and docking. Thus homology model of NK-CT1 is described in this report. The anti-proliferative activity of the protein, binding with human DNA topoisomerase-II alpha was demonstrated using docking data with AUTODOCK and AUTODOCK MGL tools. Data shows that M26, V27 and S28 of NK-CT1 is in close contact with the nucleotides of the oligonucleotide, bound with topoisomerase-II alpha complex. PMID:28149043

Mitochondrial Enzyme Plays Critical Role in Chemotherapy-Induced Heart Damage | Center for Cancer Research

Cancer.gov

Doxorubicin (DOX) is an effective drug for treating cancers ranging from leukemia and lymphoma to solid tumors, such as breast cancer. DOX kills dividing cells in two ways: inserting between the base pairs of DNA and trapping a complex of DNA and an enzyme that cuts DNA, topoisomerase 2α, preventing DNA repair. However, DOX also causes congestive heart failure in about 30 percent of adult cancer patients and delayed onset heart failure in a significant number of pediatric cancer patients. The mechanism of this DOX-mediated cardiotoxicity is not well understood since heart muscle cells neither divide nor express Top2α, and there are currently no genetic factors that identify patients who are susceptible to cardiac damage from DOX. However, a recent study showed that mice lacking another topoisomerase, Top2β, did not experience cardiac damage after treatment with DOX.

Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis.

PubMed

Djaout, Kamel; Singh, Vinayak; Boum, Yap; Katawera, Victoria; Becker, Hubert F; Bush, Natassja G; Hearnshaw, Stephen J; Pritchard, Jennifer E; Bourbon, Pauline; Madrid, Peter B; Maxwell, Anthony; Mizrahi, Valerie; Myllykallio, Hannu; Ekins, Sean

2016-06-10

There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

NASA Astrophysics Data System (ADS)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Meroterpenoids with Antitumor Activities from Guava (Psidium guajava).

PubMed

Qin, Xu-Jie; Yu, Qian; Yan, Huan; Khan, Afsar; Feng, Mi-Yan; Li, Pan-Pan; Hao, Xiao-Jiang; An, Lin-Kun; Liu, Hai-Yang

2017-06-21

Psidium guajava L., a species native to South America, has been widely cultivated in the tropical and subtropical areas of China for its popular fruits. The preliminary analysis by liquid chromatography-ultraviolet (LC-UV) indicated the presence of meroterpenoids in the fruits of P. guajava (guava). Subsequent fractionation of the petroleum ether extract resulted in the identification of two new meroterpenoids, psiguajavadials A (1) and B (2), together with 14 previously described meroterpenoids (3-16). Their structures were fully elucidated by comprehensive spectroscopic techniques and theoretical calculations. All of the meroterpenoids showed cytotoxicities against five human cancer cell lines, with guajadial B (12) being the most effective having an IC 50 value of 150 nM toward A549 cells. Furthermore, biochemical topoisomerase I (Top1) assay revealed that psiguajavadial A (1), psiguajavadial B (2), guajadial B (12), guajadial C (14), and guajadial F (16) acted as Top1 catalytic inhibitors and delayed Top1 poison-mediated DNA damage. The flow cytometric analysis indicated that the new meroterpenoids psiguajavadials A (1) and B (2) could induce apoptosis of HCT116 cells. These data suggest that meroterpenoids from guava fruit could be used for the development of antitumor agents.

Pharmacokinetically guided phase I trial of topotecan and etoposide phosphate in recurrent ovarian cancer

PubMed Central

Levitt, N C; Propper, D J; Madhusudan, S; Braybrooke, J P; Echeta, C; te Poele, R; Davies, S L; Flanagan, E; Hickson, I D; Joel, S; Ganesan, T S

2005-01-01

A pharmacokinetically guided phase I study of topotecan and etoposide phosphate was conducted in recurrent ovarian cancer. The scheduling of the topoisomerase I and II inhibitors was determined using in vitro activity data. All patients had recurrent disease following prior platinum-containing chemotherapy. Patients had a World Health Organisation performance status of 0–2 and adequate bone marrow, renal and hepatic function. Treatment was with topotecan intravenously for 5 days followed immediately by a 5-day intravenous infusion of etoposide phosphate (EP), with pharmacokinetically guided dose adjustment. Plasma etoposide levels were measured on days 2 and 4 of the infusion. A total of 21 patients entered the study. In all, 48% were platinum resistant and 71% had received prior paclitaxel. The main toxicities were haematological, short lived and reversible. A total of 29% of patients experienced grade 4 thrombocytopenia and 66% grade 4 neutropenia after the first cycle. Neutropenia and thrombocytopenia was dose limiting. The maximum-tolerated dose was topotecan 0.85 mg m−2 day−1 days 1–5 followed immediately by a 5-day infusion of EP at a plasma concentration of 1 μg ml−1. The response rate (RR) was 28% in 18 evaluable patients. There was marked interpatient variability in topoisomerase IIα levels measured from peripheral lymphocytes, with no observed increase following topotecan. This regimen of topotecan followed by EP demonstrated good activity in recurrent ovarian cancer and was noncrossresistant with paclitaxel. Both the toxicity and RR was higher than would be expected from the single agent data, in keeping with synergy of action. PMID:15956976

DNA Topoisomerase I-Targeted Therapy for Breast Cancer

DTIC Science & Technology

1997-06-01

Fort Detrick, Frederick, MD 21702-5012. 13. ABSTRACT (Maximum 200 words] Camptothecin analogues have been developed that show enhanced pre-clinical...activity against breast cancer cells. These analogues have been synthesized with two fundamental modifications that aid in their effectiveness against...activity. The new analogues , in both growth inhibition and clonogenic assays, are substantially more effective against breast cancer cells than those

Role of cytokines (TNF-alpha, IL-1beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide.

PubMed

Melo, Maria Luisa P; Brito, Gerly A C; Soares, Rudy C; Carvalho, Sarah B L M; Silva, Johan V; Soares, Pedro M G; Vale, Mariana L; Souza, Marcellus H L P; Cunha, Fernando Q; Ribeiro, Ronaldo A

2008-04-01

Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and KC ELISA. CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. These results suggest an important role of TNF-alpha, IL-1beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress.

PubMed

Preta, Giulio; de Klark, Rainier; Chakraborti, Shankhamala; Glas, Rickard

2010-08-27

Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to gamma-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer gamma-hexa-chloro-cyclohexane (gamma-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon gamma-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of gamma-H2AX in gamma-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling. Copyright 2010 Elsevier Inc. All rights reserved.

Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine.

PubMed

Nakano, Hirofumi; Omura, Satoshi

2009-01-01

Staurosporine was discovered at the Kitasato Institute in 1977 while screening for microbial alkaloids using chemical detection methods. It was during the same era that protein kinase C was discovered and oncogene v-src was shown to have protein kinase activity. Staurosporine was first isolated from a culture of Actinomyces that originated in a soil sample collected in Mizusawa City, Japan. Thereafter, indolocarbazole compounds have been isolated from a variety of organisms. The biosynthesis of staurosporine and related indolocarbazoles was finally elucidated during the past decade through genetic and biochemical studies. Subsequently, several novel indolocarbazoles have been produced using combinatorial biosynthesis. In 1986, 9 years since its discovery, staurosporine and related indolocarbazoles were shown to be nanomolar inhibitors of protein kinases. They can thus be viewed as forerunners of today's crop of novel anticancer drugs. The finding led many pharmaceutical companies to search for selective protein kinase inhibitors by screening natural products and through chemical synthesis. In the 1990s, imatinib, a Bcr-Abl tyrosine kinase inhibitor, was synthesized and, following human clinical trials for chronic myelogenous leukemia, it was approved for use in the USA in 2001. In 1992, mammalian topoisomerases were shown to be targets for indolocarbazoles. This opened up new possibilities in that indolocarbazole compounds could selectively interact with ATP-binding sites of not only protein kinases but also other proteins that had slight differences in ATP-binding sites. ABCG2, an ATP-binding cassette transporter, was recently identified as an important new target for indolocarbazoles.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo.

PubMed

Saha, Sourav; Mukherjee, Tulika; Chowdhury, Sayan; Mishra, Amartya; Chowdhury, Somenath Roy; Jaisankar, Parasuraman; Mukhopadhyay, Sibabrata; Majumder, Hemanta K

2013-12-15

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein-DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis. Copyright © 2013 Elsevier Inc. All rights reserved.

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

PubMed

Tentori, Lucio; Leonetti, Carlo; Muzi, Alessia; Dorio, Annalisa Susanna; Porru, Manuela; Dolci, Susanna; Campolo, Federica; Vernole, Patrizia; Lacal, Pedro Miguel; Praz, Françoise; Graziani, Grazia

2013-07-01

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.

Molecular-genetic profiling and high-throughput in vitro drug screening in NUT midline carcinoma—an aggressive and fatal disease

PubMed Central

Stirnweiss, Anja; Oommen, Joyce; Kotecha, Rishi S.; Kees, Ursula R.; Beesley, Alex H.

2017-01-01

NUT midline carcinoma (NMC) is a rare and aggressive cancer, with survival typically less than seven months, that can arise in people of any age. Genetically, NMC is defined by the chromosomal fusion of NUTM1 with a chromatin-binding partner, typically the bromodomain-containing protein BRD4. However, little is known about other genetic aberrations in this disease. In this study, we used a unique panel of cell lines to describe the molecular-genetic features of NMC. Next-generation sequencing identified a recurring high-impact mutation in the DNA-helicase gene RECQL5 in 75% of lines studied, and biological signals from mutation-signature and network analyses consistent with a general failure in DNA-repair. A high-throughput drug screen confirmed that microtubule inhibitors, topoisomerase inhibitors and anthracyclines are highly cytotoxic in the majority of NMC lines, and that cell lines expressing the BRD4-NUTM1 (exon11:exon2) variant are an order of magnitude more responsive to bromodomain inhibitors (iBETs) on average than those with other BRD4-NUTM1 translocation variants. We also identified a highly significant correlation between iBET and aurora kinase inhibitor efficacy in this study. Integration of exome sequencing, transcriptome, and drug sensitivity profiles suggested that aberrant activity of the nuclear receptor co-activator NCOA3 may correlate with poor response to iBETs. In conclusion, our data emphasize the heterogeneity of NMC and highlights genetic aberrations that could be explored to improve therapeutic strategies. The novel finding of a recurring RECQL5 mutation, together with recent reports of chromoplexy in this disease, suggests that DNA-repair pathways are likely to play a central role in NMC tumorigenesis. PMID:29348827

Model of DNA topology simplification has come full (supercoiled) circle after two decades of research. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

NASA Astrophysics Data System (ADS)

Stasiak, Andrzej

2016-09-01

Being a geek of DNA topology, I remember very well the stir caused by 1997 Science paper showing that DNA topoisomerases have the ability to simplify DNA topology below the topological equilibrium values [1]. In their seminal experiments Rybenkov et al. [1] started with linear double-stranded DNA molecules with cohesive ends. The mutual cohesiveness of DNA ends was due to mutual complementarity of single-stranded extensions at both ends of linear double-stranded DNA molecules. When such DNA molecules were heated up and then slowly cooled down the single-stranded ends eventually annealed with each other causing DNA circularization. This experimental protocol permitted the authors to establish topological/thermodynamic equilibrium within samples of circularized DNA molecules. Among simple unknotted circles one also observed knotted and catenated DNA molecules. The fraction of knotted molecules in DNA samples at topological equilibrium was increasing with the length of DNA molecules undergoing slow circularization. The fraction of catenated molecules was increasing with the length and the concentration of the molecules undergoing slow circularization. Rybenkov et al. incubated then such equilibrated DNA samples with type II DNA topoisomerases, which pass DNA duplex regions through each other, and observed that as the result of it the fraction of knotted and catenated DNA molecules was dramatically decreased (up to 80-fold). This elegant experiment indicated for the first time that type II DNA topoisomerases acting on knotted or catenated DNA molecules have the ability to select among many potential sites of DNA-DNA passages these that result in DNA unknotting or decatenation. Without such a selection topoisomerases could only maintain the original topological equilibrium obtained during the slow cyclization. The big question was how DNA topoisomerases can be directed to do DNA-DNA passages that preferentially result in DNA unknotting and decatenation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barros, Francisco W.A.; Bezerra, Daniel P., E-mail: danielpbezerra@gmail.com; Ferreira, Paulo M.P.

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 hallmarksmore » 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.« less

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

PubMed

Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup; Linnemann, Dorte; Stenvang, Jan; Nielsen, Hans Jørgen; Bartek, Jiri; Brünner, Nils; Nørgaard, Peter; Riis, Lene

2015-06-01

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 (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC. 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 patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according to MMR status by immunohistochemical analysis using validated antibodies for MLH1, MLH2, MSH6 and PMS2, and information on TOP1, CEN20, TOP2A and CEN17 status was previously published for this cohort. The observed TOP1 gene copy numbers in the 36 CRC test cohort were significantly greater (p < 0.01) in the pMMR subgroup (mean: 3.84, SD: 2.03) than in the dMMR subgroup (mean: 1.50, SD: 0.12). Similarly, the TOP2A copy numbers were significantly greater (p < 0.01) in the pMMR subgroup (mean: 1.99, SD: 0.52) than in the dMMR subgroup (mean: 1.52, SD: 0.10). These findings were confirmed in the validation cohort, where in the pMMR subgroup 51% had ≥2 extra TOP1 copies per cell, while all tumors classified as dMMR had diploid TOP1 status and mean TOP2A copy numbers were 2.30 (SD: 1.36) and 1.80 (SD: 0.31) (p = 0.01) in the pMMR subgroup vs. dMMR subgroup, respectively. Our results show that TOP1 and TOP2A gene copy numbers are increased in the pMMR subgroup. We propose that this preference may reflect a selective pressure to gain and/or maintain the gained extra copies of topoisomerase genes whose products are required to cope with high replication stress present in the pMMR tumors, thereby providing a survival advantage selectively in pMMR tumors. Future studies should test this concept and explore potential differences between pMMR and dMMR tumors in response to Top1 and Top2 inhibitors. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Local site preference rationalizes disentangling by DNA topoisomerases

NASA Astrophysics Data System (ADS)

Liu, Zhirong; Zechiedrich, Lynn; Chan, Hue Sun

2010-03-01

To rationalize the disentangling action of type II topoisomerases, an improved wormlike DNA model was used to delineate the degree of unknotting and decatenating achievable by selective segment passage at specific juxtaposition geometries and to determine how these activities were affected by DNA circle size and solution ionic strength. We found that segment passage at hooked geometries can reduce knot populations as dramatically as seen in experiments. Selective segment passage also provided theoretical underpinning for an intriguing empirical scaling relation between unknotting and decatenating potentials.

Harnessing Solute Carrier Transporters for Precision Oncology.

PubMed

Nyquist, Michael D; Prasad, Bhagwat; Mostaghel, Elahe A

2017-03-28

Solute Carrier (SLC) transporters are a large superfamily of transmembrane carriers involved in the regulated transport of metabolites, nutrients, ions and drugs across cellular membranes. A subset of these solute carriers play a significant role in the cellular uptake of many cancer therapeutics, ranging from chemotherapeutics such as antimetabolites, topoisomerase inhibitors, platinum-based drugs and taxanes to targeted therapies such as tyrosine kinase inhibitors. SLC transporters are co-expressed in groups and patterns across normal tissues, suggesting they may comprise a coordinated regulatory circuit serving to mediate normal tissue functions. In cancer however, there are dramatic changes in expression patterns of SLC transporters. This frequently serves to feed the increased metabolic demands of the tumor cell for amino acids, nucleotides and other metabolites, but also presents a therapeutic opportunity, as increased transporter expression may serve to increase intracellular concentrations of substrate drugs. In this review, we examine the regulation of drug transporters in cancer and how this impacts therapy response, and discuss novel approaches to targeting therapies to specific cancers via tumor-specific aberrations in transporter expression. We propose that among the oncogenic changes in SLC transporter expression there exist emergent vulnerabilities that can be exploited therapeutically, extending the application of precision medicine from tumor-specific drug targets to tumor-specific determinants of drug uptake.

Detection of functional protein domains by unbiased genome-wide forward genetic screening.

PubMed

Herzog, Mareike; Puddu, Fabio; Coates, Julia; Geisler, Nicola; Forment, Josep V; Jackson, Stephen P

2018-04-18

Establishing genetic and chemo-genetic interactions has played key roles in elucidating mechanisms by which certain chemicals perturb cellular functions. In contrast to gene disruption/depletion strategies to identify mechanisms of drug resistance, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations has been limited. Here, by demonstrating its utility in identifying chemical-genetic suppressors of sensitivity to the DNA topoisomerase I poison camptothecin or the poly(ADP-ribose) polymerase inhibitor olaparib, we detail an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast or haploid mammalian cells in a short timeframe, and with potential applications in other haploid systems. In addition to applications in molecular biology research, this protocol can be used to identify drug targets and predict drug-resistance mechanisms. Mapping suppressor mutations on the primary or tertiary structures of protein suppressor hits provides insights into functionally relevant protein domains. Importantly, we show that olaparib resistance is linked to missense mutations in the DNA binding regions of PARP1, but not in its catalytic domain. This provides experimental support to the concept of PARP1 trapping on DNA as the prime source of toxicity to PARP inhibitors, and points to a novel olaparib resistance mechanism with potential therapeutic implications.

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

PubMed Central

Saha, Sourav; Acharya, Chiranjit; Pal, Uttam; Chowdhury, Somenath Roy; Sarkar, Kahini; Maiti, Nakul C.

2016-01-01

Visceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative, N-benzyl-2,2′α-3,3′,5′,6′,7′,7α,α′-octahydro-2methoxycarbonyl-spiro[indole-3,3′-pyrrolizidine]-2-one (compound 4c), which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes of L. donovani and also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite. PMID:27503653

The role of Ca²⁺ in the activity of Mycobacterium tuberculosis DNA gyrase.

PubMed

Karkare, Shantanu; Yousafzai, Faridoon; Mitchenall, Lesley A; Maxwell, Anthony

2012-10-01

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.

The role of Ca2+ in the activity of Mycobacterium tuberculosis DNA gyrase

PubMed Central

Karkare, Shantanu; Yousafzai, Faridoon; Mitchenall, Lesley A.; Maxwell, Anthony

2012-01-01

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca2+-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca2+ cannot support topoisomerase reactions in the absence of Mg2+, but partial removal of Ca2+ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca2+. More extensive removal of Ca2+ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca2+-binding residues also leads to loss of activity. We propose that Ca2+ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca2+ binding. PMID:22844097

Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases

PubMed Central

Charvin, G.; Bensimon, D.; Croquette, V.

2003-01-01

Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of ≈2.9 s–1, Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of ≈2.4 s–1. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid. PMID:12902541

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic

PubMed Central

Perry, Kay; Hwang, Young; Bushman, Frederic D.; Van Duyne, Gregory D.

2010-01-01

Summary Poxviruses encode their own type IB topoisomerases (TopIBs) which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves a novel Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small vs. large enzyme subfamilies. PMID:20152159

Course 10: Three Lectures on Biological Networks

NASA Astrophysics Data System (ADS)

Magnasco, M. O.

1 Enzymatic networks. Proofreading knots: How DNA topoisomerases disentangle DNA 1.1 Length scales and energy scales 1.2 DNA topology 1.3 Topoisomerases 1.4 Knots and supercoils 1.5 Topological equilibrium 1.6 Can topoisomerases recognize topology? 1.7 Proposal: Kinetic proofreading 1.8 How to do it twice 1.9 The care and proofreading of knots 1.10 Suppression of supercoils 1.11 Problems and outlook 1.12 Disquisition 2 Gene expression networks. Methods for analysis of DNA chip experiments 2.1 The regulation of gene expression 2.2 Gene expression arrays 2.3 Analysis of array data 2.4 Some simplifying assumptions 2.5 Probeset analysis 2.6 Discussion 3 Neural and gene expression networks: Song-induced gene expression in the canary brain 3.1 The study of songbirds 3.2 Canary song 3.3 ZENK 3.4 The blush 3.5 Histological analysis 3.6 Natural vs. artificial 3.7 The Blush II: gAP 3.8 Meditation

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

PubMed Central

Arnò, Barbara; Coletta, Andrea; Tesauro, Cinzia; Zuccaro, Laura; Fiorani, Paola; Lentini, Sara; Galloni, Pierluca; Conte, Valeria; Floris, Barbara; Desideri, Alessandro

2013-01-01

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)–DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5′ DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism. PMID:23368812

The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes.

PubMed

Macovei, Anca; Pagano, Andrea; Sabatini, Maria Elisa; Grandi, Sofia; Balestrazzi, Alma

2018-03-28

The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 μM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold. In addition, the NSC120686-treated calli and untreated MtTdp1α -depleted calli shared a similar response in terms of programmed cell death (PCD)/necrosis and DNA damage. Interestingly, the expression profiles of MtTdp1α and MtTdp1β genes were differently affected by the NSC120686 treatment, as MtTdp1α was upregulated while MtTdp1β was downregulated. The NSC120686 treatment affected not only the MtTdp1 genes but also other genes with roles in alternative DNA repair pathways. Since the expression patterns of these genes were different than what was observed in the MtTdp1α -depleted plants, it could be hypothesized that the NSC120686 treatment exerts a different influence compared to that resulting from the lack of the MtTdp1α gene function.

CETUXIMAB AND IRINOTECAN INTERACT SYNERGISTICALLY TO INHIBIT THE GROWTH OF ORTHOTOPIC ANAPLASTIC THYROID CARCINOMA XENOGRAFTS IN NUDE MICE

PubMed Central

Kim, Seungwon; Prichard, Christopher N.; Younes, Maher N.; Yazici, Yasemin D.; Jasser, Samar A.; Bekele, B. Nebiyou; Myers, Jeffrey N.

2006-01-01

Objective Anaplastic thyroid carcinoma (ATC) remains one of the most lethal known human cancers. Targeted molecular therapy with cetuximab, a monoclonal antibody against EGFR, offers new treatment potentials for patient with ATC. Cetuximab has also been reported to have synergistic effects when combined with irinotecan, a topoisomerase inhibitor. Therefore, we hypothesized that cetuximab and irinotecan would be effective in inhibiting the growth and progression of ATC in a murine orthotopic model. Design The in vitro anti-proliferative effects of cetuximab and irinotecan on ATC cell line ARO were examined. We also studied the in vivo effects of cetuximab and irinotecan on the growth, invasion, and metastasis of orthotopic ATC tumors in nude mice. The in vivo antitumor efficacy of cetuximab/irinotecan combination was also compared with that of doxorubicin. Results Cetuximab alone did not show any anti-proliferative or pro-apoptotic effect on this cell line. However, when combined with irinotecan, cetuximab potentiated the in vitro anti-proliferative and pro-apoptotic effect of irinotecan. Cetuximab, irinotecan, and cetuximab/irinotecan combination resulted in 77%, 79%, and 93% in vivo inhibition of tumor growth, respectively. Incidences of lymph node metastasis, laryngeal invasion, and tumor microvessel density were also significantly decreased in these treatment groups. Furthermore, the cetuximab/irinotecan combination was significantly more effective than doxorubicin in inhibiting the growth of orthotopic ATC xenografts. Conclusions Combination therapy with cetuximab/irinotecan inhibits the growth and progression of orthotopic ATC xenografts in nude mice. Given the lack of curative options for patients with ATC, combination therapy with cetuximab and irinotecan treatment warrants further study. PMID:16428506

Carbohydrate linked organotin(IV) complexes as human topoisomerase Iα inhibitor and their antiproliferative effects against the human carcinoma cell line.

PubMed

Khan, Rais Ahmad; Yadav, Shipra; Hussain, Zahid; Arjmand, Farukh; Tabassum, Sartaj

2014-02-14

Dimethyltin(IV) complexes with ethanolamine (1) and biologically significant N-glycosides (2 and 3) were designed and synthesized. The structural elucidation of complexes 1-3 was done using elemental and spectroscopic methods; in addition, complex 1 was studied by single crystal X-ray diffraction studies. The in vitro DNA binding profile of complexes 2 and 3 was carried out by employing different biophysical methods to ascertain the feasibility of glycosylated complexes. Further, the cleaving ability of 2 and 3 was investigated by the agarose gel electrophoretic mobility assay with supercoiled pBR322 DNA, and demonstrated significantly good nuclease activity. Furthermore, both the complexes exhibited significant inhibitory effects on the catalytic activity of human Topo I at lower concentration than standard drugs. Computer-aided molecular docking techniques were used to ascertain the mode and mechanism of action towards the molecular target DNA and Topo I. The cytotoxicity of 2 and 3 against human hepatoma cancer cells (Huh7) was evaluated, which revealed significant regression in cancerous cells as compared with the standard drug. The antiproliferative activities of 2 and 3 were tested against human hepatoma cancer cells (Huh7), and results showed significantly good activity. Additionally, to validate the remarkable antiproliferative activity of complexes 2 and 3, specific regulatory gene expression (MMP-2 and TGF-β) was obtained by real time PCR.

Observation des cycles enzymatiques des ADN topoisomérases par micromanipulation de molécules individuelles

NASA Astrophysics Data System (ADS)

Strick, Terence R.; Charvin, Gilles; Dekker, Nynke H.; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

In this article, we describe single-molecule assays using magnetic traps and we applied these assays to topoisomerase enzymes which unwind and disentangle DNA molecules. First, the elasticity of single DNA molecule is characterized using the magnetic trap. We show that a twisting constraint may be easily applied and that its effect upon DNA may be measured accurately. Then we describe how the topoisomerase activity may be observed at the single-molecule level giving direct access to the important biological parameters of the enzyme such as velocity and processivity. Furthermore, individual cycles of unwinding can be observed in real time. This permits an accurate characterization of the enzyme's biochemical cycle. The data treatment required to identify and analyze individual topoisomerization cycles will be presented in detail. This analysis is applicable to a wide variety of molecular motors. To cite this article: T.R. Strick et al., C. R. Physique 3 (2002) 595-618.

BCR/ABL downregulates DNA-PK(CS)-dependent and upregulates backup non-homologous end joining in leukemic cells.

PubMed

Poplawski, Tomasz; Blasiak, Janusz

2010-06-01

Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK(CS); D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK(CS) (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK(CS), did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.

Mechanism of quinolone action and resistance.

PubMed

Aldred, Katie J; Kerns, Robert J; Osheroff, Neil

2014-03-18

Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone-enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells

PubMed Central

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A.; García-Pérez, Carlos A.; Guerrero-Rodríguez, Sandra L.; Ruiz, Angel J.; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M.; Velasco-Velázquez, Marco A.

2016-01-01

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists. PMID:27009862

Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

PubMed Central

Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H.; Xia, Menghang

2016-01-01

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling. PMID:26820057

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

PubMed

Aguirre-Alvarado, Charmina; Segura-Cabrera, Aldo; Velázquez-Quesada, Inés; Hernández-Esquivel, Miguel A; García-Pérez, Carlos A; Guerrero-Rodríguez, Sandra L; Ruiz-Moreno, Angel J; Rodríguez-Moreno, Andrea; Pérez-Tapia, Sonia M; Velasco-Velázquez, Marco A

2016-04-26

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

CIP-36, a novel topoisomerase II-targeting agent, induces the apoptosis of multidrug-resistant cancer cells in vitro.

PubMed

Cao, Bo; Chen, Hong; Gao, Ying; Niu, Cong; Zhang, Yuan; Li, Ling

2015-03-01

The need to overcome cancer multidrug resistance (MDR) has fueled considerable interest in the development of novel synthetic antitumor agents with cytotoxicity against cancer cell lines with MDR. In this study, we aimed to investigate CIP-36, a novel podophyllotoxin derivative, for its inhibitory effects on human cancer cells from multiple sources, particularly cells with MDR in vitro. The human leukemia cell line, K562, and the adriamycin-resistant subline, K562/A02, were exposed to CIP-36 or anticancer agents, and various morphological and biochemical properties were assessed by Hoechst 33342 staining under a fluorescence microscope. Subsequently, cytotoxicity, cell growth curves and the cell cycle were analyzed. Finally, the effects of CIP-36 on topoisomerase IIα (Topo IIα) activity were determined. Treatment with CIP-36 significantly inhibited the growth of the K562 and MDR K562/A02 cells. Our data demonstrated that CIP-36 induced apoptosis, inhibited cell cycle progression and inhibited Topo IIα activity. These findings suggest that CIP-36 has the potential to overcome the multidrug resistance of K562/A02 cells by mediating Topo IIα activity.

[Fluoroquinolone resistance mutations in topoisomerase genes of Salmonella typhimurium isolates].

PubMed

Guo, Yunchang; Pei, Xiaoyan; Liu, Xiumei

2004-09-01

Mutations in topoisomerase genes were main cause of the resistence of Salmonella typhimurium to fluoroquinolone. The MICs of three Salmonella typhimurium isolates X2, X7, X11 to ciprofloxacin were above 32 microg/ml, 0.38 microg/ml and 0.023 microg/ml, respectively. The genetic alterations in four topoisomerase genes, gyrA, gyrB, parC, and parE were detected by multiplex PCR amplimer conformation analysis in these three strains. X2 isolate showed both gyrA mutations (Ser83-->Phe, Asp87-->Asn) and parC mutation (Ser80-->Arg). X7 isolate showed a single gyrA mutation (Ser83-->Phe) and X11 isolate had no changes in all of the four quinolone resistance genes, gyrA, gyrB, parC, and parE. X7 isolate with a single gyrA mutation was less resistant to ciprofloxacin than X2 with double gyrA mutations and an additional parC mutation. GyrA and parC genes play important role of the resistance of Salmonella typhimurium to ciprofloxacin.

Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

PubMed

Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C; Heyer, Wolf-Dietrich

2015-02-19

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-04-07

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. Copyright © 2016 Elsevier Inc. All rights reserved.

UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis.

PubMed

Liao, Chunyan; Beveridge, Ryan; Hudson, Jessica J R; Parker, Jacob D; Chiang, Shih-Chieh; Ray, Swagat; Ashour, Mohamed E; Sudbery, Ian; Dickman, Mark J; El-Khamisy, Sherif F

2018-06-12

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

PubMed

Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

1995-12-01

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development.

PubMed

Klahn, Philipp; Brönstrup, Mark

The development of bacterial resistance against current antibiotic drugs necessitates a continuous renewal of the arsenal of efficacious drugs. This imperative has not been met by the output of antibiotic research and development of the past decades for various reasons, including the declining efforts of large pharma companies in this area. Moreover, the majority of novel antibiotics are chemical derivatives of existing structures that represent mostly step innovations, implying that the available chemical space may be exhausted. This review negates this impression by showcasing recent achievements in lead finding and optimization of antibiotics that have novel or unexplored chemical structures. Not surprisingly, many of the novel structural templates like teixobactins, lysocin, griselimycin, or the albicidin/cystobactamid pair were discovered from natural sources. Additional compounds were obtained from the screening of synthetic libraries and chemical synthesis, including the gyrase-inhibiting NTBI's and spiropyrimidinetrione, the tarocin and targocil inhibitors of wall teichoic acid synthesis, or the boronates and diazabicyclo[3.2.1]octane as novel β-lactamase inhibitors. A motif that is common to most clinically validated antibiotics is that they address hotspots in complex biosynthetic machineries, whose functioning is essential for the bacterial cell. Therefore, an introduction to the biological targets-cell wall synthesis, topoisomerases, the DNA sliding clamp, and membrane-bound electron transport-is given for each of the leads presented here.

The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

PubMed Central

Ferrándiz, María-José

2014-01-01

We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe2+ and Fe3+) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the Pfat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: PfatfatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the Pfatcat construction. Results suggest that topological changes are indeed involved in PfatfatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality. PMID:24145547

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells.

PubMed

Sinha, Birandra K; Kumar, Ashutosh; Mason, Ronald P

2017-07-01

Topoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide ( • NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide. Here, we have evaluated the roles of • NO/ • NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of • NO on the ATPase activity of topo II. Treatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. • NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells. PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells. As tumors express nitric oxide synthase and generate • NO, inhibition of topo II functions by • NO/ • NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, C.-P.; Fang, W.-H.; Lin, L.-I.

2008-03-15

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo II{alpha} activity through the accumulation of Topomore » II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC{sub 50} of 0.9 {mu}M, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC{sub 50} of 9.6 {mu}M, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 {mu}M. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC{sub 50} about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design.« less

Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

PubMed

Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

2013-02-01

Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes. Copyright © 2012 Elsevier Inc. All rights reserved.

F14512, a potent antitumor agent targeting topoisomerase II vectored into cancer cells via the polyamine transport system.

PubMed

Barret, Jean-Marc; Kruczynski, Anna; Vispé, Stéphane; Annereau, Jean-Philippe; Brel, Viviane; Guminski, Yves; Delcros, Jean-Guy; Lansiaux, Amélie; Guilbaud, Nicolas; Imbert, Thierry; Bailly, Christian

2008-12-01

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

NASA Astrophysics Data System (ADS)

Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

2015-12-01

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala

2010-08-27

Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an eventmore » that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.« less

Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability.

PubMed

Husain, Afzal; Begum, Nasim A; Taniguchi, Takako; Taniguchi, Hisaaki; Kobayashi, Maki; Honjo, Tasuku

2016-02-04

Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.

The water soluble peripherally tetra-substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines as new potential anticancer agents.

PubMed

Barut, Burak; Sofuoğlu, Ayşenur; Biyiklioglu, Zekeriya; Özel, Arzu

2016-09-28

In this study, [2-(2-morpholin-4-ylethoxy)ethoxy] group substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines 2-4 and their water soluble derivatives 2a, 3a and 4a were synthesized and the interactions of compounds 2a, 3a and 4a with CT-DNA and supercoiled pBR322 plasmid DNA were investigated. The results of binding experiments showed that these compounds were able to interact with CT-DNA via intercalative mode with a strong binding affinity in the order 3a > 2a > 4a. DNA-photocleavage activities of compounds 2a, 3a and 4a were determined. These compounds cleaved supercoiled pBR322 plasmid DNA efficiently under irradiation at 650 nm for 2a and 4a, and at 750 nm for 3a. These compounds displayed remarkable inhibitory activities against topoisomerase I enzyme in a dose-dependent manner. All of these results suggest that these phthalocyanines might be suitable anticancer agents due to their strong binding affinities, significant cleavage activities and effective topoisomerase I inhibition.

High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients.

PubMed

Shero, J H; Bordwell, B; Rothfield, N F; Earnshaw, W C

1986-02-14

Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme.

Marine natural products. XXXII. Absolute configurations of C-4 of the manoalide family, biologically active sesterterpenes from the marine sponge Hyrtios erecta.

PubMed

Kobayashi, M; Okamoto, T; Hayashi, K; Yokoyama, N; Sasaki, T; Kitagawa, I

1994-02-01

Cytotoxic sesterterpenes, manoalide 25-acetals (1a, 1b), seco-manoalide (2), (E)-neomanoalide (3), (Z)-neomanoalide (4), and heteronemin (6), were isolated from the marine sponge Hyrtios erecta (collected at Amami Island, Kagoshima Prefecture, Japan) by bioassay-guided separation and the absolute configurations of these manoalide family members have been determined. Manoalide 25-acetals (1a, 1b) were shown to exhibit in vivo antitumor activity and to inhibit the DNA-relaxing activity of mouse DNA topoisomerase I and the DNA-unknotting activity of calf thymus DNA topoisomerase II.

Top3-Rmi1 dissolve Rad51-mediated D-loops by a topoisomerase-based mechanism

PubMed Central

Fasching, Clare L.; Cejka, Petr; Kowalczykowski, Stephen C.; Heyer, Wolf-Dietrich

2015-01-01

Summary The displacement loop (D-loop) is the DNA strand invasion product formed during homologous recombination. Disruption of nascent D-loops represents a mechanism of anti-recombination. During Synthesis-Dependent Strand Annealing D-loop disruption after extension of the invading strand is an integral step of the pathway and ensures a non-crossover outcome. The proteins implicated in D-loop disruption are DNA motor proteins/helicases acting by migrating DNA junctions. Here we report an unanticipated mechanism of D-loop dissolution mediated by DNA topoisomerase 3 (Top3) and dependent on its catalytic activity. D-loop dissolution catalyzed by yeast Top3 is highly specific for yeast Rad51/Rad54-mediated D-loops, whereas protein-free D-loops or D-loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also the human Topoisomerase IIIα-RMI1–RMI2 complex is capable of dissolving D-loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is in part a result of unprocessed D-loops. PMID:25699708

ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure

PubMed Central

1994-01-01

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II. PMID:7929577

Development of Derivatives of 3, 3′-Diindolylmethane as Potent Leishmania donovani Bi-Subunit Topoisomerase IB Poisons

PubMed Central

Sengupta, Souvik; Mandal, Madhumita; Jaisankar, Parasuraman; D'Annessa, Ilda; Desideri, Alessandro; Majumder, Hemanta K.

2011-01-01

Background The development of 3, 3′-diindolyl methane (DIM) resistant parasite Leishmania donovani (LdDR50) by adaptation with increasing concentrations of the drug generates random mutations in the large and small subunits of heterodimeric DNA topoisomerase I of Leishmania (LdTOP1LS). Mutation of large subunit of LdTOP1LS at F270L is responsible for resistance to DIM up to 50 µM concentration. Methodology/Principal Findings In search of compounds that inhibit the growth of the DIM resistant parasite and inhibit the catalytic activity of mutated topoisomerase I (F270L), we have prepared three derivatives of DIM namely DPDIM (2,2′-diphenyl 3,3′-diindolyl methane), DMDIM (2,2′-dimethyl 3,3′-diindolyl methane) and DMODIM (5,5′-dimethoxy 3,3′-diindolyl methane) from parent compound DIM. All the compounds inhibit the growth of DIM resistant parasites, induce DNA fragmentation and stabilize topo1-DNA cleavable complex with the wild type and mutant enzyme. Conclusion The results suggest that the three derivatives of DIM can act as promising lead molecules for the generation of new anti-leishmanial agents. PMID:22174820

Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

PubMed Central

Szafran, Marcin; Skut, Patrycja; Ditkowski, Bartosz; Ginda, Katarzyna; Chandra, Govind; Zakrzewska-Czerwińska, Jolanta

2013-01-01

Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes. PMID:23913317

Ap4A induces apoptosis in human cultured cells.

PubMed

Vartanian, A; Alexandrov, I; Prudowski, I; McLennan, A; Kisselev, L

1999-07-30

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'''-P1,P3-triphosphate to diadenosine 5',5'''-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Mitochondrial Enzyme Plays Critical Role in Chemotherapy-Induced Heart Damage | Center for Cancer Research

Cancer.gov

Doxorubicin (DOX) is an effective drug for treating cancers ranging from leukemia and lymphoma to solid tumors, such as breast cancer. DOX kills dividing cells in two ways: inserting between the base pairs of DNA and trapping a complex of DNA and an enzyme that cuts DNA, topoisomerase 2α, preventing DNA repair. However, DOX also causes congestive heart failure in about 30

F14512, a polyamine-vectorized inhibitor of topoisomerase II, exhibits a marked anti-tumor activity in ovarian cancer.

PubMed

Thibault, Benoît; Clement, Emily; Zorza, Grégoire; Meignan, Samuel; Delord, Jean-Pierre; Couderc, Bettina; Bailly, Christian; Narducci, Fabrice; Vandenberghe, Isabelle; Kruczynski, Anna; Guilbaud, Nicolas; Ferré, Pierre; Annereau, Jean-Philippe

2016-01-01

Epithelial ovarian cancer is the fourth cause of death among cancer-bearing women and frequently associated with carboplatin resistance, underlining the need for more efficient and targeted therapies. F14512 is an epipodophylotoxin-core linked to a spermine chain which enters cells via the polyamine transport system (PTS). Here, we investigate this novel concept of vectorization in ovarian cancer. We compared the effects of etoposide and F14512 on a panel of five carboplatin-sensitive or resistant ovarian cancer models. We assessed the incorporation of F17073, a spermine-linked fluorescent probe, in these cells and in 18 clinical samples. We then showed that F14512 exhibits a high anti-proliferative and pro-apoptotic activity, particularly in cells with high levels of F17073 incorporation. Consistently, F14512 significantly inhibited tumor growth compared to etoposide, in a cisplatin-resistant A2780R subcutaneous model, at a dose of 1.25 mg/kg. In addition, ex vivo analysis indicated that 15 out of 18 patients presented a higher F17073 incorporation into tumor cells compared to normal cells. Overall, our data suggest that F14512, a targeted drug with a potent anti-tumor efficacy, constitutes a potential new therapy for highly PTS-positive and platinum-resistant ovarian cancer-bearing patients. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Topoisomerase II Mediates Meiotic Crossover Interference

PubMed Central

Zhang, Liangran; Wang, Shunxin; Yin, Shen; Hong, Soogil; Kim, Keun P.; Kleckner, Nancy

2014-01-01

Summary Spatial patterning is a ubiquitous feature of biological systems. Meiotic crossovers provide an interesting example, defined by the classical phenomenon of crossover interference. Here, analysis of crossover patterns in budding yeast identifies a molecular pathway for interference. Topoisomerase II (Topo II) plays a central role, thus identifying a new function for this critical molecule. SUMOylation [of TopoII and axis component Red1] and ubiquitin-mediated removal of SUMOylated proteins are also required. These and other findings support the hypothesis that crossover interference involves accumulation, relief and redistribution of mechanical stress along the protein/DNA meshwork of meiotic chromosome axes, with TopoII required to adjust spatial relationships among DNA segments. PMID:25043020

Unraveling the structure of the variola topoisomerase IB-DNA complex: a possible new twist on smallpox therapy.

PubMed

Osheroff, Neil

2006-10-01

Smallpox is a serious and highly contagious disease that is caused by the variola virus. It is one of the most severe infectious human diseases known, with mortality rates as high as 30%. A successful worldwide vaccination program led to the eradication of smallpox in 1980. However, the high transmission rate of variola virus, coupled with the deadly nature of smallpox, makes this virus a potentially devastating weapon for bioterrorism. Currently, there is no specific treatment for smallpox. However, a recent article on the structure of a variola topoisomerase IB-DNA complex provides an intriguing starting point for the rational design of drugs with potential activity against smallpox.

Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing.

PubMed

Kont, Yasemin Saygideger; Dutta, Arijit; Mallisetty, Apurva; Mathew, Jeena; Minas, Tsion; Kraus, Christina; Dhopeshwarkar, Priyanka; Kallakury, Bhaskar; Mitra, Sankar; Üren, Aykut; Adhikari, Sanjay

2016-07-01

DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non-homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced γ-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism

PubMed Central

Gubaev, Airat; Weidlich, Daniela; Klostermeier, Dagmar

2016-01-01

The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors. PMID:27557712

Enhanced malignant transformation is accompanied by increased survival recovery after ionizing radiation in Chinese hamster embryo fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boothman, D.A.

Transformed Chinese hamster embryo fibroblasts (CHEF), which gradually increase in tumor-forming ability in nude mice, were isolated from normal diploid CHEF/18 cells. Transformed CHEF cells (i.e., T30-4 > 21-2M3 > 21-2 > normal CHEF/18) showed gradual increases in potentially lethal damage (PLD) survival recovery. {beta}-Lapachone and camptothecin, modulators of topoisomerase I (Topo I) activity, not only prevented survival recovery in normal as well as in tumor cells, but enhanced unscheduled DNA synthesis. These seemingly conflicting results are due to the fact that Topo I activity can be modulated by inhibitors to convert single-stranded DNA lesions into double-stranded breaks. Increases inmore » unscheduled DNA synthesis may result from a continual supply of free ends, on which DNA repair processes may act. Altering Topo I activity with modulators appears to increase X-ray lethality via a DNA lesion modification suicide pathway. Cells down-regulate Topo I immediately after ionizing radiation to prevent Topo I-mediated lesion modification and to enhance survival recovery. 16 refs., 3 figs., 1 tab.« less

GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

PubMed

Eid, Mohammed Mansour Abbas; Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar; Shimoda, Mayuko; Sakaguchi, Nobuo

2014-06-15

RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment. Copyright © 2014 by The American Association of Immunologists, Inc.

New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

PubMed

Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Novel Indole-based Tambjamine-Analogues Induce Apoptotic Lung Cancer Cell Death through p38 Mitogen-Activated Protein Kinase Activation.

PubMed

Manuel-Manresa, Pilar; Korrodi-Gregório, Luís; Hernando, Elsa; Villanueva, Alberto; Martínez-García, David; Rodilla, Ananda M; Ramos, Ricard; Fardilha, Margarida; Moya, Juan; Quesada, Roberto; Soto-Cerrato, Vanessa; Pérez-Tomás, Ricardo

2017-07-01

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5 /survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer. Mol Cancer Ther; 16(7); 1224-35. ©2017 AACR . ©2017 American Association for Cancer Research.

[Routine chemotherapeutic drug treatment effectiveness predictive molecules and chemotherapeutic drug selection].

PubMed

Zhao, Xiao-Dong; Zhang, Yi

2006-12-01

Drug selection, the key for chemotherapy, is one of the most difficult decision-making in clinic for the treatment of malignant tumors. How to choose is undetermined. Here a new strategy--predictive molecule-targeted chemotherapy (PMTC)--is put forward to choose relatively sensitive chemotherapeutic drugs and to avoid relatively resistant traditional drugs according to the expression of predictive molecules in individual tumor tissue. For example, paclitaxel is regarded as a relatively sensitive drug and may be chosen for the tumors with high expression of p53, while it is predicted as relatively resistant drug and should be avoided for the tumors with high expression of P-glycoprotein (P-gp). Here, we reviewed the predictive values of a variety of molecules, such as p53, P-gp, topoisomerase-1, topoisomerase-2, MSI, BRCA-1, ERCC1, FANC, hMHL1/2, XPD, Bcl-2, ErbB-2, MGMT, dihydropyridine dehydrogenase (DPD), thymidylate synthetase (TS), deoxycytidine kinase (dCK), Ras, Bax, Cyclin A, tubulin proteins, and so on, for the efficacy of some traditional chemotherapeutic drugs, such as platinum, oxaliplatin, cyclophosphamide, ifosfamide, dacarbazine, methotrexate, 5-flurouracil, gemcitabine, vincristine, vinorelbine, paclitaxel, etoposide, irinotecan, topotecan, and so on.

[In vitro and in vivo antibacterial activities of pazufloxacin mesilate, a new injectable quinolone].

PubMed

Nomura, Nobuhiko; Mitsuyama, Junichi; Furuta, Yousuke; Yamada, Hisashi; Nakata, Mitsunori; Fukuda, Toshiko; Yamada, Hiroshi; Takahata, Masahiro; Minami, Shinzaburo

2002-08-01

We investigated the in vitro and in vivo antibacterial activities of pazufloxacin mesilate (PZFX mesilate), a new injectable quinolone, and obtained the following results. 1) The MIC50 and MIC90 values of PZFX against clinically isolated Gram-positive and -negative bacteria, ranged from 0.0125 to 12.5 micrograms/ml and 0.025 to 100 micrograms/ml, respectively. PZFX showed broad spectrum activity. The antibacterial activities of PZFX against quinolone-susceptible, methicillin-resistant Staphylococcus aureus, beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae, extended spectrum beta-lactamase possessing Klebsiella pneumoniae and imipenem/cilastatine (IPM/CS)-resistant Pseudomonas aeruginosa were superior to those of ceftazidime (CAZ), ceftriaxone, IPM/CS, meropenem and panipenem/betamipron. 2) PZFX showed superior bactericidal activity against S. aureus, Escherichia coli, Proteus mirabilis, Serratia marcescens and P. aeruginosa to those of CAZ and IPM/CS after treatment for 15 minutes at the drug concentration equivalent to that in human serum at clinical dose to be continued for 15 minutes. 3) CAZ and IPM/CS had no bactericidal activity at the 16 times of MIC against P. aeruginosa in human polymorphonuclear leucocytes, while PZFX exhibited potent bactericidal activity in a dose-dependent manner against such bacteria. 4) PZFX inhibited both DNA gyrase and topoisomerase IV from S. aureus at nearly the same level. PZFX showed poor inhibitory activity against topoisomerase II from human placenta and showed high selectivity to bacterial topoisomerase. 5) PZFX mesilate showed superior therapeutic activity to that of CAZ with following infection model caused by S. aureus and P. aeruginosa or each; systemic infection with cyclophosphamide-treated mice, systemic infection in mice with high challenge doses, CMC pouch infection in rat, and calculus infection in rat bladder. 6) Intravenous administration of PZFX with high plasma concentration just after administration, showed more excellent therapeutic effect against the rat intraperitoneal infection, than p.o. and s.c. administration.

Mechanisms of DNA disentangling by type II topoisomerases. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

NASA Astrophysics Data System (ADS)

Yan, Jie

2016-09-01

In this article [1] Dr. Vologodskii presents a comprehensive discussion on the mechanisms by which the type II topoisomerases unknot/disentangle DNA molecules. It is motivated by a mysterious capability of the nanometer-size enzymes to keep the steady-state probability of DNA entanglement/knot almost two orders of magnitude below that expected from thermal equilibrium [2-5]. In spite of obvious functional advantages of the enzymes, it raises a question regarding how such high efficiency could be achieved. The off-equilibrium steady state distribution of DNA topology is powered by ATP consumption. However, it remains unclear how this energy is utilized to bias the distribution toward disentangled/unknotted topological states of DNA.

Guanabenz promotes neuronal survival via enhancement of ATF4 and parkin expression in models of Parkinson disease.

PubMed

Sun, Xiaotian; Aimé, Pascaline; Dai, David; Ramalingam, Nagendran; Crary, John F; Burke, Robert E; Greene, Lloyd A; Levy, Oren A

2018-05-01

Reduced function of parkin appears to be a central pathogenic event in Parkinson disease (PD). Increasing parkin levels enhances survival in models of PD-related neuronal death and is a promising therapeutic objective. Previously, we demonstrated that the transcription factor ATF4 promotes survival in response to PD-mimetic stressors by maintaining parkin levels. ATF4 translation is up-regulated by phosphorylation of the translation initiation factor eIF2α. The small molecule guanabenz enhances eIF2α phosphorylation by blocking the function of GADD34, a regulatory protein that promotes eIF2α dephosphorylation. We tested the hypothesis that guanabenz, by inhibiting GADD34 and consequently increasing eIF2α phosphorylation and elevating ATF4, would improve survival in models of PD by up-regulating parkin. We found that GADD34 is strongly induced by 6-OHDA, and that GADD34 localization is dramatically altered in dopaminergic substantia nigra neurons in PD cases. We further demonstrated that guanabenz attenuates 6-hydroxydopamine (6-OHDA) induced cell death of differentiated PC12 cells and primary ventral midbrain dopaminergic neurons in culture, and of dopaminergic neurons in the substantia nigra of mice. In culture models, guanabenz also increases eIF2α phosphorylation and ATF4 and parkin levels in response to 6-OHDA. Furthermore, if either ATF4 or parkin is silenced, then the protective effect of guanabenz is lost. We also found similar results in a distinct model of neuronal death: primary cultures of cortical neurons treated with the topoisomerase I inhibitor camptothecin, in which guanabenz limited camptothecin-induced neuronal death in an ATF4- and parkin-dependent manner. In summary, our data suggest that guanabenz and other GADD34 inhibitors could be used as therapeutic agents to boost parkin levels and thereby slow neurodegeneration in PD and other neurodegenerative conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model.

PubMed

Horowitz, Neil S; Hua, Jun; Gibb, Randall K; Mutch, David G; Herzog, Thomas J

2004-07-01

Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum. The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin. The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01). The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01). The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil

PubMed Central

Bryant, Victoria L.; Elias, Roy M.; McCarthy, Susan M.; Yeatman, Timothy J.; Alexandrow, Mark G.

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil (5-FU) are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as back-up initiation sites under conditions of replicative stress. The current study provides definitive evidence that co-suppression of the excess/back-up MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared to drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are non-tumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when co-suppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. Implications These studies demonstrate that suppressing the back-up complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers. PMID:26063742

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil.

PubMed

Bryant, Victoria L; Elias, Roy M; McCarthy, Susan M; Yeatman, Timothy J; Alexandrow, Mark G

2015-09-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as backup initiation sites under conditions of replicative stress. The current study provides definitive evidence that cosuppression of the excess/backup MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared with drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are nontumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when cosuppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. These studies demonstrate that suppressing the backup complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers. ©2015 American Association for Cancer Research.

High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

PubMed Central

Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription.

PubMed

Gómez-Herreros, Fernando; Zagnoli-Vieira, Guido; Ntai, Ioanna; Martínez-Macías, María Isabel; Anderson, Rhona M; Herrero-Ruíz, Andrés; Caldecott, Keith W

2017-08-10

DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones

PubMed Central

Malik, Muhammad; Mustaev, Arkady; Schwanz, Heidi A.; Luan, Gan; Shah, Nirali; Oppegard, Lisa M.; de Souza, Ernane C.; Hiasa, Hiroshi; Zhao, Xilin; Kerns, Robert J.; Drlica, Karl

2016-01-01

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance. PMID:26984528

MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required in stem cell regulation and to maintain developmentally regulated gene silencing.

PubMed

Graf, Philipp; Dolzblasz, Alicja; Würschum, Tobias; Lenhard, Michael; Pfreundt, Ulrike; Laux, Thomas

2010-03-01

Maintenance of stem cells in the Arabidopsis thaliana shoot meristem is regulated by signals from the underlying cells of the organizing center, provided through the transcription factor WUSCHEL (WUS). Here, we report the isolation of several independent mutants of MGOUN1 (MGO1) as genetic suppressors of ectopic WUS activity and enhancers of stem cell defects in hypomorphic wus alleles. mgo1 mutants have previously been reported to result in a delayed progression of meristem cells into differentiating organ primordia (Laufs et al., 1998). Genetic analyses indicate that MGO1 functions together with WUS in stem cell maintenance at all stages of shoot and floral meristems. Synergistic interactions of mgo1 with several chromatin mutants suggest that MGO1 affects gene expression together with chromatin remodeling pathways. In addition, the expression states of developmentally regulated genes are randomly switched in mgo1 in a mitotically inheritable way, indicating that MGO1 stabilizes epigenetic states against stochastically occurring changes. Positional cloning revealed that MGO1 encodes a putative type IB topoisomerase, which in animals and yeast has been shown to be required for regulation of DNA coiling during transcription and replication. The specific developmental defects in mgo1 mutants link topoisomerase IB function in Arabidopsis to stable propagation of developmentally regulated gene expression.

High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

PubMed

Ngoi, Soo Tein; Thong, Kwai Lin

2014-01-01

The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

Topoisomerase expression and amplification in solid tumours: Analysis of 24,262 patients

PubMed Central

Heestand, Gregory M.; Schwaederle, Maria; Gatalica, Zoran; Arguello, David; Kurzrock, Razelle

2017-01-01

Background Topoisomerase I (TOPO1) and topoisomerase IIα (TOP2A) are specific targets of multiple chemotherapy drugs. Increased expression of TOPO1 protein and amplification of the TOP2A gene have been associated with treatment response in colorectal and breast cancers, respectively. TOPO1 and TOP2A may be potential therapeutic targets in other malignancies as well. Summary of methods We analysed TOPO1 protein expression and TOP2A gene amplification in patients (n = 24,262 specimens) with diverse cancers. Since HER2 and TOP2A co-amplification have been investigated for predictive value regarding anthracycline benefit, we analysed specimens for HER2 amplification as well. Results Overexpressed TOPO1 protein was present in 51% of the tumours. Four percent of the tumours had TOP2A amplification, with gallbladder tumours and gastroesophageal/oesophageal tumours having rates over 10%. Overall, 4903 specimens were assessed for both TOP2A and HER2 amplification; 129 (2.6%) had co-amplification. High rates (>40%) of HER2 amplification were seen in patients with TOP2A amplification in breast, ovarian, gastroesophageal/oesophageal and pancreatic cancer. Conclusion Our data indicate that increased TOPO1 expression and TOP2A amplification, as well as HER2 co-alterations, are present in multiple malignancies. The implications of these observations regarding sensitivity to chemotherapy not traditionally administered to these tumour types merits investigation. PMID:28728050

Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage

PubMed Central

Narula, Gagandeep; Tse-Dinh, Yuk-Ching

2012-01-01

Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the −4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the −4 and the −5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the −4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the −4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein–DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway. PMID:22833607

Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity against breast cancer cell line MCF-7.

PubMed

Abdelhaleem, Eman F; Abdelhameid, Mohammed K; Kassab, Asmaa E; Kandeel, Manal M

2018-01-01

A series of novel tetrahydrobenzothieno[2,3-d]pyrimidine urea derivatives was synthesized according to fragment-based design strategy. They were evaluated for their anticancer activity against MCF-7 cell line. Three compounds 9c, 9d and 11b showed 1.5-1.03 folds more potent anticancer activity than doxorubicin. In this study, a promising multi-sited enzyme small molecule inhibitor 9c, which showed the most potent anti-proliferative activity, was identified. The anti-proliferative activity of this compound appears to correlate well with its ability to inhibit topoisomerase II (IC 50  = 9.29 μM). Moreover, compound 9c showed excellent VEGFR-2 inhibitory activity, at the sub-micromolar level with IC 50 value 0.2 μM, which is 2.1 folds more potent than sorafenib. Moreover, activation of damage response pathway of the DNA leads to cell cycle arrest at G2/M phase, accumulation of cells in pre-G1 phase and annexin-V and propidium iodide staining, indicating that cell death proceeds through an apoptotic mechanism. Compound 9c showed potent pro-apoptotic effect through induction of the intrinsic mitochondrial pathway of apoptosis. This mechanistic pathway was confirmed by a significant increase in the expression of the tumor suppressor gene p53, elevation in Bax/BCL-2 ratio and a significant increase in the level of active caspase-3. Quantitative structure-activity relationship (QSAR) studies delivered equations of five 3D descriptors with R 2  = 0.814. This QSAR model provides an effective technique for understanding the observed antitumor properties and thus could be adopted for developing effective lead structures. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Novel cholinesterase modulators and their ability to interact with DNA

NASA Astrophysics Data System (ADS)

Janockova, Jana; Gulasova, Zuzana; Musilek, Kamil; Kuca, Kamil; Kozurkova, Maria

2013-11-01

In the present work, an interaction of four cholinesterase modulators (1-4) with calf thymus DNA was studied via spectroscopic techniques (UV-Vis, fluorescent spectroscopy and circular dichroism). From UV-Vis spectroscopic analysis, the binding constants for DNA-pyridinium oximes complexes were calculated (K = 3.5 × 104 to 1.4 × 105 M-1). All these measurements indicated that the compounds behave as effective DNA-interacting agents. Electrophoretic techniques proved that ligand 2 inhibited topoisomerase I at a concentration 5 μM.

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus.

PubMed

Voisset, Cécile; Daskalogianni, Chrysoula; Contesse, Marie-Astrid; Mazars, Anne; Arbach, Hratch; Le Cann, Marie; Soubigou, Flavie; Apcher, Sébastien; Fåhraeus, Robin; Blondel, Marc

2014-04-01

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8(+) T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II-DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II-DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.

Phase Ib study of the mitochondrial inhibitor ME-344 plus topotecan in patients with previously treated, locally advanced or metastatic small cell lung, ovarian and cervical cancers.

PubMed

Diamond, Jennifer R; Goff, Barbara; Forster, Martin D; Bendell, Johanna C; Britten, Carolyn D; Gordon, Michael S; Gabra, Hani; Waterhouse, David M; Poole, Mark; Ross Camidge, D; Hamilton, Erika; Moore, Kathleen M

2017-10-01

Background This multicenter, open-label, phase Ib study was designed to assess the safety, pharmacokinetics and preliminary efficacy of ME-344, a mitochondrial inhibitor, administered in combination with the topoisomerase I inhibitor, topotecan, in patients with previously treated, locally advanced or metastatic small cell lung (SCLC), ovarian and cervical cancers. Patients and methods In Part 1, patients received ME-344 10 mg/kg intravenously weekly on days 1, 8, 15 and 22 in combination with topotecan 4 mg/m 2 on days 1, 8, and 15 of a 28 day cycle. Cycles were repeated until disease progression or unacceptable toxicity. Patients were evaluated for dose-limiting toxicity (DLT) in cycle 1 and ME-344 pharmacokinetic samples were obtained. In Part 2, patients with locally advanced or metastatic SCLC and ovarian cancer were enrolled in expansion cohorts treated at the recommended phase II dose (RP2D) determined in Part 1. Results Fourteen patients were enrolled in Part 1 and no DLTs were observed. The RP2D of ME-344 in combination with topotecan was established as 10 mg/kg. In Part 2, 32 patients were enrolled. The most common treatment-emergent all-grade and grade 3/4 toxicities included fatigue (65.2%, 6.5%), neutropenia (56.5%, 43.5%) and thrombocytopenia (50%, 23.9%). One patient with recurrent ovarian cancer experienced a partial response by RECIST 1.1 and 21 patients achieved stable disease as best response. Conclusions The combination of ME-344 10 mg/kg weekly and topotecan 4 mg/m 2 was tolerable, however, the degree of anti-cancer activity does not support further investigation of the combination in unselected patients with SCLC, ovarian and cervical cancers.

Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress.

PubMed

Desmarais, Joëlle A; Hoffmann, Michele J; Bingham, Gregg; Gagou, Mary E; Meuth, Mark; Andrews, Peter W

2012-07-01

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability. Copyright © 2012 AlphaMed Press.

Recent developments in the antiprotozoal and anticancer activities of the 2-alkynoic fatty acids

PubMed Central

Carballeira, Néstor M.

2013-01-01

The 2-alkynoic fatty acids are an interesting group of synthetic compounds that display antimycobacterial, antifungal, anticancer, and pesticidal activities but their antiprotozoal activity has received little attention until recently. In this review we have summarized our present knowledge of the biomedical potential of the 2-hexadecynoic acid (2-HDA) and 2-octadecynoic acid (2-ODA) together with several mechanistic pieces of work attesting to the fact that these compounds, and their metabolites, are good fatty acid biosynthesis inhibitors. The antiprotozoal activity of 2-HDA and 2-ODA against Leishmania donovani and Plasmodium falciparum, parasites responsible for visceral leishmaniasis and malaria, respectively, is also reviewed. The evidence obtained so far supports the fact that these fatty acids are good inhibitors of the L. donovani DNA topoisomerase IB enzyme (LdTopIB) and the potency of LdTopIB inhibition is chain length dependent. We also demonstrate the generality of the antiprotozoal activity of 2-HDA and 2-ODA against P. falciparum, and review our present knowledge of their inhibition of key P. falciparum enzymes such as PfFabZ, PfFabG, and PfFabI together with some possible modes of inhibition. Recent research by our group has also demonstrated that 2-ODA displays antineoplastic activity, specifically against the neuroblastoma SH-SY5Y cell line via lactate dehydrogenase (LDH) release, which is a cell death mechanism principally associated to necrosis. This is the first comprehensive review of the medicinal chemistry of this interesting group of acetylenic fatty acids. PMID:23727443

Recent developments in the antiprotozoal and anticancer activities of the 2-alkynoic fatty acids.

PubMed

Carballeira, Néstor M

2013-01-01

The 2-alkynoic fatty acids are an interesting group of synthetic compounds that display antimycobacterial, antifungal, anticancer, and pesticidal activities but their antiprotozoal activity has received little attention until recently. In this review we have summarized our present knowledge of the biomedical potential of the 2-hexadecynoic acid (2-HDA) and 2-octadecynoic acid (2-ODA) together with several mechanistic pieces of work attesting to the fact that these compounds, and their metabolites, are good fatty acid biosynthesis inhibitors. The antiprotozoal activity of 2-HDA and 2-ODA against Leishmania donovani and Plasmodium falciparum, parasites responsible for visceral leishmaniasis and malaria, respectively, is also reviewed. The evidence obtained so far supports the fact that these fatty acids are good inhibitors of the L. donovani DNA topoisomerase IB enzyme (LdTopIB) and the potency of LdTopIB inhibition is chain length dependent. We also demonstrate the generality of the antiprotozoal activity of 2-HDA and 2-ODA against P. falciparum, and review our present knowledge of their inhibition of key P. falciparum enzymes such as PfFabZ, PfFabG, and PfFabI together with some possible modes of inhibition. Recent research by our group has also demonstrated that 2-ODA displays antineoplastic activity, specifically against the neuroblastoma SH-SY5Y cell line via lactate dehydrogenase (LDH) release, which is a cell death mechanism principally associated to necrosis. This is the first comprehensive review of the medicinal chemistry of this interesting group of acetylenic fatty acids. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

TNF Signaling through RIP1 Kinase Enhances SN38-Induced Death in Colon Adenocarcinoma.

PubMed

Cabal-Hierro, Lucia; O'Dwyer, Peter J

2017-04-01

Elucidation of TNF-directed mechanisms for cell death induction and maintenance of tumor growth has revealed a role for receptor-interacting protein kinases 1 and 3 (RIPK1/RIP1 and RIPK3/RIP3), components of the necrosome complex, as determinants of cell fate. Here, the participation of TNF signaling was analyzed with regard to the cytotoxic action of different DNA-damaging agents in a panel of colon cancer cells. While most of these cell lines were insensitive to TNF, combination with these drugs increased sensitivity by inducing cell death and DNA damage, especially in the case of the topoisomerase inhibitor SN38. Changes in levels of RIP1 and RIP3 occurred following monotherapy with SN38 or in combination with TNF. Downregulation of RIP1 resulted in increased resistance to SN38, implying a requirement for RIP1 in mediating cytotoxicity through the TNF/TNFR signaling pathway. Downregulation of RIP1 in a xenograft model impaired tumor growth inhibition from SN38 treatment, suggesting the potential of RIP1 to determine the clinical outcome of irinotecan treatment. These results indicate that TNF plays a key role in determining the cytotoxic effectiveness of SN38 in colorectal cancer and suggests a re-evaluation of TNF-based interventions to enhance therapeutic efficacy. Implications: The capacity of RIP1 to influence drug sensitivity suggests RIP1 may have biomarker potential. Mol Cancer Res; 15(4); 395-404. ©2017 AACR . ©2017 American Association for Cancer Research.

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

PubMed

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.

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

PubMed Central

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

Effects of an unusual poison identify a lifespan role for Topoisomerase 2 in Saccharomyces cerevisiae.

PubMed

Tombline, Gregory; Millen, Jonathan I; Polevoda, Bogdan; Rapaport, Matan; Baxter, Bonnie; Van Meter, Michael; Gilbertson, Matthew; Madrey, Joe; Piazza, Gary A; Rasmussen, Lynn; Wennerberg, Krister; White, E Lucile; Nitiss, John L; Goldfarb, David S

2017-01-05

A progressive loss of genome maintenance has been implicated as both a cause and consequence of aging. Here we present evidence supporting the hypothesis that an age-associated decay in genome maintenance promotes aging in Saccharomyces cerevisiae (yeast) due to an inability to sense or repair DNA damage by topoisomerase 2 (yTop2). We describe the characterization of LS1, identified in a high throughput screen for small molecules that shorten the replicative lifespan of yeast. LS1 accelerates aging without affecting proliferative growth or viability. Genetic and biochemical criteria reveal LS1 to be a weak Top2 poison. Top2 poisons induce the accumulation of covalent Top2-linked DNA double strand breaks that, if left unrepaired, lead to genome instability and death. LS1 is toxic to cells deficient in homologous recombination, suggesting that the damage it induces is normally mitigated by genome maintenance systems. The essential roles of yTop2 in proliferating cells may come with a fitness trade-off in older cells that are less able to sense or repair yTop2-mediated DNA damage. Consistent with this idea, cells live longer when yTop2 expression levels are reduced. These results identify intrinsic yTop2-mediated DNA damage as potentially manageable cause of aging.

Clinical associations of anticentromere antibodies and antibodies to topoisomerase I. A study of 355 patients.

PubMed

Weiner, E S; Earnshaw, W C; Senécal, J L; Bordwell, B; Johnson, P; Rothfield, N F

1988-03-01

Anticentromere antibodies (ACA) and anti-topoisomerase I (anti-topo I) were assayed in serum samples from 355 patients: 89 with proximal scleroderma; 54 with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias), without proximal scleroderma; 154 with primary and secondary Raynaud's disease; and 58 with other rheumatic diseases, without Raynaud's disease. Sera from healthy control subjects were also assayed. Using immunoblotting techniques, anti-topo I was detected in 28% of the patients with proximal scleroderma; using immunodiffusion techniques, this antibody was found in only 20% of the same group of patients. Anti-topo I and ACA were found primarily in patients with scleroderma, CREST syndrome, and Raynaud's phenomenon. ACA identified patients with less severe disease, whereas anti-topo I identified patients with skin and cardiac involvement and patients with malignancies.

Arabidopsis thaliana GYRB3 Does Not Encode a DNA Gyrase Subunit

PubMed Central

Evans-Roberts, Katherine M.; Breuer, Christian; Wall, Melisa K.; Sugimoto-Shirasu, Keiko; Maxwell, Anthony

2010-01-01

Background DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. Methodology/Principal Findings We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer. Conclusions/Significance These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen. PMID:20360860

Arabidopsis thaliana GYRB3 does not encode a DNA gyrase subunit.

PubMed

Evans-Roberts, Katherine M; Breuer, Christian; Wall, Melisa K; Sugimoto-Shirasu, Keiko; Maxwell, Anthony

2010-03-26

DNA topoisomerases are enzymes that control the topology of DNA in all cells. DNA gyrase is unique among the topoisomerases in that it is the only enzyme that can actively supercoil DNA using the free energy of ATP hydrolysis. Until recently gyrase was thought to be unique to bacteria, but has now been discovered in plants. The genome of the model plant, Arabidopsis thaliana, is predicted to encode four gyrase subunits: AtGyrA, AtGyrB1, AtGyrB2 and AtGyrB3. We found, contrary to previous data, that AtGyrB3 is not essential to the survival of A. thaliana. Bioinformatic analysis suggests AtGyrB3 is considerably shorter than other gyrase B subunits, lacking part of the ATPase domain and other key motifs found in all type II topoisomerases; but it does contain a putative DNA-binding domain. Partially purified AtGyrB3 cannot bind E. coli GyrA or support supercoiling. AtGyrB3 cannot complement an E. coli gyrB temperature-sensitive strain, whereas AtGyrB2 can. Yeast two-hybrid analysis suggests that AtGyrB3 cannot bind to AtGyrA or form a dimer. These data strongly suggest that AtGyrB3 is not a gyrase subunit but has another unknown function. One possibility is that it is a nuclear protein with a role in meiosis in pollen.

Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2‧-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity

NASA Astrophysics Data System (ADS)

Hasan, Md. Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

2016-01-01

Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2 = 3-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2 = 4-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy = 2,2‧-bipyridine, Phen = 1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH ∼ 7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4 > 3 > 1 > 2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4‧,6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87 μM respectively.

State of the art and perspectives in the treatment of glioblastoma.

PubMed

Grimm, Sean A; Chamberlain, Marc C

2012-09-01

Glioblastoma is the most common malignant primary brain tumor. Cures are rare and median survival varies from several to 22 months. Standard treatment for good performance patients consists of maximal safe surgical resection followed by radiotherapy with concurrent temozolomide (TMZ) chemotherapy and six cycles of postradiotherapy TMZ. At recurrence, treatment options include repeat surgery (with or without Gliadel wafer placement), reirradiation or systemic therapy. Most patients with good performance status are treated with cytotoxic chemotherapy or targeted biologic therapy following or in lieu of repeat surgery. Cytotoxic chemotherapy options include nitrosoureas, rechallenge with TMZ, platins, phophoramides and topoisomerase inhibitors, although efficacy is limited. Despite the intense effort of developing biologic agents that target angiogenesis and growth and proliferative pathways, bevacizumab is the only agent that has shown efficacy in clinical trials. It was awarded accelerated approval in the USA after demonstrating an impressive radiographic response in two open-label, prospective Phase II studies. Two randomized, Phase III trials of upfront bevacizumab have completed and may demonstrate survival benefit; however, results are pending at this time. Given the limited treatment options at tumor recurrence, consideration for enrollment on a clinical trial is encouraged.

Monocyte-specific Accessibility of a Matrix Attachment Region in the Tumor Necrosis Factor Locus*

PubMed Central

Biglione, Sebastian; Tsytsykova, Alla V.; Goldfeld, Anne E.

2011-01-01

Regulation of TNF gene expression is cell type- and stimulus-specific. We have previously identified highly conserved noncoding regulatory elements within DNase I-hypersensitive sites (HSS) located 9 kb upstream (HSS−9) and 3 kb downstream (HSS+3) of the TNF gene, which play an important role in the transcriptional regulation of TNF in T cells. They act as enhancers and interact with the TNF promoter and with each other, generating a higher order chromatin structure. Here, we report a novel monocyte-specific AT-rich DNase I-hypersensitive element located 7 kb upstream of the TNF gene (HSS−7), which serves as a matrix attachment region in monocytes. We show that HSS−7 associates with topoisomerase IIα (Top2) in vivo and that induction of endogenous TNF mRNA expression is suppressed by etoposide, a Top2 inhibitor. Moreover, Top2 binds to and cleaves HSS−7 in in vitro analysis. Thus, HSS−7, which is selectively accessible in monocytes, can tether the TNF locus to the nuclear matrix via matrix attachment region formation, potentially promoting TNF gene expression by acting as a Top2 substrate. PMID:22027829

Frequent topoisomerase IV mutations associated with fluoroquinolone resistance in Ureaplasma species.

PubMed

Song, Jingjuan; Qiao, Yingli; Kong, Yingying; Ruan, Zhi; Huang, Jun; Song, Tiejun; Zhang, Jun; Xie, Xinyou

2015-11-01

This study aimed to investigate the role of quinolone resistance-determining regions (QRDRs) of DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) associated with fluoroquinolone resistance. A total of 114 Ureaplasma spp. strains, isolated from clinical female patients with symptomatic infection, were tested for species distribution and susceptibility to four fluoroquinolones. Moreover, we analysed the QRDRs and compared these with 14 ATCC reference strains of Ureaplasma spp. serovars to identify mutations that caused antimicrobial resistance. Our study indicated that moxifloxacin was the most effective fluoroquinolone against Ureaplasma spp. (MIC range: 0.125-32 μg ml⁻¹). However, extremely high MICs were estimated for ciprofloxacin (MIC range: 1-256 μg ml⁻¹) and ofloxacin (MIC range: 0.5-128 μg ml⁻¹), followed by levofloxacin (MIC range: 0.5-64 μg ml⁻¹). Seven amino acid substitutions were discovered in GyrB, ParC and ParE, but not in GyrA. Ser-83 → Leu/Trp (C248T/G) in ParC and Arg-448 → Lys (G1343A) in ParE, which were potentially responsible for fluoroquinolone resistance, were observed in 89 (77.2 %) and three (2.6 %) strains, respectively. Pro-462 → Ser (C1384T), Asn-481 → Ser (A1442G) and Ala-493 → Val (C1478T) in GyrB and Met-105 → Ile (G315T) in ParC seemed to be neutral polymorphisms, and were observed and occurred along with the amino acid change of Ser-83 → Leu (C248T) in ParC. Interestingly, two novel mutations of ParC and ParE were independently found in four strains. These observations suggest that amino acid mutation in topoisomerase IV appears to be the leading cause of fluoroquinolone resistance, especially the mutation of Ser-83 → Leu (C248T) in ParC. Moxifloxacin had the best activity against strains with Ser-83 → Leu mutation.

Quercetin exerts synergetic anti-cancer activity with 10-hydroxy camptothecin.

PubMed

Tang, Qin; Ji, Fangling; Wang, Jingyun; Guo, Lianying; Li, Yachen; Bao, Yongming

2017-11-15

Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤10μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser 139 of γH2A.X in MCF7 cells starts to increase significantly (P<0.05) at 6h when treated by the combination of 10μM Qu and 0.62μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12h to 48h via up-regulation of p21 and induces apoptosis at 24h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage. Copyright © 2017. Published by Elsevier B.V.

A HER2-targeting antibody-drug conjugate, trastuzumab deruxtecan (DS-8201a), enhances antitumor immunity in a mouse model.

PubMed

Iwata, Tomomi Nakayama; Ishii, Chiaki; Ishida, Saori; Ogitani, Yusuke; Wada, Teiji; Agatsuma, Toshinori

2018-04-27

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate with a topoisomerase I inhibitor exatecan derivative (DX-8951 derivative, DXd), has been reported to exert potent antitumor effects in xenograft mouse models and clinical trials. In this study, the immune system-activating ability of DS-8201a was assessed. DS-8201a significantly suppressed tumor growth in an immunocompetent mouse model with human HER2-expressing CT26.WT (CT26.WT-hHER2) cells. Cured immunocompetent mice rejected not only re-challenged CT26.WT-hHER2 cells, but also CT26.WT-mock cells. Splenocytes from the cured mice responded to both CT26.WT-hHER2 and CT26.WT-mock cells. Further analyses revealed that DXd up-regulated CD86 expression on bone marrow-derived DCs in vitro, and that DS-8201a increased tumor-infiltrating DCs and up-regulated their CD86 expression in vivo. DS-8201a also increased tumor-infiltrating CD8+ T cells and enhanced PD-L1 and MHC class I expression on tumor cells. Furthermore, combination therapy with DS-8201a and anti-PD-1 antibody was more effective than either monotherapy. In conclusion, DS-8201a enhanced antitumor immunity, as evidenced by the increased expression of DC markers, augmented expression of MHC class I in tumor cells, and rejection of re-challenged tumor cells by adaptive immune cells, suggesting that DS-8201a enhanced tumor recognition by T cells. Furthermore, DS-8201a treatment benefited from combination with anti-PD-1 antibody, possibly due to increased T cell activity and up-regulated PD-L1 expression induced by DS-8201a. Copyright ©2018, American Association for Cancer Research.

The benzene metabolite, hydroquinone and etoposide both induce endoreduplication in human lymphoblastoid TK6 cells

PubMed Central

Ji, Zhiying; Zhang, Luoping; Guo, Weihong; McHale, Cliona M.; Smith, Martyn T.

2009-01-01

Both occupational exposure to the leukemogen benzene and in vitro exposure to its metabolite hydroquinone (HQ) lead to the induction of numerical and structural chromosome changes. Several studies have shown that HQ can form DNA adducts, disrupt microtubule assembly and inhibit DNA topoisomerase II (topo II) activity. As these are potential mechanisms underlying endoreduplication (END), a phenomenon that involves DNA amplification without corresponding cell division, we hypothesized that HQ could cause END. We measured END in the human lymphoblastoid cell line, TK6, treated with HQ (0–20 μM) and etoposide (0–0.2 μM) for 48 h. Etoposide was used as a positive control as it is a topo II poison and established human leukemogen that has previously been shown to induce END in Chinese hamster ovary cells. Both HQ and etoposide significantly induced END in a dose-dependent manner (Ptrend < 0.0001 and Ptrend = 0.0003, respectively). Since END may underlie the acquisition of high chromosome numbers by tumour cells, it may play a role in inducing genomic instability and subsequent carcinogenesis from HQ and etoposide. In order to further explore the cytogenetic effects of HQ and etoposide, we also examined specific structural changes. HQ did not induce translocations of chromosome 11 [t(11;?)] but significantly induced translocations of chromosome 21 [t(21;?)] and structural chromosome aberrations (SCA) (Ptrend = 0.0415 and Ptrend < 0.0001, respectively). Etoposide potently induced all these structural changes (Ptrend < 0.0001). The lack of an effect of HQ on t(11;?) and the reduced ability of HQ to induce t(21;?) and SCA, compared with etoposide, further suggests that HQ acts primarily as a topo II catalytic inhibitor rather than as a topo II poison in intact human cells. PMID:19491217

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway.

PubMed

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata , was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately.

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway

PubMed Central

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Background Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. Materials and methods U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Results Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. Conclusion The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately. PMID:29785137

Effective conversion of irinotecan to SN-38 after intratumoral drug delivery to an intracranial murine glioma model in vivo. Laboratory investigation.

PubMed

Wang, Weijun; Ghandi, Alex; Liebes, Leonard; Louie, Stan G; Hofman, Florence M; Schönthal, Axel H; Chen, Thomas C

2011-03-01

Irinotecan (CPT-11), a topoisomerase I inhibitor, is a cytotoxic agent with activity against malignant gliomas and other tumors. After systemic delivery, CPT-11 is converted to its active metabolite, SN-38, which displays significantly higher cytotoxic potency. However, the achievement of therapeutically effective plasma levels of CPT-11 and SN-38 is seriously complicated by variables that affect drug metabolism in the liver. Thus the capacity of CPT-11 to be converted to the active SN38 intratumorally in gliomas was addressed. For in vitro studies, 2 glioma cell lines, U87 and U251, were tested to determine the cytotoxic effects of CPT-11 and SN-38 in a dose-dependent manner. In vivo studies were performed by implanting U87 intracranially into athymic/nude mice. For a period of 2 weeks, SN-38, CPT-11, or vehicle was administered intratumorally by means of an osmotic minipump. One series of experiments measured the presence of SN-38 or CPT-11 in the tumor and surrounding brain tissues after 2 weeks' exposure to the drug. In a second series of experiments, after 2 weeks' exposure to the drug, the animals were maintained, in the absence of drug, until death. The survival curves were then calculated. The results show that the animals that had CPT-11 delivered intratumorally by the minipump expressed SN-38 in vivo. Furthermore, both CPT-11 and SN-38 accumulated at higher levels in tumor tissues compared with uninvolved brain. Intratumoral delivery of CPT-11 or SN-38 extended the average survival time of tumor-bearing animals from 22 days to 46 and 65 days, respectively. These results demonstrate that intratumorally administered CPT-11 can be effectively converted to SN-38 and this method of drug delivery is effective in extending the survival time of animals bearing malignant gliomas.

Dysregulation of RNA Interference in Breast Cancer

DTIC Science & Technology

2007-07-01

of miRNA complementary elements in 3-UTR of target mRNAs, the concentration in the seed (6–8 bp) of continuous Watson - Crick base pairing in the 5...treated the transfected cells with the anticancer drug topotecan (TPT) that is known to inhibit DNA topoisomerase I and cause DNA damage (Tanizawa et...as DNA damage caused by TPT, can increase the inhibitory effect mediated by R el at iv e C el l G ro w th R el at iv e C el l G ro w th Negative

Biomolecular structure manipulation using tailored electromagnetic radiation: a proof of concept on a simplified model of the active site of bacterial DNA topoisomerase.

PubMed

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay

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 analysesmore » 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.« less

Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases

PubMed Central

Pavlov, Andrey R.; Belova, Galina I.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2002-01-01

Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes. PMID:12368475

Triggering autophagic cell death with a di-manganese(II) developmental therapeutic.

PubMed

Slator, Creina; Molphy, Zara; McKee, Vickie; Kellett, Andrew

2017-08-01

There is an unmet need for novel metal-based chemotherapeutics with alternative modes of action compared to clinical agents such as cisplatin and metallo-bleomycin. Recent attention in this field has focused on designing intracellular ROS-mediators as powerful cytotoxins of human cancers and identifying potentially unique toxic mechanisms underpinning their utility. Herein, we report the developmental di-manganese(II) therapeutic [Mn 2 (μ-oda)(phen) 4 (H 2 O) 2 ][Mn 2 (μ-oda)(phen) 4 (oda) 2 ]·4H 2 O (Mn-Oda) induces autophagy-promoted apoptosis in human ovarian cancer cells (SKOV3). The complex was initially identified to intercalate DNA by topoisomerase I unwinding and circular dichroism spectroscopy. Intracellular DNA damage, detected by γH2AX and the COMET assay, however, is not linked to direct Mn-Oda free radical generation, but is instead mediated through the promotion of intracellular reactive oxygen species (ROS) leading to autophagic vacuole formation and downstream nuclear degradation. To elucidate the cytotoxic profile of Mn-Oda, a wide range of biomarkers specific to apoptosis and autophagy including caspase release, mitochondrial membrane integrity, fluorogenic probe localisation, and cell cycle analysis were employed. Through these techniques, the activity of Mn-Oda was compared directly to i.) the pro-apoptotic clinical anticancer drug doxorubicin, ii.) the multimodal histone deacetylase inhibitor suberoyanilide hydroxamic acid, and iii.) the autophagy inducer rapamycin. In conjunction with ROS-specific trapping agents and established inhibitors of autophagy, we have identified autophagy-induction linked to mitochondrial superoxide production, with confocal image analysis of SKOV3 cells further supporting autophagosome formation. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of an unusual poison identify a lifespan role for Topoisomerase 2 in Saccharomyces cerevisiae

PubMed Central

Polevoda, Bogdan; Rapaport, Matan; Baxter, Bonnie; Van Meter, Michael; Gilbertson, Matthew; Madrey, Joe; Piazza, Gary A.; Rasmussen, Lynn; Wennerberg, Krister; White, E. Lucile; Nitiss, John L.; Goldfarb, David S.

2017-01-01

A progressive loss of genome maintenance has been implicated as both a cause and consequence of aging. Here we present evidence supporting the hypothesis that an age-associated decay in genome maintenance promotes aging in Saccharomyces cerevisiae (yeast) due to an inability to sense or repair DNA damage by topoisomerase 2 (yTop2). We describe the characterization of LS1, identified in a high throughput screen for small molecules that shorten the replicative lifespan of yeast. LS1 accelerates aging without affecting proliferative growth or viability. Genetic and biochemical criteria reveal LS1 to be a weak Top2 poison. Top2 poisons induce the accumulation of covalent Top2-linked DNA double strand breaks that, if left unrepaired, lead to genome instability and death. LS1 is toxic to cells deficient in homologous recombination, suggesting that the damage it induces is normally mitigated by genome maintenance systems. The essential roles of yTop2 in proliferating cells may come with a fitness trade-off in older cells that are less able to sense or repair yTop2-mediated DNA damage. Consistent with this idea, cells live longer when yTop2 expression levels are reduced. These results identify intrinsic yTop2-mediated DNA damage as a potentially manageable cause of aging. PMID:28077781

Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

PubMed

Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J; Reifenberger, Guido; Büsselberg, Dietrich

2017-04-04

Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 μM) or TOPO (0.1 nM-1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

Genome-wide association study of chemotherapeutic agent-induced severe neutropenia/leucopenia for patients in Biobank Japan.

PubMed

Low, Siew-Kee; Chung, Suyoun; Takahashi, Atsushi; Zembutsu, Hitoshi; Mushiroda, Taisei; Kubo, Michiaki; Nakamura, Yusuke

2013-08-01

Chemotherapeutic agents are notoriously known to have a narrow therapeutic range that often results in life-threatening toxicity. Hence, it is clinically important to identify the patients who are at high risk for severe toxicity to certain chemotherapy through a pharmacogenomics approach. In this study, we carried out multiple genome-wide association studies (GWAS) of 13 122 cancer patients who received different chemotherapy regimens, including cyclophosphamide- and platinum-based (cisplatin and carboplatin), anthracycline-based (doxorubicin and epirubicin), and antimetabolite-based (5-fluorouracil and gemcitabine) treatment, antimicrotubule agents (paclitaxel and docetaxel), and topoisomerase inhibitors (camptothecin and etoposide), as well as combination therapy with paclitaxel and carboplatin, to identify genetic variants that are associated with the risk of severe neutropenia/leucopenia in the Japanese population. In addition, we used a weighted genetic risk scoring system to evaluate the cumulative effects of the suggestive genetic variants identified from GWAS in order to predict the risk levels of individuals who carry multiple risk alleles. Although we failed to identify genetic variants that surpassed the genome-wide significance level (P < 5.0 × 10(-8) ) through GWAS, probably due to insufficient statistical power and complex clinical features, we were able to shortlist some of the suggestive associated loci. The current study is at the relatively preliminary stage, but does highlight the complexity and problematic issues associated with retrospective pharmacogenomics studies. However, we hope that verification of these genetic variants through local and international collaborations could improve the clinical outcome for cancer patients. © 2013 Japanese Cancer Association.

Effectiveness of imatinib mesylate over etoposide in the treatment of sensitive and resistant chronic myeloid leukaemia cells in vitro.

PubMed

Husaini, Roslina; Ahmad, Munirah; Zakaria, Zubaidah

2017-06-01

Chronic myeloid leukaemia (CML) is a form of leukaemia derived from the myeloid cell lineage. Imatinib mesylate, the breakpoint cluster region-abelson murine leukeamia kinase inhibitor, is a specific reagent used in the clinical treatment of CML. The DNA topoisomerase II inhibitor, etoposide, is also employed as a therapeutic, though it is used to a lesser extent. The present study aims to evaluate the effects of CML-targeted therapy, utilising imatinib mesylate and etoposide in the in vitro treatment of parental sensitive and adriamycin-resistant CML in the K562 and K562/ADM cell lines, respectively. Preliminary work involved the screening of multidrug resistant (MDR) gene expression, including MDR1, MRP1 and B-cell lymphoma 2 (BCL-2) at the mRNA levels. The sensitive and resistant CML cell lines expressed the MRP1 gene, though the sensitive K562 cells expressed low, almost undetectable levels of MDR1 and BCL-2 genes relative to the K562/ADM cells. Following treatment with imatinib mesylate or etoposide, the IC50 for imatinib mesylate did not differ between the sensitive and resistant cell lines (0.492±0.024 and 0.378±0.029, respectively), indicating that imatinib mesylate is effective in the treatment of CML regardless of cell chemosensitivity. However, the IC50 for etoposide in sensitive K562 cells was markedly lower than that of K562/ADM cells (50.6±16.5 and 194±8.46 µM, respectively), suggesting that the higher expression levels of MDR1 and/or BCL-2 mRNA in resistant cells may be partially responsible for this effect. This is supported by terminal deoxynucleotidyl transferase dUTP nick-end labeling data, whereby a higher percentage of apoptotic cells were found in the sensitive and resistant K562 cells treated with imatinib mesylate (29.3±0.2 and 31.9±16.7%, respectively), whereas etoposide caused significant apoptosis of sensitive K562 cells (18.3±8.35%) relative to K562/ADM cells (5.17±3.3%). In addition, the MDR genes in K562/ADM cells were knocked down by short interfering RNAs. The percentage knockdowns were 15.4% for MRP1, 17.8% for MDR and 30.7% for BCL-2, which resulted in a non-significant difference in the half maximal inhibitory concentration value of K562/ADM cells relative to K562 cells upon treatment with etoposide.

Multidrug-resistant Staphylococcus haemolyticus isolates from infected eyes and healthy conjunctivae in India.

PubMed

Panda, Sasmita; Kar, Sarita; Sharma, Savitri; Singh, Durg V

2016-09-01

This study aimed to determine the presence of antibiotic resistance genes (ARGs), SCCmec elements and genetic relatedness among Staphylococcus haemolyticus isolated from patients with a variety of eye infections (n=11) and from healthy conjunctiva (n=7). Minimum inhibitory concentrations were determined for 14 antimicrobials according to BSAC guidelines. PCR was used to identify the presence of mecA, mecC, SCCmec type and ARGs. Sequencing was used to determine mutations in gyrA, gyrB, topoisomerase IVA and IVB genes. Genetic relatedness was determined by PFGE. Of the 18 isolates, 17 showed resistance to at least one antibiotic, but none showed resistance to vancomycin or rifampicin. Ten isolates were oxacillin-resistant and carried the mecA gene, eight of which belonged to SCCmec type V. The presence of non-mec SCC elements in two meticillin-susceptible isolates and untypeable SCC elements in meticillin-resistant isolates suggests the involvement of S. haemolyticus in the diversification of SCC elements. Sequence analysis revealed point mutations in gyrA (Ser-84→Leu) and topoisomerase IVA genes (Ser-80→Leu) in 13 isolates, and additional variation in the QRDR (Asp-84→Asn) of two isolates, showing good correlation between mutations in gyrA and topoisomerase IV genes and the level of resistance to fluoroquinolones. PFGE analysis showed distinct pulsotypes forming two major clusters, indicating the existence of diversity among isolates, irrespective of the source of isolation. This study suggests that S. haemolyticus isolates from infected eyes and healthy conjunctivae invariably carried ARGs and SCCmec elements and showed diversity in their genomic content, irrespective of the source of isolation. Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

[Fluoroquinolones and Gram-negative bacteria: antimicrobial activity and mechanisms of resistance].

PubMed

Luzzaro, F

2008-04-01

Fluoroquinolones acts by interacting with type II topoisomerases (DNA gyrase and topoisomerases IV). Related to this mechanism of action, bacteria have developed resistance mechanisms consisting in some target mutations (GyrA/GyrB for DNA gyrase and ParC/ParE for topoisomerase IV) or in a reduced access to the target itself, by either decreased permeability or augmented expression of efflux pumps, such as AcrAB and MexAB. Along with these classical mechanisms of chromosomal resistance, the presence of fluoroquinolones resistant proteins (Qnr) has been recently evidenced, codified by transmissible genes by means of plasmids, especially in Enterobacter spp., Escherichia coli and Klebsiella pneumoniae, whereas Proteus mirabilis and non fermenter Gram-negative, like Acinetobacter spp. and Pseudomonas aeruginosa, are not involved in such a kind of resistance. Qnr proteins determine a slight increase in MIC values, which often remains below the susceptibility breakpoint. More relevant is their impact on MPC values. Additionally, new specific resistance mechanisms have been described. AAC(6')-Ib-cr represents the first enzyme able to inactivate, by acetylation, antimicrobials of two different classes, aminoglycosides and fluoroquinolones. However, ciprofloxacin and norfloxacin, but not levofloxacin, are susceptible to this enzyme action. Finally, the presence of another resistance mechanism has been reported, an efflux-pump plasmid-mediated, codified by the QepA gene, which acts by a selective mechanism. Only hydrophilic fluoroquinolones, i.e. norfloxacin and ciprofloxacin, but not all the other ones, i.e. levofloxacin, moxifloxacin, etc, are affected by this mechanism. In the light of these new information, it is clear that, in terms of bacterial resistance, it is not any more possible to assimilate one fluoroquinolones to another, since different molecules can be diversely active, due to the specific resistance mechanism.

Mechanistic review of drug-induced steatohepatitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schumacher, Justin D., E-mail: Justin.d.schumacher@rutgers.edu; Guo, Grace L.

Drug-induced steatohepatitis is a rare form of liver injury known to be caused by only a handful of compounds. These compounds stimulate the development of steatohepatitis through their toxicity to hepatocyte mitochondria; inhibition of beta-oxidation, mitochondrial respiration, and/or oxidative phosphorylation. Other mechanisms discussed include the disruption of phospholipid metabolism in lysosomes, prevention of lipid egress from hepatocytes, targeting mitochondrial DNA and topoisomerase, decreasing intestinal barrier function, activation of the adenosine pathway, increasing fatty acid synthesis, and sequestration of coenzyme A. It has been found that the majority of compounds that induce steatohepatitis have cationic amphiphilic structures; a lipophilic ring structuremore » with a side chain containing a cationic secondary or tertiary amine. Within the last decade, the ability of many chemotherapeutics to cause steatohepatitis has become more evident coining the term chemotherapy-associated steatohepatitis (CASH). The mechanisms behind drug-induced steatohepatitis are discussed with a focus on cationic amphiphilic drugs and chemotherapeutic agents. - Highlights: • Reviewed the mechanisms underlying drug-induced steatohepatitis for many compounds • Mitochondrial dysfunction is critical in the development of drug-induced steatohepatitis. • Majority of drugs that induce steatohepatitis are cationic amphiphilic drugs. • Chemotherapeutics that induce CASH are cationic amphiphilic drugs. • Majority of drugs that induce steatohepatitis are carnitine palmitoyltransferase-I inhibitors.« less

Discovery of novel immunostimulants by dendritic-cell–based functional screening

PubMed Central

Mizumoto, Norikatsu; Gao, Jimin; Matsushima, Hironori; Ogawa, Yasushi; Tanaka, Hiroaki; Takashima, Akira

2005-01-01

Immunostimulants represent an emerging class of drugs for the treatment of infectious disorders and cancer. CpG oligonucleotides and imiquimod, prototypic drugs in this category, are now known to activate dendritic cells (DCs). Here we report the development of a highly sensitive, unbiased functional screen to detect DC-stimulatory signals. Because interleukin-1β (IL-1β) mRNA expression is closely associated with DC activation, we engineered DCs to stably express a fluorescent marker gene under the control of IL-1β promoter. By screening about 3000 compounds with the resulting DC biosensor clone, we identified DC-stimulatory potentials of topoisomerase I inhibitors (camptothecin derivatives) and microtubule depolymerizing drugs (colchicine and podophyllotoxin). In response to treatment with each agent, bone marrow–derived DC preparations exhibited characteristic phenotypic and/or functional changes associated with DC activation. All of these agents also triggered nuclear factor–κB (NFκB) activation in DCs, suggesting a common pharmacologic mechanism of action. Furthermore, locally administered colchicine induced in situ maturation and migration of DCs and augmented both humoral and cellular immune responses. These results support the practical utility of the DC-based biosensor system to discover novel DC-targeted immunostimulants and unveil previously unrecognized (and totally unexpected) pharmacologic activities of several drugs that are commonly used for the treatment of various disorders. PMID:16002424

Mechanisms and chemical induction of aneuploidy in rodent germ cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mailhes, J B; Marchetti, F

The objective of this review is to suggest that the advances being made in our understanding of the molecular events surrounding chromosome segregation in non-mammalian and somatic cell models be considered when designing experiments for studying aneuploidy in mammalian germ cells. Accurate chromosome segregation requires the temporal control and unique interactions among a vast array of proteins and cellular organelles. Abnormal function and temporal disarray among these, and others to be inidentified, biochemical reactions and cellular organelles have the potential for predisposing cells to aneuploidy. Although numerous studies have demonstrated that certain chemicals (mainly those that alter microtubule function) canmore » induce aneuploidy in mammalian germ cells, it seems relevant to point out that such data can be influenced by gender, meiotic stage, and time of cell-fixation post-treatment. Additionally, a consensus has not been reached regarding which of several germ cell aneuploidy assays most accurately reflects the human condition. More recent studies have shown that certain kinase, phosphatase, proteasome, and topoisomerase inhibitors can also induce aneuploidy in rodent germ cells. We suggest that molecular approaches be prudently incorporated into mammalian germ cell aneuploidy research in order to eventually understand the causes and mechanisms of human aneuploidy. Such an enormous undertaking would benefit from collaboration among scientists representing several disciplines.« less

Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

PubMed

Lebel, Michel

2002-01-01

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

Randomized study of sequential cisplatin-topotecan/carboplatin-paclitaxel versus carboplatin-paclitaxel: effects on quality of life.

PubMed

Brotto, Lori; Brundage, Michael; Hoskins, Paul; Vergote, Ignace; Cervantes, Andres; Casado, Herraez A; Poveda, A; Eisenhauer, Elizabeth; Tu, Dongsheng

2016-03-01

A recent phase III trial compared the efficacy of cisplatin-topotecan (a topoisomerase I inhibitor) followed by carboplatin-paclitaxel (Arm 1) versus paclitaxel-carboplatin (Arm 2) in women with newly diagnosed stage IIB or greater ovarian cancer. There was a significantly lower response rate in the experimental arm compared to standard treatment, and less likelihood of normalized CA125 within the first 3 months. At 43 months follow-up, there were no significant group differences in progression-free survival. There were also significantly more side effects in the experimental arm. The current study examined quality of life (QoL) endpoints using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC QLQ-C30) and the ovarian cancer module, QLQ-OV28, administered prior to randomization, at day 1 of treatment cycles 3, 5, and 7, at completion of the last cycle, and at 3 and 6 months following completion of chemotherapy. Global QoL, physical symptoms, fatigue, and role, emotional, cognitive and social function (all from the EORTC QLQ-C30) significantly improved in both treatment arms, with no significant between-arm differences. Between-group differences in pain, insomnia, and peripheral neuropathy reported while on treatment did not differ at follow-up. Nausea and vomiting improved more with standard treatment both during and after treatment. Body image significantly differed between the groups only at cycle 5 (more deterioration in Arm 2) but group differences disappeared at follow-up. A stratified analysis of global QoL by debulking surgery status found no greater effect indicating that overall improvements in QoL were unrelated to surgical recovery. There was no significant QoL advantage of cisplatin-topotecan. This finding, combined with no progression-free survival conferred by this combination, reaffirms carboplatin-paclitaxel as the standard of care for women with newly diagnosed ovarian cancer.

Synthesizing topological structures containing RNA

NASA Astrophysics Data System (ADS)

Liu, Di; Shao, Yaming; Chen, Gang; Tse-Dinh, Yuk-Ching; Piccirilli, Joseph A.; Weizmann, Yossi

2017-03-01

Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. Synthetic RNA topological structures are significant for understanding the physical and biological properties pertaining to RNA topology, and these properties in turn could facilitate identifying naturally occurring topologically nontrivial RNA molecules. Here we show that topological structures containing single-stranded RNA (ssRNA) free of strong base pairing interactions can be created either by configuring RNA-DNA hybrid four-way junctions or by template-directed synthesis with a single-stranded DNA (ssDNA) topological structure. By using a constructed ssRNA knot as a highly sensitive topological probe, we find that Escherichia coli DNA topoisomerase I has low RNA topoisomerase activity and that the R173A point mutation abolishes the unknotting activity for ssRNA, but not for ssDNA. Furthermore, we discover the topological inhibition of reverse transcription (RT) and obtain different RT-PCR patterns for an ssRNA knot and circle of the same sequence.

mir-24 activity propagates stress-induced senescence by down regulating DNA topoisomerase 1.

PubMed

Bu, Huajie; Baraldo, Giorgia; Lepperdinger, Günter; Jansen-Dürr, Pidder

2016-03-01

MicroRNAs (miRNAs) are a group of small non-coding executor RNAs. Their function as key modulators of cellular senescence has been widely recognized recently. By cross-comparing several human aging models we previously identified dozens of miRNAs being differentially regulated during aging. Here the functions of two miRNAs, mir-24 and mir-424, were investigated in an oxidative stress-induced fibroblast premature senescence model. Using pre-miRNA precursors, miRNAs were overexpressed in cells undergoing premature senescence induced by oxidative stress. More senescent cells were observed in mir-24 transfected cells. p53 was upregulated in mir-24 overexpressing cells, but downregulated in mir-424 overexpressing cells. DNA topoisomerase I (TOP1), an enzyme controlling DNA topology, was identified as a target of mir-24, whose expression was induced by oxidative stress. Knocking down TOP1 induced cellular senescence. These results suggest that mir-24 activity propagates stress-induced senescence by down regulating TOP1. Copyright © 2016 Elsevier Inc. All rights reserved.

Enhanced fluorescence norfloxacin substituted naphthalimide derivatives: Molecular docking and antibacterial activity

NASA Astrophysics Data System (ADS)

Kumar, Santosh; Kumar, Gaurav; Tripathi, Amit Kumar; Seena, Sahadevan; Koh, Joonseok

2018-04-01

Hybrid derivatives are a fascinating and challenging process in the area of drug discovery. Naphthalimide derivatives with modified norfloxacin moiety were designed and synthesized. Docking simulations were done to assess the interactions of the derivatives with the E. coli type II topoisomerases Gyrase B and ParE ATP-binding pocket by taking novobiocin as a standard molecule. Results suggested that the norfloxacin substituted naphthalimide derivatives indicate red-shift emission maxima when compared to 4-bromo 1,8-naphthalic anhydride. The molecular docking simulation study revealed that the derivatives have similar interaction but a different mode of binding with the gyrase B ATP-binding pocket as compare to novobiocin. However, they bound to ParE ATP-binding pocket similarly to novobiocin. The antibacterial property was confirmed with disc diffusion method. Our study indicated that the norfloxacin substituted naphthalimide novel derivatives have pronounced fluorescence, anti-topoisomerase activity, and antibacterial properties; therefore, they could be developed into new drug candidates.

Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes.

PubMed

Katyal, Sachin; Lee, Youngsoo; Nitiss, Karin C; Downing, Susanna M; Li, Yang; Shimada, Mikio; Zhao, Jingfeng; Russell, Helen R; Petrini, John H J; Nitiss, John L; McKinnon, Peter J

2014-06-01

DNA damage is considered to be a prime factor in several spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. We observed the endogenous accumulation of pathogenic topoisomerase-1 (Top1)-DNA cleavage complexes (Top1ccs) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We found that the defective DNA damage response factors in these two diseases cooperatively modulated Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single-strand break repair factors, including tyrosyl-DNA phosphodiesterase-1 or XRCC1, resulted in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Notably, direct Top1 poisoning to elevate Top1cc levels phenocopied the neuropathology of the mouse models described above. Our results identify a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating that genome integrity is important for preventing disease in the nervous system.

Characterization of a point mutation in the parC gene of Mycoplasma bovirhinis associated with fluoroquinolone resistance.

PubMed

Hirose, K; Kawasaki, Y; Kotani, K; Abiko, K; Sato, H

2004-05-01

Quinolone-resistant (QR) mutants of Mycoplasma bovirhinis strain PG43 (type strain) were generated by stepwise selection in increasing concentrations of enrofloxacin (ENR). An alteration was found in the quinolone resistance-determining region (QRDR) of the parC gene coding for the ParC subunit of topoisomerase IV from these mutants, but not in the gyrA, gyrB, and parE gene coding for the GyrA and GyrB subunits of DNA gyrase and the ParE subunit of topoisomerase IV. Similarly, such an alteration in QRDR of parC was found in the field isolates of M. bovirhinis, which possessed various levels of QR. The substitution of leucine (Leu) by serine (Ser) at position 80 of QRDR of ParC was observed in both QR-mutants and QR-isolates. This is the first report of QR based on a point mutation of the parC gene in M. bovirhinis.

Alteration of Escherichia coli topoisomerase IV conformation upon enzyme binding to positively supercoiled DNA.

PubMed

Crisona, Nancy J; Cozzarelli, Nicholas R

2006-07-14

Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left-handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction.

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

PubMed Central

Aoyama, M; Grabowski, D R; Dubyak, G R; Constantinou, A I; Rybicki, L A; Bukowski, R M; Ganapathi, M K; Hickson, I D; Ganapathi, R

1998-01-01

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons. PMID:9841887

NPRL-Z-1, as a new topoisomerase II poison, induces cell apoptosis and ROS generation in human renal carcinoma cells.

PubMed

Wu, Szu-Ying; Pan, Shiow-Lin; Xiao, Zhi-Yan; Hsu, Jui-Ling; Chen, Mei-Chuan; Lee, Kuo-Hsiung; Teng, Che-Ming

2014-01-01

NPRL-Z-1 is a 4β-[(4"-benzamido)-amino]-4'-O-demethyl-epipodophyllotoxin derivative. Previous reports have shown that NPRL-Z-1 possesses anticancer activity. Here NPRL-Z-1 displayed cytotoxic effects against four human cancer cell lines (HCT 116, A549, ACHN, and A498) and exhibited potent activity in A498 human renal carcinoma cells, with an IC50 value of 2.38 µM via the MTT assay. We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells. NPRL-Z-1 induced ataxia telangiectasia-mutated (ATM) protein kinase phosphorylation at serine 1981, leading to the activation of DNA damage signaling pathways, including Chk2, histone H2AX, and p53/p21. By ICE assay, the data suggested that NPRL-Z-1 acted on and stabilized the topoisomerase II (TOP2)-DNA complex, leading to TOP2cc formation. NPRL-Z-1-induced DNA damage signaling and apoptotic death was also reversed by TOP2α or TOP2β knockdown. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator. Thus, NPRL-Z-1 may present a significant potential anticancer candidate against renal carcinoma.

Archaeal Genome Organization and Stress Responses: Implications for the Origin and Evolution of Cellular Life

NASA Astrophysics Data System (ADS)

Musgrave, David; Zhang, Xiaoying; Dinger, Marcel

2002-08-01

For DNA to be used as an informational molecule it must exist in the cell on the edge of stability because all genomic processes require local controlled melting. This presents mechanistic opportunities and problems for genomic DNA from hyperthermophilic organisms, whose unpackaged DNA could melt at optimal temperatures for growth. Hyperthermophiles are suggested to employ the novel positively supercoiling topoisomerase enzyme reverse gyrase (RG) to form positively supercoiled DNA that is intrinsically resistant to thermal denaturation. RG is presently the only archaeal gene that is uniquely found in hyperthermophiles and therefore is central to hypotheses suggesting a hypothermophilic origin of life. However, the suggestion that RG has evolved by the fusion of two pre-existing enzymes has led to hypotheses for a lower temperature for the origin of life. In addition to the action of topoisomerases, DNA packaging and the intracellular ionic environment can also manipulate DNA topology significantly. In the Euryarchaeota, nucleosomes containing minimal histones can adopt two alternate DNA topologies in a salt-dependent manner. From this we hypothesize that since internal salt concentrations are increased following an increase in temperature, the genomic effects of temperature fluctuations could also be accommodated by changes in nucleosome organization. In addition, stress-induced changes in the nucleoid proteins could also play a role in maintaining the genome in the optimal topological state in changing environments. The function of these systems could therefore be central to temperature adaptation and thus be implicated in origin of life scenarios involving hyperthermophiles.

Occult nodal metastasis in solid carcinomata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moloy, P.J.; Nicolson, G.L.

1987-01-01

This book contains 23 selections. Some of the titles are: Rationale for radiotherapy in subclinical nodal disease; rationale of chemotherapy for nodal disease: The stabilization of topoisomerase II-DNA complexes as a mechanism of antineoplastic drug action; magnetic resonance imaging of malignant cervical adenopathy; and local and regional immune function in cancer patients.

Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

Semisynthesis, Cytotoxic Activity, and Oral Availability of New Lipophilic 9-Substituted Camptothecin Derivatives

PubMed Central

2013-01-01

Despite that 9-substituted camptothecins are promising candidates in cancer therapy, the limited accessibility to this position has reduced the studies of these derivatives to a few standard modifications. We report herein a novel semisynthetic route based on the Tscherniac–Einhorn reaction to synthesize new lipophilic camptothecin derivatives with amidomethyl and imidomethyl substitutions in position 9. Compounds were evaluated for their antiproliferative activity, topoisomerase I inhibition, and oral availability. Preliminary data demonstrated that bulky imidomethyl modification is an appropriate lipophilic substitution for an effective oral administration relative to topotecan. In addition, this general procedure paves the way for obtaining new camptothecin derivatives. PMID:24900725

Bcl-xL is overexpressed in hormone-resistant prostate cancer and promotes survival of LNCaP cells via interaction with proapoptotic Bak.

PubMed

Castilla, Carolina; Congregado, Belén; Chinchón, David; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

2006-10-01

Androgen-sensitive prostate cancer cells turn androgen resistant through complex mechanisms that involve dysregulation of apoptosis. We investigated the role of antiapoptotic Bcl-xL in the progression of prostate cancer as well as the interactions of Bcl-xL with proapoptotic Bax and Bak in androgen-dependent and -independent prostate cancer cells. Immunohistochemical analysis was used to study the expression of Bcl-xL in a series of 139 prostate carcinomas and its association with Gleason grade and time to hormone resistance. Expression of Bcl-xL was more abundant in prostate carcinomas of higher Gleason grades and significantly associated with the onset of hormone-refractory disease. In vivo interactions of Bcl-xL with Bax or Bak in untreated and camptothecin-treated LNCaP and PC3 cells were investigated by means of coimmunoprecipitation. In the absence of any stimuli, Bcl-xL interacts with Bax and Bak in androgen-independent PC3 cells but only with Bak in androgen-dependent LNCaP cells. Interactions of Bcl-xL with Bax and Bak were also evidenced in lysates from high-grade prostate cancer tissues. In LNCaP cells treated with camptothecin, an inhibitor of topoisomerase I, the interaction between Bcl-xL and Bak was absent after 36 h, Bcl-xL decreased gradually and Bak increased coincidentally with the progress of apoptosis. These results support a model in which Bcl-xL would exert an inhibitory effect over Bak via heterodimerization. We propose that these interactions may provide mechanisms for suppressing the activity of proapoptotic Bax and Bak in prostate cancer cells and that Bcl-xL expression contributes to androgen resistance and progression of prostate cancer.

Activation of a PGC-1-related Coactivator (PRC)-dependent Inflammatory Stress Program Linked to Apoptosis and Premature Senescence*

PubMed Central

Gleyzer, Natalie; Scarpulla, Richard C.

2013-01-01

PGC-1-related coactivator (PRC), a growth-regulated member of the PGC-1 coactivator family, contributes to the expression of the mitochondrial respiratory apparatus. PRC also orchestrates a robust response to metabolic stress by promoting the expression of multiple genes specifying inflammation, proliferation, and metabolic reprogramming. Here, we demonstrate that this PRC-dependent stress program is activated during apoptosis and senescence, two major protective mechanisms against cellular dysfunction. Both PRC and its targets (IL1α, SPRR2D, and SPRR2F) were rapidly induced by menadione, an agent that promotes apoptosis through the generation of intracellular oxidants. Menadione-induced apoptosis and the PRC stress program were blocked by the antioxidant N-acetylcysteine. The PRC stress response was also activated by the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN-38), an inducer of premature senescence in tumor cells. Cells treated with SN-38 displayed morphological characteristics of senescence and express senescence-associated β-galactosidase activity. In contrast to menadione, the SN-38 induction of the PRC program occurred over an extended time course and was antioxidant-insensitive. The potential adaptive function of the PRC stress response was investigated by treating cells with meclizine, a drug that promotes glycolytic energy metabolism and has been linked to cardio- and neuroprotection against ischemia-reperfusion injury. Meclizine increased lactate production and was a potent inducer of the PRC stress program, suggesting that PRC may contribute to the protective effects of meclizine. Finally, c-MYC and PRC were coordinately induced under all conditions tested, implicating c-MYC in the biological response to metabolic stress. The results suggest a general role for PRC in the adaptive response to cellular dysfunction. PMID:23364789

Cellular Pharmacokinetics and Intracellular Activity of Gepotidacin against Staphylococcus aureus Isolates with Different Resistance Phenotypes in Models of Cultured Phagocytic Cells

PubMed Central

Peyrusson, Frédéric

2018-01-01

ABSTRACT Gepotidacin (GSK2140944), a novel triazaacenaphthylene bacterial topoisomerase inhibitor, is currently in clinical development for the treatment of bacterial infections. This study examined in vitro its activity against intracellular Staphylococcus aureus (involved in the persistent character of skin and skin structure infections) by use of a pharmacodynamic model and in relation to cellular pharmacokinetics in phagocytic cells. Compared to oxacillin, vancomycin, linezolid, daptomycin, azithromycin, and moxifloxacin, gepotidacin was (i) more potent intracellularly (the apparent bacteriostatic concentration [Cs] was reached at an extracellular concentration about 0.7× its MIC and was not affected by mechanisms of resistance to the comparators) and (ii) caused a maximal reduction of the intracellular burden (maximum effect) of about −1.6 log10 CFU (which was better than that caused by linezolid, macrolides, and daptomycin and similar to that caused by moxifloxacin). After 24 h of incubation of infected cells with antibiotics at 100× their MIC, the intracellular persisting fraction was <0.1% with moxifloxacin, 0.5% with gepotidacin, and >1% with the other drugs. The accumulation and efflux of gepotidacin in phagocytes were very fast (kin and kout, ∼0.3 min−1; the plateau was reached within 15 min) but modest (intracellular concentration-to-extracellular concentration ratio, ∼1.6). In cell fractionation studies, about 40 to 60% of the drug was recovered in the soluble fraction and ∼40% was associated with lysosomes in uninfected cells. In infected cells, about 20% of cell-associated gepotidacin was recovered in a sedimentable fraction that also contained bacteria. This study highlights the potential for further study of gepotidacin to fight infections where intracellular niches may play a determining role in bacterial persistence and relapses. PMID:29358297

Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma

PubMed Central

Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E.; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J.; Reifenberger, Guido; Büsselberg, Dietrich

2017-01-01

Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1−10 μM) or TOPO (0.1 nM−1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment. PMID:28206967

Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling.

PubMed

Artico, M; Di Santo, R; Costi, R; Novellino, E; Greco, G; Massa, S; Tramontano, E; Marongiu, M E; De Montis, A; La Colla, P

1998-10-08

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2, 4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1, 3-indandione, and others. While active against both 3'-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the cinnamoyl residue C=C-C=O in a syn disposition, differently from flavone derivatives characterized by an anti arrangement about the same fragment. Certain inhibitors, lacking one of the two pharmacophoric catechol hydroxyls, retain moderate potency thanks to nonpharmacophoric fragments (i.e., a m-methoxy group in curcumin) which favorably interact with an "accessory" region of IN. This region is supposed to be located adjacent to the binding site accommodating the pharmacophoric dihydroxycinnamoyl moiety. Disruption of coplanarity in the inhibitor structure abolishes activity owing to poor shape complementarity with the target or an exceedingly high strain energy of the coplanar conformation.

Prevention of Post-Radiotherapy Failure in Prostate Cancer by Vitamin D

DTIC Science & Technology

2005-03-01

cur- 55. Mitchell MF, Hittelman WN, Hong WK et al. The natural history cumin , a chemo preventive agent, in patients with high-risk or of cervical...chromosomal DNA loops during oxidative stress . Genes & Dev 13:1553-1560. 16 1999 Chen AY, Choy H and Rothenberg ML. DNA topoisomerase I-targeting drugs as

Antagonism between curcumin and the topoisomerase II inhibitor etoposide

PubMed Central

Saleh, Ekram M.; El-awady, Raafat A; Eissa, Nadia A.; Abdel-Rahman, Wael M.

2012-01-01

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program. PMID:22895066

Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

PubMed Central

Gandapu, Upendhar; Chaitanya, R. K.; Kishore, Golla; Reddy, Raju C.; Kondapi, Anand K.

2011-01-01

Background Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research. Methodology/Principal Findings In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI50 = 15.6 µM) is twice more toxic than nano-curcumin (GI50 = 32.5 µM), nano-curcumin (IC50<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC50 = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis. Conclusion Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness. PMID:21887247

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

PubMed

Dinh, Thanh Theresa; Gao, Lei; Liu, Xigang; Li, Dongming; Li, Shengben; Zhao, Yuanyuan; O'Leary, Michael; Le, Brandon; Schmitz, Robert J; Manavella, Pablo A; Manavella, Pablo; Li, Shaofang; Weigel, Detlef; Pontes, Olga; Ecker, Joseph R; Chen, Xuemei

2014-07-01

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

A rapid high-resolution method for resolving DNA topoisomers.

PubMed

Mitchenall, Lesley A; Hipkin, Rachel E; Piperakis, Michael M; Burton, Nicolas P; Maxwell, Anthony

2018-01-16

Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.

Human topoisomerase I poisoning: docking protoberberines into a structure-based binding site model

NASA Astrophysics Data System (ADS)

Kettmann, Viktor; Košt'álová, Daniela; Höltje, Hans-Dieter

2004-12-01

Using the X-ray crystal structure of the human topoisomerase I (top1) - DNA cleavable complex and the Sybyl software package, we have developed a general model for the ternary cleavable complex formed with four protoberberine alkaloids differing in the substitution on the terminal phenyl rings and covering a broad range of the top1-poisoning activities. This model has the drug intercalated with its planar chromophore between the -1 and +1 base pairs flanking the cleavage site, with the nonplanar portion pointing into the minor groove. The ternary complexes were geometry-optimized and relative interaction energies, computed by using the Tripos force field, were found to rank in correct order the biological potency of the compounds; in addition, the model is also consistent with the top1-poisoning inactivity of berberine, a major prototype of the protoberberine alkaloids. The model might serve as a rational basis for elaboration of the most active compound as a lead structure, in order to develop more potent top1 poisons as next generation anti-cancer drugs.

TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage.

PubMed

Ye, Jing; Lenain, Christelle; Bauwens, Serge; Rizzo, Angela; Saint-Léger, Adelaïde; Poulet, Anaïs; Benarroch, Delphine; Magdinier, Frédérique; Morere, Julia; Amiard, Simon; Verhoeyen, Els; Britton, Sébastien; Calsou, Patrick; Salles, Bernard; Bizard, Anna; Nadal, Marc; Salvati, Erica; Sabatier, Laure; Wu, Yunlin; Biroccio, Annamaria; Londoño-Vallejo, Arturo; Giraud-Panis, Marie-Josèphe; Gilson, Eric

2010-07-23

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres. Copyright 2010 Elsevier Inc. All rights reserved.

Virtual Combinatorial Library Design, Synthesis and In vitro Anticancer Assessment of -2-Amino-3-Cyanopyridine Derivatives.

PubMed

Dev, Sanal; Dhaneshwar, Sunil R; Mathew, Bijo

2018-01-01

For the development of new class of anticancer agents, a series of novel 2-amino-3-cyanopyridine derivatives were designed from virtual screening with Glide program by setting Topoisomerase II as the target. The top ranked ten molecules from the virtual screening were synthesized by microwave assisted technique and investigated for their cytotoxic activity against MCF-7 and A- 549 cell lines by using sulforhodamine B assay method. The most active compound 2-amino-4-(3,5-dibromo-4-hydroxyphenyl)-6-(2,4- dichlorophenyl) nicotinonitrile (CG-5) showed significant cytotoxic profile with (LC50 = 97.1, TGI = 29.9 and GI50 = <0.1 µM) in MCF-7 and (LC50= 93.0, TGI= 50.0 and GI50= <7 µM) in A-549 cell lines. A molecular docking study was performed to explore the binding interaction of CG-5with the active site of Topoisomerase II. It can be concluded that halogen substituent pyridine ring was benefit for cytotoxicity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Acriflavine enhances the antitumor activity of the chemotherapeutic drug 5-fluorouracil in colorectal cancer cells.

PubMed

Zargar, Parisa; Ghani, Esmaeel; Mashayekhi, Farideh Jalali; Ramezani, Amin; Eftekhar, Ebrahim

2018-06-01

5-Fluorouracil (5-FU)-based chemotherapy improves the overall survival rates of patients with colorectal cancer (CRC). However, only a small proportion of patients respond to 5-FU when used as a single agent. The aim of the present study was to investigate whether the anticancer property of 5-FU is potentiated by combination treatment with acriflavine (ACF) in CRC cells. Additionally, the potential underlying molecular mechanisms of the cytotoxic effect of ACF were determined. The cytotoxic effects of ACF, 5-FU and irinotecan on different CRC cell lines with different p53 status were investigated using an MTT assay. SW480 cells that express a mutated form of p53 and two other CRC cell lines were used, HCT116 and LS174T, with wild-type p53. To determine the effect of ACF on the sensitivity of cells to 5-FU, cells were co-treated with the 30% maximal inhibitory concentration (IC 30 ) of ACF and various concentrations of 5-FU, or pretreated with the IC 30 of ACF and various concentrations of 5-FU. To assess the mechanism of action of ACF, cells were treated with IC 30 values of the compound and then the reverse transcription-quantitative polymerase chain reaction was used to evaluate mRNA levels of hypoxia-inducible factor-1α (HIF-1α) and topoisomerase 2. Results indicate that pretreatment with ACF markedly sensitized CRC cells to the cytotoxic effects of 5-FU, whereas simultaneous treatment with ACF and 5-FU were not able to alter the resistance of CRC cells to 5-FU. In comparison with irinotecan, ACF was a more potent agent for enhancing the antitumor activity of 5-FU. ACF did not alter the mRNA levels of either HIF-1α or topoisomerase 2. The results of the present study reveal for the first time that pretreatment of CRC cells with ACF markedly increases the cytotoxic effects of 5-FU, regardless of the p53 status of cells.

TOPOISOMERASE1α Acts through Two Distinct Mechanisms to Regulate Stele and Columella Stem Cell Maintenance.

PubMed

Zhang, Yonghong; Zheng, Lanlan; Hong, Jing Han; Gong, Ximing; Zhou, Chun; Pérez-Pérez, José Manuel; Xu, Jian

2016-05-01

TOPOISOMERASE1 (TOP1), which releases DNA torsional stress generated during replication through its DNA relaxation activity, plays vital roles in animal and plant development. In Arabidopsis (Arabidopsis thaliana), TOP1 is encoded by two paralogous genes (TOP1α and TOP1β), of which TOP1α displays specific developmental functions that are critical for the maintenance of shoot and floral stem cells. Here, we show that maintenance of two different populations of root stem cells is also dependent on TOP1α-specific developmental functions, which are exerted through two distinct novel mechanisms. In the proximal root meristem, the DNA relaxation activity of TOP1α is critical to ensure genome integrity and survival of stele stem cells (SSCs). Loss of TOP1α function triggers DNA double-strand breaks in S-phase SSCs and results in their death, which can be partially reversed by the replenishment of SSCs mediated by ETHYLENE RESPONSE FACTOR115 In the quiescent center and root cap meristem, TOP1α is epistatic to RETINOBLASTOMA-RELATED (RBR) in the maintenance of undifferentiated state and the number of columella stem cells (CSCs). Loss of TOP1α function in either wild-type or RBR RNAi plants leads to differentiation of CSCs, whereas overexpression of TOP1α mimics and further enhances the effect of RBR reduction that increases the number of CSCs Taken together, these findings provide important mechanistic insights into understanding stem cell maintenance in plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

PubMed Central

Scanlon, K J; Jiao, L; Funato, T; Wang, W; Tone, T; Rossi, J J; Kashani-Sabet, M

1991-01-01

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression. Images PMID:1660142

Synthesis, structures, nuclease activity, cytotoxicity, DFT and molecular docking studies of two nitrato bridged homodinuclear (Cu-Cu, Zn-Zn) complexes containing 2,2'-bipyridine and a chalcone derivative.

PubMed

Gaur, Ruchi; Choubey, Diksha Kumari; Usman, Mohammad; Ward, Benzamin D; Roy, Jagat Kumar; Mishra, Lallan

2017-08-01

Nitrato briged dinuclear complexes of type [Cu 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 1 and [Zn 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 2 (L=deprotonated form of free ligand LH, [1-(2-hydroxyphenyl)-3-(9-anthracenyl) propenone; bpy=2,2'bipyridine] are synthesized and characterized using a battery of physicochemical techniques and X-ray crystallography. A distorted square pyramidal geometry is assigned to them with N 2 O 3 coordination core around the metal ion. The co-ligand L binds the metal ions through its O,O' atoms in anti-syn mode. The metal centers in complexes 1 and 2 are separated via bridging nitrato group at a distance of 6.073Å and 5.635Å respectively. Their structures and absorption spectra are supported by the computational studies using density functional theory (DFT) and TD-DFT. Both complexes exhibit nuclease activity and cleave supercoiled (form I) DNA. The complex 1 preferentially binds major groove of DNA and follows an oxidative pathway whereas complex 2 binds with minor groove of DNA via hydrolytic pathway. Both complexes inhibit topoisomerase I relaxation activity with IC 50 values of 7 and 35μM. Molecular docking studies support the groove binding and topoisomerase I binding of the complexes. The complex 1 showed a significant cytotoxicity against HeLa cell lines (a cervical cancer cell lines) in vitro with IC 50 value calculated as 2.9±0.021μM as compared to 28.2±0. 044μΜ for complex 2. Complex 2 induces the cell apoptosis at a later-stage as compared to complex 1. The cell apoptosis and topoisomerase inhibition by complexes enable them to be potential candidates as future anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

A phase II single institution single arm prospective study with paclitaxel, ifosfamide and cisplatin (TIP) as first-line chemotherapy in high-risk germ cell tumor patients with more than ten years follow-up and retrospective correlation with ERCC1, Topoisomerase 1, 2A, p53 and HER-2 expression.

PubMed

Ligia Cebotaru, Cristina; Zenovia Antone, Nicoleta; Diana Olteanu, Elena; Bejinariu, Nona; Buiga, Rares; Todor, Nicolae; Ioana Iancu, Dana; Eliade Ciuleanu, Tudor; Nagy, Viorica

2016-01-01

One half of high-risk germ cell tumor (HRGCT) patients relapse after standard chemotherapy. This phase II study evaluated prospectively the toxicity and efficacy in first-line of the paclitaxel-ifosfamide-cisplatin combination (TIP) in HRGCT patients and tried to identify biomarkers that may allow patient-tailored treatments. Between October 1997- September 2000, 28 chemo-naive HRGCT patients were enrolled. Patients received 4 cycles of TIP (paclitaxel 175 mg/m(2) day 1/; ifosfamide 1.2 g/m(2)/day, days 1-5; Mesna 1.2 g/m(2)/day, days 1-5; and cisplatin 20 mg/m(2)/day, days 1-5 every 3 weeks). A non-randomized comparison was made between HRGCT patients treated in the same period with first-line TIP and bleomycin-etoposide-cisplatin (BEP) (28 patients vs 20). In 17 HRGCT patients treated between 1998-2006, ERCC1, Topoisomerase 1 and 2A, p53 and HER-2 expression was retrospectively analysed by immunohistochemistry (IHC) (7 patients with TIP, 10 with BEP), and correlations were made with response to chemotherapy and survival. With a median follow-up of 72 months [range 48+...89+], 5-year disease free survival (DFS) was 55%, with 95% CI 36-72, and the overall survival (OS) was 63%, with 95% CI 44-78. In June 2015, with a median follow-up of 196.47 months (range 177.30-209.27) (>15 years), 12 [%?] patients were alive and disease-free, and 16 [%?] had died (12 specific causes). There was no significant correlation between the expression of ERCC1, Topoisomerase 1 and 2A, HER-2 and p53 and response to treatment. Long-term follow-up showed no difference in OS between TIP vs BEP as first-line therapy. Both regimens had mild toxicity.

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

PubMed

Neri, L M; Bortul, R; Zweyer, M; Tabellini, G; Borgatti, P; Marchisio, M; Bareggi, R; Capitani, S; Martelli, A M

1999-06-01

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.

Simple and efficient liposomal encapsulation of topotecan by ammonium sulfate gradient: stability, pharmacokinetic and therapeutic evaluation.

PubMed

Liu, Jun-Jen; Hong, Ruey-Long; Cheng, Wen-Fang; Hong, Keelung; Chang, Fu-Hsiung; Tseng, Yun-Long

2002-08-01

Topotecan (TPT), a topoisomerase I inhibitor, is presently undergoing clinical evaluation worldwide. Previous studies have shown that entrapping TPT within multi-lamellar vesicle liposome can stabilize the lactone moiety, which is structurally important for biological activity. However, low drug:lipid ratios due to the amphipathic character and small entrapment volume in the unilamellar vesicle limits the development of pharmaceutically acceptable liposomal formulation. With an aim to improve on this drawback, we herein describe a method that utilizes the ammonium sulfate gradient to entrap TPT into liposomes. By this method, the encapsulation efficiency was over 90% and a drug:lipid molar ratio as high as 1:5.4 was reached. In comparison with free drug, liposome-encapsulated TPT is more stable in physiological conditions and shows higher in vitro cytotoxicity. Because of increased blood circulation time, the initial plasma concentration and area under the plasma concentration of liposomal drugs were 14 and 40 times, respectively, of those of free drug. Furthermore, liposome encapsulation enhanced the antitumor activity of TPT in syngeneic murine C-26 and human HTB-9 xenograft models in vivo. At a dose of 5 mg/kg, the tumor growth delay of liposomal formulation was significantly than that of free TPT. Based on these results, we believe that this liposomal TPT formulation is worthy of further clinical study. Copyright 2002 Lippincott Williams & Wilkins.

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.

PubMed

Oropesa, Manuel; de la Mata, Mario; Maraver, Juan Garrido; Cordero, Mario D; Cotán, David; Rodríguez-Hernández, Angeles; Domínguez-Moñino, Irene; de Miguel, Manuel; Navas, Plácido; Sánchez-Alcázar, José A

2011-04-01

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.

Canine spontaneous glioma: A translational model system for convection-enhanced delivery

PubMed Central

Dickinson, Peter J.; LeCouteur, Richard A.; Higgins, Robert J.; Bringas, John R.; Larson, Richard F.; Yamashita, Yoji; Krauze, Michal T.; Forsayeth, John; Noble, Charles O.; Drummond, Daryl C.; Kirpotin, Dmitri B.; Park, John W.; Berger, Mitchel S.; Bankiewicz, Krystof S.

2010-01-01

Canine spontaneous intracranial tumors bear striking similarities to their human tumor counterparts and have the potential to provide a large animal model system for more realistic validation of novel therapies typically developed in small rodent models. We used spontaneously occurring canine gliomas to investigate the use of convection-enhanced delivery (CED) of liposomal nanoparticles, containing topoisomerase inhibitor CPT-11. To facilitate visualization of intratumoral infusions by real-time magnetic resonance imaging (MRI), we included identically formulated liposomes loaded with Gadoteridol. Real-time MRI defined distribution of infusate within both tumor and normal brain tissues. The most important limiting factor for volume of distribution within tumor tissue was the leakage of infusate into ventricular or subarachnoid spaces. Decreased tumor volume, tumor necrosis, and modulation of tumor phenotype correlated with volume of distribution of infusate (Vd), infusion location, and leakage as determined by real-time MRI and histopathology. This study demonstrates the potential for canine spontaneous gliomas as a model system for the validation and development of novel therapeutic strategies for human brain tumors. Data obtained from infusions monitored in real time in a large, spontaneous tumor may provide information, allowing more accurate prediction and optimization of infusion parameters. Variability in Vd between tumors strongly suggests that real-time imaging should be an essential component of CED therapeutic trials to allow minimization of inappropriate infusions and accurate assessment of clinical outcomes. PMID:20488958

Problem-solving test: catalytic activities of a human nuclear enzyme.

PubMed

Szeberényi, József

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5′-end and 3′-end, bacteriophage, polyacrylamide gel electrophoresis, urea, autoradiography, proofreading, telomerase, endonucleases, exonucleases, primase, topoisomerases, and excinuclease.

Feasibility of Screening for Antibiotic Resistance-Part II

DTIC Science & Technology

2005-08-01

fluoroquinolones were introduced on the market. The first two generations are not widely used anymore, because of widespread resistance or ineffectiveness...Mutations that confer resistance against ciprofloxacin have been found in all four genes encoding subunits of Topoisomerase H and IV, and also in an entirely...aerogenes, Erwinia carotovora, Escherichia coli K12, Francisella tularensis, Klebsiella pneumoniae , Neisseria gonorrhoeae, Providencia stuartii

ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype.

PubMed Central

Muñoz, R; De La Campa, A G

1996-01-01

The genes encoding the ParC and ParE subunits of topoisomerase IV of Streptococcus pneumoniae, together with the region encoding amino acids 46 to 172 (residue numbers are as in Escherichia coli) of the pneumococcal GyrA subunit, were partially characterized. The gyrA gene maps to a physical location distant from the gyrB and parC loci on the chromosome, whereas parC is closely linked to parE. Ciprofloxacin-resistant (Cpr) clinical isolates of S. pneumoniae had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level Cpr) or in both quinolone resistance-determining regions of ParC and GyrA (high-level Cpr). Mutations were found in residue positions equivalent to the serine at position 83 and the aspartic acid at position 87 of the E. coli GyrA subunit. Transformation experiments suggest that ParC is the primary target of ciprofloxacin. Mutation in parC appears to be a prerequisite before mutations in gyrA can influence resistance levels. PMID:8891124

The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes.

PubMed

Nikalayevich, Elvira; Ohkura, Hiroyuki

2015-02-01

Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. Here, we took advantage of Drosophilaoocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, we found severe undercondensation of chromosomes after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. We show that the main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. We further show that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes.

The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes

PubMed Central

Nikalayevich, Elvira; Ohkura, Hiroyuki

2015-01-01

ABSTRACT Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. Here, we took advantage of Drosophila oocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, we found severe undercondensation of chromosomes after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. We show that the main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. We further show that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes. PMID:25501812

Investigating the potential for toxicity from long-term use of the herbal products, goldenseal and milk thistle.

PubMed

Dunnick, June K; Singh, Bhanu; Nyska, Abraham; Peckham, John; Kissling, Grace E; Sanders, J Michael

2011-02-01

Two-year toxicity studies were conducted on the widely used herbal products, goldenseal and milk thistle, in male and female F344/N rats and B6C3F1 mice. With goldenseal root powder, the primary finding was an increase in liver tumors in rats and mice, and with milk thistle extract, a decrease in spontaneous background tumors including mammary gland tumors in female rats and liver tumors in male mice. Increased tumorigenicity in rodents exposed to goldenseal root powder may be due in part to the topoisomerase inhibition properties of berberine, a major alkaloid constituent in goldenseal, or its metabolite, berberrubine. In the clinic, use of topoisomerase-inhibiting agents has been associated with secondary tumor formation and inhibition in DNA repair processes. In contrast, the radical-scavenging and antioxidant properties of silibinin and other flavonolignans in milk thistle extract may have contributed to the decrease in background tumors in rodents in the present studies. The fate of the active constituents of goldenseal and milk thistle is similar in humans and rodents; therefore, the modes of action may translate across species. Further studies are needed to extrapolate the findings to humans.

CENP-A and topoisomerase-II antagonistically affect chromosome length.

PubMed

Ladouceur, A-M; Ranjan, Rajesh; Smith, Lydia; Fadero, Tanner; Heppert, Jennifer; Goldstein, Bob; Maddox, Amy Shaub; Maddox, Paul S

2017-09-04

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening. © 2017 Ladouceur et al.

Tetramethylpyrazine promotes SH-SY5Y cell differentiation into neurons through epigenetic regulation of Topoisomerase IIβ.

PubMed

Yan, Y; Zhao, J; Cao, C; Jia, Z; Zhou, N; Han, S; Wang, Y; Xu, Y; Zhao, J; Yan, Y; Cui, H

2014-10-10

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Recently, it has been reported that TMP enhances neurogenesis, and promotes neural stem cell differentiation toward neurons. However, its molecular basis remains unknown. Topoisomerase IIβ (TopoIIβ) is a nuclear enzyme with an essential role in neuronal development. This study aimed to investigate whether TopoIIβ is involved in TMP-induced neuronal differentiation. We examined the effect of TMP on neuronal differentiation of SH-SY5Y cells. It was found that TMP inhibited cell proliferation and induced G0/G1 cell cycle arrest. TMP promoted SH-SY5Y cells to differentiate toward post-mitotic neurons characterized by long, out-branched neurites and up-regulated neuronal markers, microtubule-associated protein 2 (MAP2) and tau. Meanwhile, we demonstrated that TopoIIβ was highly expressed following TMP treatment. To unravel how TMP affects TopoIIβ expression, two chromatin active markers, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4) were examined in this study. Our data showed that the levels of Ac-H3 and Ac-H4 were positively correlated with TopoIIβ expression in the processes of neuronal differentiation. We further performed chromatin immunoprecipitation (ChIP) analysis and identified that TMP enhanced the recruitment of Ac-H3 and Ac-H4 to the TopoIIβ gene promoter region. Therefore, we concluded that TMP may stimulate neuronal differentiation of SH-SY5Y cells through epigenetic regulation of TopoIIβ. These results suggest a novel molecular mechanism underlying TMP-promoted neuronal differentiation. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

What Is Old Is New Again: Delafloxacin, a Modern Fluoroquinolone.

PubMed

Cho, Jonathan C; Crotty, Matthew P; White, Bryan P; Worley, Marylee V

2018-01-01

Delafloxacin is a new fluoroquinolone antimicrobial approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs) in adults using dosage regimens of 300 mg intravenously every 12 hours, 450 mg orally every 12 hours, or switching from intravenous to oral regimens for a 5- to 14-day treatment duration. Dosage adjustments in patients with severe renal dysfunction (estimated glomerular filtration rate [eGFR] = 15-29 ml/min/1.73 m 2 ) are not required for oral doses but should be decreased to 200 mg intravenously every 12 hours in patients requiring parenteral therapy. Due to insufficient data, use of delafloxacin is not recommended for patients on hemodialysis or with end-stage renal disease (eGFR < 15 ml/min/1.73 m 2 ). Delafloxacin works through inhibition of DNA gyrase (topoisomerase II) and topoisomerase IV, which are essential enzymes for bacterial DNA transcription, replication, repair, and recombination and exhibits bactericidal activity against gram-positive and gram-negative organisms through a concentration-dependent matter. Delafloxacin has a very broad spectrum of activity against atypical, anaerobic, and resistant gram-negative and gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. During phase 3 trials, the most common side effects associated with delafloxacin were gastrointestinal (nausea, diarrhea). Unlike other fluoroquinolones, there does not seem to be a risk of QTc prolongation or phototoxicity with delafloxacin. The availability of both parenteral and oral formulations for delafloxacin distinguishes it from many of the currently available agents approved for ABSSSIs. Phase 3 studies for the treatment of respiratory infections are currently under way, and future results of these studies will further help delineate the role of delafloxacin. © 2017 Pharmacotherapy Publications, Inc.

Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.

PubMed

Ogitani, Yusuke; Hagihara, Katsunobu; Oitate, Masataka; Naito, Hiroyuki; Agatsuma, Toshinori

2016-07-01

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

ERIC Educational Resources Information Center

Tweedie, John W.; Stowell, Kathryn M.

2005-01-01

A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene

PubMed Central

Macovei, Anca; Faè, Matteo; Biggiogera, Marco; de Sousa Araújo, Susana; Carbonera, Daniela; Balestrazzi, Alma

2018-01-01

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5′-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells. PMID:29868059

Role of Choline in the Modulation of Degenerative Processes: In Vivo and In Vitro Studies.

PubMed

Merinas-Amo, Tania; Tasset-Cuevas, Inmaculada; Díaz-Carretero, Antonio M; Alonso-Moraga, Ángeles; Calahorro, Fernando

2017-03-01

The purpose of the present study was to examine the nutraceutical potential of choline as an added value to its well-known brain nutrient role. Several toxicity, antitoxicity, genotoxicity, antigenotoxicity, and longevity endpoints were checked in the somatic mutation and recombination test in in vivo Drosophila animal model. Cytotoxicity in human leukemia-60 cell line (HL-60) promyelocytic and NIH3T3 mouse fibroblast cells, proapoptotic DNA fragmentation, comet assay, methylation status, and macroautophagy (MA) activity were tested in in vitro assays. Choline is not only safe but it is also able to protect against the DNA damage caused by an oxidative genotoxin. Moreover, it improves the life extension in the animal model. The in vitro results show that it is able to exhibit genetic damage against leukemia HL-60 cells. Single-strand breaks in DNA are observed at the molecular level in treatments with choline, although only a significant hypermethylation on the long interspersed elements-1 and a hypomethylation on the satellite-alpha DNA repetitive DNA sequences of HL-60 cells at the lowest concentration (0.447 mM) were observed. Besides, choline decreased MA at the lower assayed concentration and the MA response to topoisomerase inhibitor (etoposide) is maintained in the presence of treatment with 0.22 mM choline. Taking into account the hopeful results obtained in the in vivo and in vitro assays, choline could be proposed as a substance with an important nutraceutical value for different purposes.

Etoposide; colchicine; mitomycin C and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster lung (CHL) cells at Covance laboratories; Harrogate UK in support of OECD draft Test Guideline 487.

PubMed

Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

2010-10-29

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the Chinese hamster lung cell line CHL. Etoposide (a topoisomerase inhibitor), colchicine (an aneugen), mitomycin C (a DNA cross linking agent) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.