Eberhard Unger

Publications (25)121.32 Total impact

• Article: Phenylimino-10H-anthracen-9-ones as novel antimicrotubule agents-synthesis, antiproliferative activity and inhibition of tubulin polymerization.

  Helge Prinz, Peter Schmidt, Konrad J Böhm, Silke Baasner, Klaus Müller, Matthias Gerlach, Eckhard G Günther, Eberhard Unger
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  ABSTRACT: A novel series of phenylimino-10H-anthracen-9-ones and 9-(phenylhydrazone)-9,10-anthracenediones were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. The 10-(3-hydroxy-4-methoxy-phenylimino)-10H-anthracen-9-one 15h and its dichloro analog 16b were identified as potent inhibitors of tumor cell growth (16b, IC(50) K562 0.11 μM), including multidrug resistant phenotypes. Compound 15h had excellent activity as an inhibitor of tubulin polymerization. Concentration-dependent cell cycle analyzes by flow cytometry confirmed that KB/HeLa cells treated by 15h and 16b were arrested in the G2/M phases of the cell cycle. In competition experiments, 15h strongly displaced radiolabeled colchicine from its binding site on tubulin, showing IC(50) values similar to that of colchicine. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.
  Bioorganic & medicinal chemistry 06/2011; 19(14):4183-91. · 2.82 Impact Factor
• Article: Synthesis, antiproliferative activity and inhibition of tubulin polymerization by 1,5- and 1,8-disubstituted 10H-anthracen-9-ones bearing a 10-benzylidene or 10-(2-oxo-2-phenylethylidene) moiety.

  Holger C Nickel, Peter Schmidt, Konrad J Böhm, Silke Baasner, Klaus Müller, Matthias Gerlach, Eberhard Unger, Eckhard G Günther, Helge Prinz
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  ABSTRACT: A novel series of 1,5- and 1,8-disubstituted 10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones was synthesized to assess the substituent effects on biological activity. The 3-hydroxy-2,4-dimethoxy-benzylidene analogue 16 h displayed strong antiproliferative activity against several tumor cell lines, including multi-drug resistant phenotypes. Flow cytometric studies showed that KB/HeLa cells treated by elected compounds were arrested in the G2/M phases of the cell cycle. Among the compounds tested for inhibition of tubulin polymerization, 14 compounds proved to be exceptionally active with IC(50) values < 1 microM. In the 1,5-dichloro-derived series of benzylideneanthracenones, E/Z isomers were separated and biological effects were monitored. We found that the olefinic geometry had no significant effect on biological activity. Furthermore, the E isomeric 1,5-dichloro-substituted phenacylidenes entirely proved to be more potent inhibitors of tubulin polymerization than the recently described 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones. In conclusion, the present study improves understanding of the action of anthracenone-based tubulin polymerization inhibitors and contributes to the design of further potent anti-tubulin drugs.
  European journal of medicinal chemistry 08/2010; 45(8):3420-38. · 3.27 Impact Factor
• Article: Containment of extended length polymorphisms in silk proteins.

  Alberto Chinali, Wolfram Vater, Baerbel Rudakoff, Alexander Sponner, Eberhard Unger, Frank Grosse, Karl-Heinz Guehrs, Klaus Weisshart
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  ABSTRACT: The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes. Due to limited numbers of individuals screened to date little is known about the extent of these heterogeneities and how they are finally manifested in the proteins. Using expanded sample sizes, we show that sequence variations expressed as deletions or insertions of tri-nucleotides lead to different sized and structured repetitive units throughout a silk protein. Moreover, major ampullate spidroins 1 can quite dramatically differ in their overall lengths; however, extreme variants do not spread widely in a spider population. This suggests that a certain size range stabilized by purifying selection is important for spidroin 1 gene integrity and protein function. More than one locus for spidroin 1 genes possibly exist within one individual genome, which are homogenized in size, are differentially expressed and give a spider a certain degree of adaptation on silk's composition and properties. Such mechanisms are shared to a lesser extent by the second major ampullate spidroin gene.
  Journal of Molecular Evolution 03/2010; 70(4):325-38. · 2.27 Impact Factor
• Article: 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones as highly active antimicrotubule agents: synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

  Helge Prinz, Peter Schmidt, Konrad J Böhm, Silke Baasner, Klaus Müller, Eberhard Unger, Matthias Gerlach, Eckhard G Günther
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  ABSTRACT: A series of 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones were synthesized and evaluated for interactions with tubulin and for antiproliferative activity against a panel of human and rodent tumor cell lines. The 4-methoxy analogue 17b was most potent, displaying IC(50) values ranging from 40 to 80 nM, including multidrug resistant phenotypes, and had excellent activity as an inhibitor of tubulin polymerization (IC(50) = 0.52 microM). Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with 17b were arrested in the G2/M phases of the cell cycle (EC(50) = 90 nM). In competition experiments, 17b strongly displaced [(3)H]-colchicine from its binding site in the tubulin. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.
  Journal of Medicinal Chemistry 03/2009; 52(5):1284-94. · 4.80 Impact Factor
• Article: Hydrophobic interaction of organic chemicals with microtubule assembly in vitro.

  Thomas Stoiber, Eberhard Unger, Susanne B Dorn, Gisela H Degen, Hermann M Bolt
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  ABSTRACT: A recent concept connecting the lipophilicity of organic chemicals with their genotoxicity on a chromosomal level implies that the lipophilic character of organic chemicals determines a certain background of chromosomal genotoxicity that can be addressed as "non-specific". This is opposed to compounds with more "specific" modes of action. Such mechanisms influence the processes of karyokinesis and cytokinesis. A critical partial process for the chromosomal segregation is the dynamics of assembly and disassembly of microtubules. To broaden the present database for such interactions, chemicals were selected based on their lipophilicity (log P between -1.5 and +1.0) and on hints from the literature pointing to possibilities of interaction with the tubulin-microtubule system. Thus, acetamide, acrylamide, methylmethane sulfonate, acetonitrile, acrylonitrile and cyclohexanone were assessed as to their potencies to influence the dynamic processes of microtubule assembly and disassembly in a cell-free system in vitro. These compounds covered a range of log P between -1.5 and 1.0, complementary to compounds investigated earlier. The entire body of data supports the general concept that hydrophobic interactions are connected with non-specific processes, which contribute to a background genotoxicity on a chromosomal level. It also points to the dynamics of microtubule assembly and disassembly as a decisive partial process involved.
  Archive für Toxikologie 03/2008; 82(9):601-6. · 4.67 Impact Factor
• Article: Sulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

  Anne Zuse, Peter Schmidt, Silke Baasner, Konrad J Böhm, Klaus Müller, Matthias Gerlach, Eckhard G Günther, Eberhard Unger, Helge Prinz
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  ABSTRACT: Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of <100 nM. The methylamino analogue 14i was the most active compound in this assay (14i, IC50 K562: 0.05 muM). Antiproliferative activities of selected compounds were additionally evaluated against a panel of 12 tumor cell lines, including multi-drug-resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with selected compounds were arrested in the G2/M phases of the cell cycle. In competition experiments, these compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values lower than that of colchicine. The results demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.
  Journal of Medicinal Chemistry 11/2007; 50(24):6059-66. · 5.25 Impact Factor
• Source

  Available from: PubMed Central

  Article: Composition and hierarchical organisation of a spider silk.

  Alexander Sponner, Wolfram Vater, Shamci Monajembashi, Eberhard Unger, Frank Grosse, Klaus Weisshart
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  ABSTRACT: Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.
  PLoS ONE 02/2007; 2(10):e998. · 4.09 Impact Factor
• Article: 9-Benzylidene-naphtho[2,3-b]thiophen-4-ones as novel antimicrotubule agents-synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

  Anne Zuse, Peter Schmidt, Silke Baasner, Konrad J Böhm, Klaus Müller, Matthias Gerlach, Eckhard G Günther, Eberhard Unger, Helge Prinz
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  ABSTRACT: A novel series of 9-benzylidene-naphtho[2,3-b]thiophen-4-ones and structurally related compounds were synthesized and evaluated for their ability to inhibit tubulin polymerization. The 4-hydroxy-3,5-dimethoxy-benzylidene analogue 15d was identified as a potent cytotoxic agent in an assay based on K562 leukemia cells. Antiproliferative activity of 15d and the 2,4-dimethoxy-3-hydroxy-benzylidene analogue 15e was additionally evaluated against a panel of 12 tumor cell lines, including multidrug resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that K562 cells as well as KB/HeLa cells treated by 15d were arrested in the G2/M phases of the cell cycle. Moreover, four compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds. In competition experiments, the most active compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values virtually 3- to 4-fold lower than that of colchicine.
  Journal of Medicinal Chemistry 01/2007; 49(26):7816-25. · 5.25 Impact Factor
• Article: 9-Benzylidene-naphtho[2,3-b]thiophen-4-ones as Novel Antimicrotubule AgentsSynthesis, Antiproliferative Activity, and Inhibition of Tubulin Polymerization

  Anne Zuse, Peter Schmidt, Silke Baasner, Konrad J. Böhm, Klaus Müller, Matthias Gerlach, Eckhard G. Günther, Eberhard Unger, Helge Prinz
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  ABSTRACT: A novel series of 9-benzylidene-naphtho[2,3-b]thiophen-4-ones and structurally related compounds were synthesized and evaluated for their ability to inhibit tubulin polymerization. The 4-hydroxy-3,5-dimethoxy-benzylidene analogue 15d was identified as a potent cytotoxic agent in an assay based on K562 leukemia cells. Antiproliferative activity of 15d and the 2,4-dimethoxy-3-hydroxy-benzylidene analogue 15e was additionally evaluated against a panel of 12 tumor cell lines, including multidrug resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that K562 cells as well as KB/HeLa cells treated by 15d were arrested in the G2/M phases of the cell cycle. Moreover, four compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds. In competition experiments, the most active compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values virtually 3- to 4-fold lower than that of colchicine.
  11/2006;
• Article: Design, synthesis, and biological evaluation of 3,4-diarylmaleimides as angiogenesis inhibitors.

  Christian Peifer, Thomas Stoiber, Eberhard Unger, Frank Totzke, Christoph Schächtele, Dieter Marmé, Ruth Brenk, Gerhard Klebe, Dieter Schollmeyer, Gerd Dannhardt
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  ABSTRACT: The new analogue 2 of combretastatin A-4 was discovered to be an inhibitor of tubulin polymerization with an IC50 of 7.6 microM and reduced angiogenesis in the in vivo chick embryo model. Interestingly, in a series of 2,3-diarylmaleimides closely related to this lead, no other compound was found to be active in the tubulin polymerization assay. However, by screening in the in vivo chick embryo assay 10 was identified as a potent angiogenesis inhibitor indicating an alternative target. Indeed, molecular modeling studies suggest a reasonable binding mode of 10 at the ATP-binding site of the model kinase CDK2. Motivated by these results, analogues of 10 were screened for inhibitory activity in a panel of 12 selected protein kinases and a high affinity of 10 to VEGF-R2 was found showing an IC50 of 2.5 nM. Structure-activity relationships (SAR) for this compound series with the isolated enzyme and equivalent antiangiogenic activity in the chick embryo assay are presented herein.
  Journal of Medicinal Chemistry 03/2006; 49(4):1271-81. · 5.25 Impact Factor
• Article: The conserved C-termini contribute to the properties of spider silk fibroins.

  Alexander Sponner, Wolfram Vater, Winfried Rommerskirch, Fritz Vollrath, Eberhard Unger, Frank Grosse, Klaus Weisshart
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  ABSTRACT: Spider silk fibroins can adopt different structural states at high protein concentrations. They are soluble within the spinning dope of the glands, but readily converted into insoluble polymers upon extrusion. A contribution of the C-termini to the maintenance and conversion of these states is suggested by their predicted secondary structures and biochemical behavior in vitro. Special sequence parts endow the C-termini with the capability to promote both the solubility and aggregation of the fibroins depending on the environmental conditions.
  Biochemical and Biophysical Research Communications 01/2006; 338(2):897-902. · 2.48 Impact Factor
• Article: Differential polymerization of the two main protein components of dragline silk during fibre spinning.

  Alexander Sponner, Eberhard Unger, Frank Grosse, Klaus Weisshart
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  ABSTRACT: Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3, 6, 7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk.
  Nature Material 11/2005; 4(10):772-5. · 32.84 Impact Factor
• Article: Genotoxicity of inorganic lead salts and disturbance of microtubule function.

  Daniela Bonacker, Thomas Stoiber, Konrad J Böhm, Irina Prots, Minsheng Wang, Eberhard Unger, Ricarda Thier, Hermann M Bolt, Gisela H Degen
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  ABSTRACT: Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 microM lead chloride and 0.05 microM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 microM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 microM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 microM and reached half-maximal inhibition of motility at about 50 microM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.
  Environmental and Molecular Mutagenesis 06/2005; 45(4):346-53. · 3.71 Impact Factor
• Article: Characterization of the protein components of Nephila clavipes dragline silk.

  Alexander Sponner, Bernhard Schlott, Fritz Vollrath, Eberhard Unger, Frank Grosse, Klaus Weisshart
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  ABSTRACT: Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.
  Biochemistry 04/2005; 44(12):4727-36. · 3.42 Impact Factor
• Article: Genotoxicity of inorganic mercury salts based on disturbed microtubule function.

  Daniela Bonacker, Thomas Stoiber, Minsheng Wang, Konrad J Böhm, Irina Prots, Eberhard Unger, Ricarda Thier, Hermann M Bolt, Gisela H Degen
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  ABSTRACT: This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 microM, and inhibition is complete at about 10 microM. In this range, the tubulin assembly is fully (up to 6 microM) or partially (~6-10 microM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 microM Hg2+, the IC50 5.8 microM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 microM and a complete inhibition is reached at 1 microM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 microM HgCl2. Between 15 and 20 microM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 microM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 microM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.
  Archive für Toxikologie 11/2004; 78(10):575-83. · 4.67 Impact Factor
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  Available from: scienceformulas.com

  Article: Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II).

  Thomas Stoiber, Daniela Bonacker, Konrad J Böhm, Hermann M Bolt, Ricarda Thier, Gisela H Degen, Eberhard Unger
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  ABSTRACT: Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 microM of complexed Hg(II), and for inhibition of motility it was 0.05 microM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 microM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.
  Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/2004; 563(2):97-106. · 2.85 Impact Factor
• Article: Interaction of mercury(II) with the microtubule cytoskeleton in IMR-32 neuroblastoma cells.

  Thomas Stoiber, Gisela H Degen, Hermann M Bolt, Eberhard Unger
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  ABSTRACT: On the background of the neurotoxicity of mercury compounds, the interaction of mercury(II) with the cytoskeleton was investigated in vitro using IMR-32 neuroblastoma cells. Conditions for culture of these cells on microscopic slides and procedures for immunofluorescence staining of the microtubule network were optimised. Both morphology and viability of IMR-32 cells were affected by mercury(II) at concentrations higher than 15 microM. Pronounced disintegration of the microtubule cytoskeleton was detected at 30 microM mercury(II). Compared to previous studies with fibroblasts, the no-observed-effect concentration was markedly lower, pointing to a particular sensitivity of nerve cells to mercury. This could be due to disturbed information transfer processes depending on an intact microtubule system.
  Toxicology Letters 07/2004; 151(1):99-104. · 3.23 Impact Factor
• Article: Chromosomal genotoxicity of nitrobenzene and benzonitrile.

  Daniela Bonacker, Thomas Stoiber, Konrad J Böhm, Eberhard Unger, Gisela H Degen, Ricarda Thier, Hermann M Bolt
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  ABSTRACT: In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 micro M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 micro M, and no-effect-concentrations were between 0.001 and 0.005 micro M. Clearly enhanced MN rates were found at 0.1 micro M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37 degrees C was seen above the no-effect-concentration of 2 mM, with an IC(50) of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 micro M and reaching complete inhibition of motility at 30 micro M, whereas benzonitrile up to 200 micro M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 micro M. This points to the relevance of interactions with the cellular spindle apparatus.
  Archive für Toxikologie 02/2004; 78(1):49-57. · 4.67 Impact Factor
• Article: Chromosomal genotoxicity of nitrobenzene and benzonitrile

  Daniela Bonacker, Thomas Stoiber, KonradJ. Böhm, Eberhard Unger, GiselaH. Degen, Ricarda Thier, HermannM. Bolt
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  ABSTRACT: In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18h treatment with up to 1mM nitrobenzene or 1mM benzonitrile. Subsequently, a concentration range up to 100M was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01M, and no-effect-concentrations were between 0.001 and 0.005M. Clearly enhanced MN rates were found at 0.1M and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1mM nitrobenzene without addition of DMSO. Higher concentrations (5mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15mM. For benzonitrile in presence of DMSO, a clear dose–response of inhibition of tubulin assembly at 37C was seen above the no-effect-concentration of 2mM, with an IC50 of 13mM and protein denaturation starting above a level of about 20mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5mM nitrobenzene or 13mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin–kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5M and reaching complete inhibition of motility at 30M, whereas benzonitrile up to 200M did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1M. This points to the relevance of interactions with the cellular spindle apparatus.
  Archive für Toxikologie 12/2003; 78(1):49-57. · 4.67 Impact Factor
• Article: Novel benzylidene-9(10H)-anthracenones as highly active antimicrotubule agents. Synthesis, antiproliferative activity, and inhibition of tubulin polymerization.

  Helge Prinz, Yukihito Ishii, Takeo Hirano, Thomas Stoiber, Juan A Camacho Gomez, Peter Schmidt, Heiko Düssmann, Angelika M Burger, Jochen H M Prehn, Eckhard G Günther, Eberhard Unger, Kazuo Umezawa
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  ABSTRACT: A novel series of 10-benzylidene-9(10H)-anthracenones and 10-(phenylmethyl)-9(10H)-anthracenones were synthesized and evaluated for antiproliferative activity in an assay based on K562 leukemia cells. The 3-hydroxy-4-methoxybenzylidene analogue 9h was found to be the most active compound (IC(50) K562: 20 nM). Structure-activity relationships are also considered. The highly active compound 9h and the 2,4-dimethoxy-3-hydroxybenzylidene analogue 9l were tested against five tumor cell lines using the XTT assay, including multidrug resistant phenotypes. Induction of cell death in a variety of tumor cell lines was determined in a monolayer assay using propidium iodide. Noteworthy, all compounds within the series induced elongations in K562 cells similar to vinblastine-treated cells. The effect of the lead compound 9h on K562 cell growth was associated with cell cycle arrest in G2/M. Concentrations for 50% KB/HeLa cells arrested in G2/M after treatment with 9h and 9l were determined and found to be in the range of 0.2 microM. Additionally, we monitored the dose dependent caspase-3-like protease activity in K562 cells and MCF-7/Casp-3 cells treated with 9h, indicating induction of apoptosis. Western blotting analysis demonstrated that 9h caused a shift in tubulin concentration from the polymerized state found in the cell pellet to the unpolymerized state found in the cell supernatant. Seven compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds such as colchicine, podophyllotoxin, and nocodazole. In general, the antiproliferative activity correlated with inhibition of tubulin polymerization. The most active compounds strongly displaced [(3)H]colchicine from its binding site in the tubulin, yielding IC(50) values 3- to 4-fold lower than that of colchicine. The novel benzylidene-9(10H)-anthracenones described in the present study constitute an interesting group of highly active and easily accessible antimitotic agents that inhibit tubulin polymerization.
  Journal of Medicinal Chemistry 08/2003; 46(15):3382-94. · 5.25 Impact Factor