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The compounds reducing tumor cell viability and disrupting DNA topoisomerase reactions have been widely used in anticancer drug development. Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a potent intercalating agent that interferes with nucleic acid processing through interaction with DNA topoisomerase II. Although ellipticine is a well-characterized compound, it is not a widely-accepted drug due to the adverse effects detected upon administration. We have previously reported two novel ellipticine derivatives, N-methyl-5-demethyl ellipticine (ET-1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2) as potent compounds targeting DNA topoisomerase II. This study covers an extended synthesis, characterization, and activity data for five new salts of N-methyl 5-demetyl ellipticine (Z-1, Z-2, Z-4, Z-5 and Z-6) having several organic halides and their effects on human topoisomerase II enzymes. Moreover, combined in silico studies were conducted for better understanding of modes of action of studied molecules at the binding pocket of target. Our results showed that three of the derivatives (Z-1, Z-2, and Z-6) have considerable effect on the catalytic activity of human topoisomerase II implying the influence of alkyl groups added to the parental structure of ellipticine. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway.Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells.Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.

Background DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway. Methods Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells. Results and conclusions Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors., Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349], This research was supported by Grant 115Z349 (Z.T. and S.Z.) from the Turkish Scientific and Technical Research Assembly. the cell lines used in this study were provided by Drs. Shu-Chun Teng and Tsai-Kun Li (College of Medicine, National Taiwan University, Taipei, Taiwan).

Objective:Kidney transplantation is an important treatment option in end stage renal failure. Tissue death may be an important problem when a kidney is removed from a cadaver and transported to a donor a long distance away. The purpose of this study is to determine the critical time point for apoptotic cell death in a renal ischemia/reperfusion model and determine the effects of N-acetylcystein on apoptosis induced by ischemia injury.Methods:Apoptotic cell death after induced renal ischemia followed by reperfusion, was estimated in a group of Wistar albino rats by immunoflourescence and ELISA techniques. N-acetylcystein, an antioxidant agent, was given to the rats to study the effect on apoptosis. Tissues were examined immunohistochemically at 0, 1 h, 24 h, 5 days and 10 days for detection of apoptotic cells.Results:Our results showed that an ischemia for 60 min followed by reperfusion for 60 min triggered apoptosis. Moreover, N-acetylcystein significantly diminished both the ischemia/reperfusion damage and apoptosis.Conclusion:We anticipate our results would be important for kidney transplantation in estimating the critical time point for apoptosis and administration of N-acetylcystein prior to removal of the organ may be important in delaying the onset of apoptosis.

WOS: 000417698200025, PubMed ID: 29232376, General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA-and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes., Turkish Scientific and Technological Research Assembly [112T429]; Hungarian NRDIO [GINOP-2.3.2-15-2016-00020], This work was supported by the Turkish Scientific and Technological Research Assembly (112T429) (Dr. Sevil Zencir), and Hungarian NRDIO-GINOP-2.3.2-15-2016-00020 (Prof Imre M. Boros). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Summary What is known and Objective: Imatinib is a specific BCR/ABL inhibitor, commonly used for the treatment of chronic myeloid leukaemia (CML), a hematological malignancy resulting from a chromosomal translocation that generates the BCR/ABL fusion protein. Recent studies showed that the imatinib has cytotoxic and apoptotic effects on many BCR/ABL-negative cancers. Numerous compounds with cytotoxic potential exert their functions by interfering with the DNA topoisomerase. In this study, we examined the effects of imatinib on tumour cell-killing in relation to DNA topoisomerase enzyme inhibition. Methods: We determined the cytotoxicity by cell proliferation assay (XTT; tetrazolium hydroxide), using the human K562 CML cells, and loss of mitochondrial membrane potential by monitoring the changes in caspase-3 enzyme activity. Type I and II topoisomerase activities were measured by supercoiled plasmid relaxation and minicircle DNA decatenation assays respectively. Results and Discussion: Imatinib-induced apoptosis and inhibited cell proliferation in a dose-dependent manner. We also found that the imatinib was effective in both type I and type II topoisomerase reactions to a varying degree between 94% and 7% for the concentration range of 1 mm–0·02 mm in a dose-dependent manner. What is new and Conclusion: Our results suggest that the inhibition of topoisomerases may be a significant factor in imatinib-induced apoptosis in CML.

Telomeres are specialized DNA-protein complexes found at the tips of linear chromosomes. In this study, we investigated the effects of oxidative stress on telomeric length distribution of proliferating vascular smooth muscle cells following balloon injury in single or combined treatment of rabbits with either buthionine sulfoximine or taurine. Exposure to oxidative stress increased the balloon injury whereas taurine treatment significantly diminished l-buthionine-sulfoximine-related intimal hyperplasia. Our results also showed that both variables had a significant influence on mean telomeric length distribution.

Benzimidazoles are important compounds because of their antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Some benzimidazole derivatives also interfere with the reactions of DNA topoisomerases, enzymes functioning at almost all stages of the cell cycle. In this study, nine 1H-benzimidazole derivatives with substituents at positions 2 and 5 were synthesized and the structure of the compounds was elucidated by instrumental methods. The characterized compounds were screened to identify if they interfered with mammalian type I DNA topoisomerase activity via in vitro supercoil relaxation assays. Selected compounds were subjected to cytostatic assays using HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells. Our results showed that 5-chloro-2-(2-hydroxyphenyl)-1H-benzimidazole exerted the most profound topoisomerase I inhibition and cytotoxicity. © 2008.

WOS: 000262609100002, PubMed ID: 19029249, Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3' overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5' end degradation., National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM 61645]; American Cancer SocietyAmerican Cancer Society [024255-01]; The Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [SBAG-2791], This work was supported by grants to M.J.M. from the National Institutes of Health (GM 61645) and the American Cancer Society (024255-01) and to Z.T. from The Scientific and Technological Research Council of Turkey (TUBITAK) (SBAG-2791).; We thank Elizabeth Blackburn, in whose lab M.J.M. constructed the ter1-24T(SnaB)/ter1-19A(Acc) heteroallelic strains. We also thank Evelina Basenko for helpful comments on the manuscript and Rolf Kooistra for providing us with the wild-type strain CBS 2359 and the ku80 Delta strain.

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.

WOS: 000372677900019, PubMed ID: 26894873, Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot., DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIa were assessed. GE-2 enhanced DNA cleavage similar to 4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIa construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIa except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIa may play a role in mediating the cytotoxic effects of these fungal metabolites., National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM033944]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109S361]; EBILTEMEge University [2012/BIL/028]; Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R25 GM062459, T32 GM008320], This research was supported by grant GM033944 (N.O.) from the National Institutes of Health, grants from TUBITAK (109S361) and EBILTEM (2012/BIL/028) (E.B.), and grant 115Z349 (Z.T.) from the Turkish Scientific and Technical Research Assembly. K.R.V. was a trainee under NIH grants R25 GM062459 and T32 GM008320 and was supported in part by a Research Supplement to Grant GM033944 to Promote Diversity in Health-Related Research.

WOS: 000371344600033, PubMed ID: 26906637, Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an antineoplastic agent that intercalates into DNA and alters topoisomerase II activity. Unfortunately, this compound displays a number of adverse properties. Therefore, to investigate new ellipticine-based compounds for their potential as topoisomerase II-targeted drugs, we synthesized two novel derivatives, N-methyl-5-demethyl ellipticine (ET1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2). As determined by DNA decatenation and cleavage assays, ET-1 and ET-2 act as catalytic inhibitors of human topoisomerase II alpha and are both more potent than the parent compound. Neither compound impairs the ability of the type II enzyme to bind its DNA substrate. Finally, the potency of ET-1 and ET-2 as catalytic inhibitors of topoisomerase II alpha appears to be related to their ability to intercalate into the double helix. (C) 2016 Elsevier Ltd. All rights reserved., National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM033944, R25-GM062459, T32-GM08320]; Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349], The authors declare no competing financial interests. This research was supported by grant GM033944 (N.O.) from the National Institutes of Health and grant 115Z349 (Z.T.) from the Turkish Scientific and Technical Research Assembly. K.R.V. was a trainee under National Institutes of Health grants R25-GM062459 and T32-GM08320 and was supported in part by a Research Supplement to Grant GM033944 to Promote Diversity in Health-Related Research. We are grateful to Rachel E. Ashley, Lorena Infante Lara, and Elizabeth G. Gibson for critical reading of the manuscript.

Summary Objective: To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. Method: Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. Results: Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. Conclusions: These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.

Benzimidazole is one of the most important heterocyclic groups manifesting various biological properties, such as antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Several benzimidazole derivatives are also active as inhibitors of type I DNA topoisomerases. In this study, three 1H-benzimidazole derivatives with different electronic characteristics at position 5-, namely 5-chloro-4-(1H-benzimidazole-2-yl)phenol (Cpd I), 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) and 4-(1H-benzimidazole-2-yl)phenol (Cpd III), were synthesized and evaluated for their effects on mammalian type I DNA topoisomerase activity using quantitative in vitro plasmid supercoil relaxation assays. For the structure elucidation of the compounds, melting points, UV, IR, 1H NMR, 13C NMR, mass spectral data and elemental analyses were interpreted. Among the compounds, 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) manifested relatively potent topoisomerase I inhibition.

The metabolic activation of numerous exogenous and endogenous chemicals is catalysed by cytochrome P450 enzymes (CYPs). The aim of this study was to analyse the expression of the individual forms of CYP at the mRNA level in human larynx and quantitatively to compare their expressions in human liver, the main organ of CYP expression. Individual forms of CYP mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CYPs -1A1, -1A2, -2A6, -2B6, -2C, -2D6, -2E1, -3A3/4, -3A5, -3A7 and -4B1. An RNA competitor of known copy number, covering the primer sequences necessary to amplify the entire object CYPs within a single molecule, was used as reference. This study reports a consistent detection of mRNAs for the CYPs -1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 in the human larynx tissue. The data indicate that the human larynx highly resembles the lung tissue in CYP content, as a comparable subset of CYP mRNAs was detected in the larynx previously reported for human lung with the exception of CYP1A2. The results are discussed in quantitative ratios of the detected CYP mRNAs in relation to the hepatic CYP expression.

PubMed ID: 15713803, Eukaryotic cells, including some human cancers, that lack telomerase can sometimes maintain telomeres by using recombination. It was recently proposed that recombinational telomere elongation (RTE) in a telomerase-deletion mutant of the yeast Kluyveromyces lactis occurs through a roll-and-spread mechanism as described in our previous work. According to this model, a tiny circle of telomeric DNA is copied by a rolling-circle mechanism to generate one long telomere, the sequence of which is then spread to all other telomeres by gene-conversion events. In support of this model, we demonstrate here that RTE in K. lactis occurs by amplification of a sequence originating from a single telomere. When a mutationally tagged telomere is of normal length, its sequence is spread to all other telomeres at a frequency (?10%) consistent with random selection among the 12 telomeres in the cell. However, when the mutationally tagged telomere is considerably longer than other telomeres, cellular senescence is partially suppressed, and the sequence of the tagged telomere is spread to all other telomeres in >90% of cells. Strikingly, the transition between a state resistant to recombination and a state capable of initiating recombination is abrupt, typically occurring when telomeres are ?3-4 repeats long. Last, we show that mutant repeats that are defective at regulating telomerase are also defective at regulating telomere length during RTE. © 2005 by The National Academy of Sciences of the USA.

Although natural products have been used by man since ancient times, it was only in the past century that active components of medicinal plants became available in chemically pure form. An impressive number of natural products have been introduced into medical practice and also used as lead or model molecules for structure optimization and for the development of more potent or better-tolerated drugs. A recent comprehensive review found that only 20.2% of the anticancer agents approved in the period 1981–2010 were purely synthetic, whereas the remaining 79.8% were natural products or inspired by natural products (D. J. Newman and G. M. Cragg, “Natural products as sources of new drugs over the 30 years from 1981 to 2010” Journal of Natural Products, vol. 75, no. 3, pp. 311–335, 2012), indicating that structures from nature are indispensable in anticancer lead-finding research. In this special issue, we present a set of original research papers concerning various aspects of the antiproliferative properties of plant extracts or isolated natural products. Three of them describe investigations with promising extracts. The methanolic root extract of Sclerocarya birrea gave rise to oxidative stress and apoptosis in HepG2 cells (M. F. Armentano et al.). Fractions of the methanolic extract of Croton sphaerogynus exhibited antiproliferative activity against adherent cancer cell lines (K. P. dos Santos et al.). The mechanism of the anticancer action of the ethyl acetate extract of the traditional Chinese medicinal plant Euphorbia helioscopia was explored through the use of a hepatocellular carcinoma xenograft model in nude mice. The treatment with the extract resulted in apoptosis induction, decreased tumor growth, and invasion (J. Cheng et al.). Two papers in this issue concentrate on the isolation and subsequent investigation of natural products. Various antiproliferative xanthones were isolated from Garcinia species and two of them directly inhibited JAK kinase (L. Xu et al.). The natural products hamigerone and radicinol, isolated from fungi by means of a bioassay-guided procedure, exhibited antiproliferative and proapoptotic properties in adherent cancer cell lines without affecting the viability of normal cells (P. Giridharan et al.). Two further publications deal with previously identified compounds that were earlier not investigated in detail oncopharmacologically. Vitamin K1 displays antiproliferative activity, causes cell cycle arrest, and induces apoptosis in human colon cancer cell lines (A. Orlando et al.). The sesterterpene heteronemin exerted considerable growth-inhibiting activity against human cancer cell lines, including A498 renal carcinoma cells, resulting in apoptosis through the mitochondrial pathway and also autophagy in A498 cells (S.-Y. Wu et al.). Two papers describe potentially relevant interactions between natural products and therapeutically utilized anticancer agents. Curcumin and one of its analogues exhibited additive properties with the proteasome inhibitor bortezomib against HL60 cells (L. I. Nagy et al.). Three ecdysteroids sensitized both multidrug-resistant and nonresistant adherent cell lines towards traditional anticancer agents, including doxorubicin (A. Martins et al.). One of the presented papers reports on the validation of an in vitro method for the screening of potential anticancer extracts or compounds. Instead of the use of cultured cancer cell lines, slices of breast cancer tissues obtained from surgery were incubated with antiproliferative agents and the viabilities of the slices were determined (I. E. Carranza-Torres et al.). We strongly trust that these publications in this special issue will contribute to the development of natural product-based and innovative drug candidates.

DNA topoisomerases are essential enzymes that regulate the conformational changes in DNA topology by catalysing the concerted breakage and rejoining of DNA strands during normal cellular growth. Over the past few years there has been considerable pharmacological interest in these enzymes because inhibitors of DNA topoisomerases represent a major class of anticancer drugs. This review highlights topoisomerase-targeting drugs that have shown promising anticancer activities. The mechanisms by which those drugs interfere with the catalytic cycles of type I and type II DNA topoisomerases and the factors involved in the development of resistance to these drugs are discussed.

The detected phenotypes in many diseases are caused from dysfunction in protein-protein, protein-DNA and receptor-ligand interactions. Therefore, determination of these molecular interactions followed by designing or screening the compounds to target these interactions provides a significant challenge in drug development. This review aims to highlight the yeast two-hybrid system in determination of protein-protein interactions and its possible outcomes in pharmaceutical research. The variations of the basic methodology as one- and three-hybrid systems are also disussed in relation to their potential pharmaceutical applications.

PubMed ID: 10597310, Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML if disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis., Leukemia Research Foundation RO1 CA80728-01, MT-13608, The mAb 5E10 antibody was a kind gift of L de Jong. We are grateful for technical assistance and advice from Graeme W Carlile and Melanie J Dobson and for critical reading of the manuscript by Ari Melnick. KLB Borden acknowledges support from MRC MT-13608, RO1 CA80728-01 and Leukemia Research Foundation.

Kidney failure is an important health issue all over the world. Therefore effective treatment strategies need to be developed to cure acute kidney failure. Ischemia initiates a series of biochemical reactions that may end up with cellular dysfunction and ultimate cell death via diminishing cellular energy level and accumulating toxic subtances. Tissue and organ death is therefore an inevitable result due to added harms exerted by neutrophils, which arose following reperfusion and released mediators. In this study we estimated the percentage of poliferative, necrotic and apoptotic cell death in a kidney ischemia/reprfusion model using Wistar albino rats. Ischemia was conducted in left kidney while the right ones served as control. The presence of the apoptotic cell was investigated in reperfused kidney. Our study was extended to cover testing the effect of antioxidant agent, N-acetylcystein on apoptosis. We showed that a reperfusion with a 60 mins duration following a 60 mins of ischemia triggered apoptosi...

WOS: 000316038500003, PubMed ID: 23395680, Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. (C) 2013 Elsevier Inc. All rights reserved., United States Department of AgricultureUnited States Department of Agriculture (USDA) [2003-35302-12930, 2011-65503-20030]; National Science FoundationNational Science Foundation (NSF) [IOS-0628064]; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; [TUBITAK 108T945], This research was financially supported by United States Department of Agriculture PECASE award 2003-35302-12930, and AFRI award 2011-65503-20030, National Science Foundation Grant IOS-0628064, and National Institutes of Health Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu.

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks., Scientific and Technological Research Council of Turkey (TBAG-108T548)

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase IImediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners. © 2013 Biochemical Society.

WOS: 000294317300024, PubMed ID: 21787750, The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-I, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, beta-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kit 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP. (C) 2011 Elsevier Inc. All rights reserved., USDA PECASE [2003-35302-12930]; NSFNational Science Foundation (NSF) [IBN-0628064]; USDAUnited States Department of Agriculture (USDA) [2011-65503-20030]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108T945], This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003-35302-12930, NSF Grant IBN-0628064, USDA Grant 2011-65503-20030 and NIH Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu. We thank Dr. David L. Zoetewey for critical reading of this manuscript.

PubMedID: 20824388 We investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trains exposure on lipid peroxidation, and, hence, oxidative stress in the rat liver tissue. The parameters that we measured were the levels of plasma alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as plasma albumin, bilirubin, and total protein levels in 30 adult male Wistar rats exposed to ELF. We also determined the percentage of apoptotic and necrotic cells of the kidney extracts from the animals by flow cytometry method. Apoptotic cell death was further characterized by monitoring DNA degradation using gel electrophoresis. The results showed an increase in the levels of oxidative stress indicators, and the flow cytometric data suggested a possible relationship between the exposure to magnetic field and the cell death. We showed significantly lower necrotic cell percentages in experimental animals compared to either unexposed or sham control groups. However, DNA ladder analyses did not differentiate between the groups. Our results were discussed in relation to the response of biological systems to EMF. © 2010 Springer Science+Business Media, LLC. Acknowledgment The authors acknowledge with thanks the support provided by the Biochemistry department of Cukurova Medical School by way of valuable contributions in the determination of oxidative stress parameters.

Human Cytochrome P450 (CYP) comprises a multigene family of microsomal enzymes that metabolize a wide variety of xenobiotics, including drugs and carcinogens. Although the a number of CYP enzymes were also detected in epithelial cells along the gastrointestinal tract, little is known about the expression of CYP genes in gastric tissue.In this study, the expression patterns of CYP isoforms was investigated in a total of 14 antral biopsy tissues obtained from the patients with either chronic gastritis (n = 6) or cancer (n = 8) by gene-specific real-time reverse transcriptase -PCR analyses. We employed primer sets specific for CYPs -1A1, -1A2, -2A6, -2B6, -2C, -2D6, -2E1, and -3A5.Among the isoforms CYP1A1, CYP2C and CYP2D6 gave rise to detectable mRNAs in all 14 gastric tissues while the mRNAs for the other CYPs were detected in some of the tissues. The expression patterns were compared to clinical parameters. There were no significant differences in the parameters between the two groups; however the mRNA expression of CYP2A6 was significantly higher in women than man (p0.05).Our data suggests that the CYP isoforms were independently expressed with respect to the pathological status in human gastric tissue.

1a A number of studies reported Mannich bases to manifest antimicrobial, cytotoxic, anticancer, anti-inflammatory, and anticonvulsant activities. A considerable number of therapeutically important cytotoxic compounds are active on DNA topoisomerases that regulate the DNA topology. In the present study we evaluated the biological activity of mono- Mannich bases, 1-aryl-3-phenethylamino-1-propanone hydrochlorides (- 10a), and semicyclic mono- Mannich bases, 3-aroyl-4-aryl-1-phenethyl-4-piperidinols (1b - 9b), synthesized in our laboratory. We employed androgen-independent human prostate cancer cells (PC-3) to assess the cytotoxicity of the compounds and extended the biological activity evaluation to cover supercoil relaxation assays of mammalian type I topoisomerases. Our results showed that the compounds had cytotoxicity within the 8.2 - 32.1 μM range, while two compounds gave rise to a comparable average value in topo I interference of 42% and 40% for 10a (with a hydroxy substituent on the phenyl ring from mono-Mannich bases) and 5b (with a fluoro substituent on the phenyl ring from the semicyclic mono-Mannich base series, piperidinols), respectively

Intimal hyperplasia due to smooth muscle cell proliferation and migration has been reported to be responsible for the pathogenesis of atherosclerosis and restenosis, manifested following balloon angioplasty. In this study, we employed the balloon angioplasty model to study telomere length regulation in proliferating vascular smooth muscle cells. Our results showed that balloon angioplasty in iliac arteries resulted in intimal hyperplasia due to proliferation of the smooth muscle cells and small size telomeric restrictional fragments were evident in injured arteries.

Some acetophenone (CAS 98-86-2) derived bis Mannich bases, bis-(3-aryl-3-oxo-propyl)-methylamine hydrochlorides (B1-B5) and representative piperidinols, 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlorides (C1, C2 and C5), which are the structural isomers of B1, B2 and B5, were synthesized and their effects on DNA topoisomerase I were tested. Aryl part was phenyl in B1 and C1, p-methylphenyl in B2 and C2, p-methoxyphenyl in B3, p-chlorophenyl in B4, and 2-thienyl in B5 and C5. The compounds' chemical structures were confirmed by UV, IR, 1H NMR, 13C NMR, ESI-MS spectra. The purity levels of the compounds were determined by elemental analysis. Among the compounds, B1-B5 and C5 were found to inhibit DNA topo isomerase I at varying degrees. The compounds B1-B5 and C5 manifested an average of 46%, 20%, 40%, 22%, 24% and 22% inhibition on topoisomerase I, respectively, suggesting that the cytotoxic actions of the compounds may be linked to DNA topoisomerase I inhibition. There was a significant negative correlation between the LC50 values reported and topoisomerase I inhibition among the compounds studied. The topoisomerase inhibiting effects of the compounds of the B series may be attributed to the linear structures of the compounds and the possible formation of the hydrogen bonds with the DNA nucleotides. Among the compounds studied, the most potent topoisomerase I inhibiting compounds B1 (46%) and B3 (40%) may serve as model compounds to develop new more potent topoisomerase inhibitors in future studies.

Chalcones (1,3-diaryl-2-propen-1-ones) are ?, ß-unsaturated ketones with cytotoxic and anticancer properties. Several reports have shown that compounds with cytotoxic properties may also interfere with DNA topoisomerase functions. Five derivatives of 4'-hydroxychalcones were examined for cytotoxicity against transformed human T (Jurkat) cells as well as plasmid supercoil relaxation experiments using mammalian DNA topoisomerase I. The compounds were 3-phenyl-1-(4'-hydroxyphenyl)-2-propen-1-one (I), 3-(p-methylphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (II), 3-(p-methoxyphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (III), 3-(p-chlorophenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (IV), and 3-(2- thienyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (V). The order of the cytotoxicity of the compounds was; IV > III > II > I > V. Compound IV, had the highest Hammett and log P values (0.23 and 4.21, respectively) and exerted both highest cytotoxicity and strongest DNA topoisomerase I inhibition. Compounds I and II gave moderate interference with the DNA topoisomerase I while III & V did not interfere with the enzyme. © 2009 Informa UK Ltd.

PubMed ID: 19040121, Cytochrome P450 (CYP) is a heme-containing enzyme superfamily metabolizing a wide variety of xenobiotics, including drugs and carcinogens. The majority of CYP genes are expressed in the liver, however, some CYP isoforms are also reported for a number of extra hepatic tissues. We analyzed Cytochrome P450-2A6, -3A5 and -4B1 mRNAs using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) in a total of 21 homogenized prostate tissues with or without malignancy. We detected a consistent expression of CYP2A6 and CYP3A5 in all, and of CYP4B1 in some (11/21) of the samples at mRNA level. Neither the histopathological status nor the smoking habit of the individuals affected CYP4B1 expression. Our results reflect possible roles for these particular CYPs in therapy and protection of prostate tissue. © 2008 Verlag der Zeitschrift für Naturforschung.

Benzimidazole is one of the most important heterocyclic groups manifesting various biological properties, such as antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Several benzimidazole derivatives are also active as inhibitors of type I DNA topoisomerases. In this study, three 1H-benzimidazole derivatives with different electronic characteristics at position 5-, namely 5-chloro-4-(1H-benzimidazole-2-yl)phenol (Cpd I), 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) and 4-(1H-benzimidazole-2-yl)phenol (Cpd III), were synthesized and evaluated for their effects on mammalian type I DNA topoisomerase activity using quantitative in vitro plasmid supercoil relaxation assays. For the structure elucidation of the compounds, melting points, UV, IR, 1H NMR, 13C NMR, mass spectral data and elemental analyses were interpreted. Among the compounds, 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) manifested relatively potent topoisomerase I inhibition.

WOS: 000237848000007, DNA topoisomerases are essential enzymes that regulate the conformational changes in DNA topology by catalyzing the concerted breakage and rejoining of DNA strands during normal cellular growth. During the past few years, there has been considerable pharmacological interest in these enzymes because the inhibitors of DNA topoisomerases represent a major class of anticancer drugs. In this study, we investigated the effects of the extracts, prepared from a number of Helichrysum taxa, on mammalian DNA topoisomerase I via in vitro supercoil relaxation assays using plasmid substrate, pBR322. Cytotoxic alkaloid, camptothecin, an inhibitor of eukaryotic topoisomerase I, was used as reference compound throughout the assays. Quantitative comparisons of the supercoiled and relaxed DNA band intensities on agarose gels suggested that the extracts from H. pallasii (Sprengel) Ledeb., H. armenium DC subsp. araxinum (Kirp.) Takht, and H. plicatum DC subsp. plicatum manifested a considerable inhibition on the enzyme in a dose-dependent manner. The inhibition of mammalian DNA topoisomerase I by Helichrysum extracts is a significant result because they can be potential sources of anticancer drugs.

This study investigates the contribution of deformational strain imposed by topological interconversions of DNA in ethidium bromide-binding on agarose gels. Closed-circular plasmid DNAs were nicked using UV exposure and the DNA bands were quantified by densitometry. The results show that the closed circular DNA binds the same amount of the dye as its nicked counterpart. The relationship between the band intensity on X-ray films of chemiluminescence-detected Southern blots and DNA concentration was shown to be linear.

This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.

Selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors and used as substrates in a kinetic analysis of the highly purified bovine mitochondrial type I topoisomerase. Recombinant plasmids containing the bovine mtDNA heavy and light strand origins of replication (pZT-Hori and pZT-Lori, respectively), a major transcription termination region (pZT-Term) and a portion of cytochrome b gene (pZT-Cytb) were prepared. Southern hybridization using probes specific for either control or mtDNA-containing plasmid indicated a relative preference by the mitochondrial topoisomerase I to relax supercoils in pZT-Hori and pZT-Term. Quantitative determination of kinetic parameters derived from double-reciprocal Lineweaver-Burk plots showed that recombinant plasmids containing the heavy and light strand origins and the transcription termination region were preferentially relaxed by the mitochondrial enzyme with Km values 2.3- to 3.3-fold lower than controls. The Km values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 +/- 0.9 microM, 25.2 +/- 1.0 microM and 17.0 +/- 0.8 microM, respectively, while those for control plasmids were 57.5 +/- 2.1 microM and 56.3 +/- 2.3 microM. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km = 53.4 +/- 2.0 microM vs. 56.3 +/- 2.3 microM, respectively) indicating that mitochondrial topoisomerase I preferentially interacts with certain mtDNA sequences but not others. Identical experiments with the purified nuclear enzyme did not differentiate between control or mtDNA containing plasmids.

Developing small molecule inhibitors in anti-cancer drug research is an integral part of clinical oncology. Gefitinib (Iressa) is a promising small molecule inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. It was shown to have the potential to improve the clinical effectiveness of EGFR-targeted therapies and gained a significant impact in treatment of non-small-cell lung cancer (NSCLC). Over the past years, several naturally occurring and synthetic compounds with anti-cancer activities were shown to exert their actions via interfering with normal DNA topoisomerase reactions. DNA topoisomerases are ubiquitous enzymes that regulate DNA topology by making transient breakages at nucleic acid backbone and rejoining of DNA strands. Topoisomerases carry out their reactions by two different mechanisms; Type I topoisomerases make a single-stranded break in a DNA duplex while type II topoisomerases create transient breaks in both strands of a duplex. In this study we aimed to examine the effects of Gefitinib on these enzymes to extend the evaluation of its biological activities by plasmid supercoil relaxation and kinetoplast decatenation assays for type I and type II topoisomerases, respectively. Our results are discussed in relation to the outcomes of Gefitinib reported in clinical trials, which together would contribute in identifying the mechanisms of Gefitinib-induced tumor cell-killing. Key Words; Gefitinib; DNA topoisomerases; anti-cancer drugs Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1688.

WOS: 000175163500008, PubMed ID: 11960911, We investigated the relationship between inter-individual difference in CYP2A6 genotype and susceptibility to oral cancer among habitual betel quid chewers in a Sri Lanka population. A total of 286 subjects showing oral malignant or premalignant lesions and 135 control subjects with no lesions were analyzed. The frequency of homozygotes for CYP2A6*4C mutation, a gene deletion type of polymorphism, was significantly lower in the case subjects than the controls. The odds ratio (OR) of the group homozygous for the deletion was significantly lower and calculated to be 0.14 (95% CI; 0.03-0.72). In the allelic base analysis, there was also a significant decrease in the OR of the deletion allele. Our data suggest that deficient CYP2A6 activity due to genetic polymorphism reduces oral cancer risk in betel quid chewers.

Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several “hits” were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs.; La résistance acquise aux traitements anticancéreux représente un problème clinique majeur. Les voies de réparation de l'ADN fournissent un mécanisme de résistance, mais celle-ci peut aussi résulter de mutations ou d'une expression réduite de la protéine ciblée. La surexpression ou la localisation nucléaire de la Y-Box binding (YB-1) protéine est considérée comme un marqueur pronostique de chimiorésistance de la tumeur. YB-1 interagit avec plusieurs partenaires ; dans cette étude, nous nous sommes concentrés sur son interaction avec l'enzyme de réparation de l'ADN NTH1 (hNTH1) et l'ADN topoisomérase I (hTopoI), deux enzymes stimulées par YB-1. L'abondance du complexe hNTH1/YB-1 est accrue dans les cellules tumorales résistantes au cisplatine. La TopoI humaine est une enzyme essentielle impliquée dans la régulation cellulaire du surenroulement de l'ADN et est la cible de plusieurs agents anticancéreux. YB-1 augmente la sensibilité à l'inhibiteur de TopoI, la camptothécine, dans les tumeurs. Nous avons caractérisé les complexes YB-1/hNTH1 et YB-1/hTopoI in vitro et in vivo en utilisant des mesures de transfert d'énergie par résonance en fluorescence (ou FRET) pour identifier et développer de nouvelles stratégies pour le traitement de tumeurs chimio-résistantes. Nous avons développé et optimisé un biosenseur original basé sur le FRET pour cribler deux chimiothèques de taille moyenne afin d’identifier des inhibiteurs potentiels du complexe hNTH1/YB-1. Plusieurs «hits» ont été identifiés qui réduisent de façon significative le niveau de FRET de notre biosenseur. Pour certains de ces composés, nous avons reproduit ces résultats à partir de poudres, effectué des courbes dose-réponse et validé leurs actions en tant qu'inhibiteurs de l'interface hNTH1/YB-1 en utilisant d'autres tests d’interactions. Ensemble nos résultats démontrent que YB-1 interagit directement et stimule des enzymes de la réparation de l'ADN et du relaxation de l’ADN, et que cibler l’interface YB-1/hNTH1 représente une nouvelle stratégie intéressante pour le développement de traitements anticancéreux.

Acquired resistance to anti-cancer therapy is common and is a major clinical issue. Functional DNA repair pathways provide a common mechanism for drug resistance, but it can also result from mutations or reduced expression of the targeted protein. The overexpression or nuclear localisation of the multifunctional Y-box binding protein (YB-1) is considered as a prognostic marker for drug resistance in tumours. YB-1 has several interaction partners in cells; in this study, we have focused on its interaction with the human DNA repair enzyme NTH1 (hNTH1) and human DNA topoisomerase I (hTopoI), two enzymes that have been shown to be stimulated by YB-1. The abundance of the hNTH1/YB-1 complex was shown to increase in cisplatin-resistant tumour cells. Human TopoI is an essential enzyme involved in cellular regulation of DNA supercoiling and is the target of several anti-cancer agents. YB-1 enhances the activity of hTopoI and its sensitivity to hTopoI inhibitor, camptothecin in tumour cells. We have characterised the YB-1/hNTH1 and YB-1/hTopoI complexes in vitro and in vivo using Fluorescence Resonance Energy Transfer (FRET) measurements to identify and develop new strategies for the treatment of drug-resistant tumours. We also designed and optimised an original FRET-based biosensor to screen two medium-sized chemical libraries in order to find potential inhibitors of the hNTH1/YB-1 complex. Several "hits" were identified that significantly reduced the FRET level of our biosensor. For some of these compounds, we have reproduced these results starting from powders, have performed dose-response curves and have validated their actions as inhibitors of the hNTH1/YB-1 interface using alternative binding assays. Taken together, our results demonstrate that YB-1 directly interacts and stimulates a DNA repair and a DNA relaxing enzyme and targeting the YB-1/hNTH1 interface represents an interesting new strategy for the development of anti-cancer drugs., Anti-kanser tedavisine karşı kazanılan direnç yaygın ve önemli bir klinik sorundur. Fonksiyonel DNA onarım yolları, ilaç direnci için yaygın bir mekanizma sağlamaktadır, ancak aynı zamanda, hedeflenen proteinin mutasyonlarından veya ekspresyonunun azalmasından da kaynaklanabilmektedir. Çok işlevli Y-box bağlanma proteinin (YB-1) aşırı ekspresyonu veya nükleer lokalizasyonu, tümörlerde ilaç direnci için bir prognostik belirteç olarak kabul edilmektedir. YB-1'in hücrelerde birkaç etkileşim partneri vardır; bu çalışmada, YB-1 ile aktiviteleri uyarıldığı gösterilen insan DNA onarım enzimi NTH1 (hNTH1) ve insan DNA topoizomeraz I (hTopoI) ile olan etkileşimleri üzerine odaklandık. Cisplatin-dirençli tümör hücrelerinde hNTH1/YB-1 kompleks oluşumunun arttığı gösterilmiştir. İnsan TopoI, DNA süpersarmallarının hücresel regülasyonunda yer alan ve birkaç anti-kanser ajanının hedefi olan önemli bir enzimdir. YB-1, hTopoI aktivitesini ve tümör hücrelerinin hTopoI inhibitörü Campthotecin'e olan duyarlılığını artırmaktadır. Bu çalışmada YB-1/hNTH1 ve YB-1/hTopoI komplekslerini in vitro ve in vivo olarak, ilaca dirençli tümörlerin tedavisi için yeni stratejiler tanımlamak ve geliştirmek için Fluoresans Rezonans Enerji Transferi (FRET) ölçümlerini kullanarak karakterize ettik. Ayrıca, hNTH1/YB-1 kompleksinin potansiyel inhibitörlerini bulmak için iki orta ölçekli kimyasal kütüphaneyi taramak üzere orijinal bir FRET-bazlı biyosensör tasarladık ve optimize ettik. Biyosensörümüzün FRET seviyesini önemli ölçüde azaltan birkaç "hit" tespit ettik. Bu bileşiklerin bazıları için, toz bileşiklerden başlayarak bu sonuçları tekrar ürettik, doz-cevap eğrileri gerçekleştirdik ve alternatif bağlama deneyleri kullanarak hNTH1/YB-1 arayüzünün inhibitörleri olarak onayladık. Birlikte ele alındığında, sonuçlarımız YB-1'in DNA onarımı ve DNA relakse edici enzimleriyle doğrudan etkileşime girip uyardığını ve YB-1/hNTH1 arayüzünü hedef almanın, anti-kanser ilaçlarının geliştirilmesi için ilginç yeni bir stratejiyi temsil ettiğini göstermektedir.