title: Role of JNK and NF-κB in mediating the effect of combretastatin A-4 and brimamin on endothelial and carcinoma cells.
abstract: OBJECTIVE
  The 4,5-diarylimidazole brimamin is an analog of the natural vascular-disrupting agent combretastatin A-4 (CA-4) with improved water solubility, tolerance by animals and efficacy in multidrug-resistant tumors. Here, we aimed at identifying the major mechanisms underlying the in vitro and in vivo actions of brimamin on endothelial and carcinoma cells, including vascularization.
  
  
  METHODS
  The contribution of specific signaling kinases to the effects of brimamin on cytoskeleton organization and the viability and differentiation of endothelial cells was assessed by MTT and tube formation assays in the presence or absence of specific kinase inhibitors. Changes in DNA affinity and expression of NF-κB in endothelial and carcinoma-derived cells and their solid tumors (xenografts) treated with brimamin were ascertained by electrophoretic mobility shift assays and Western blotting. The anti-vascular effect of brimamin in solid tumors was verified by CD31 immunostaining.
  
  
  RESULTS
  We found that brimamin can inhibit tubulin polymerization and cause a reorganization of F-actin in Ea.hy926 endothelial cells. Its inhibitory effect on tube formation was found to depend on functional Rho kinase and JNK. JNK inhibition was found to suppress the induction of endothelial cell apoptosis by brimamin. In CA-4-refractory human BxPC-3 pancreas carcinoma-derived and triple-negative MDA-MB-231 breast carcinoma-derived cells brimamin was found to inhibit growth and to induce apoptosis at low nanomolar concentrations by blocking NF-κB activation in a dose-dependent manner. Brimamin was also found to reduce the in vivo growth rate and vascularization of MDA-MB-231 xenografts in mice. Residual tumor cells of these treated xenografts showed a relatively low expression of the p65 subunit of NF-κB.
  
  
  CONCLUSIONS
  Our data indicate that cellular JNK and Rho kinase activities are crucial for the cytotoxic and cytoskeleton reorganizing effects of brimamin on endothelial cells. In addition, we found that in resistant carcinoma cells and xenografts brimamin can induce down-regulation of anti-apoptotic NF-κB expression and signaling. Its chemical properties and efficacy against clinically relevant cancer entities make brimamin a promising candidate vascular-disrupting agent.
authors.affiliation: Organic Chemistry Laboratory, University Bayreuth, Universitaetsstrasse 30, 95440, Bayreuth, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Mahal
authors.affiliation: Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, 4100 John R. Street, Detroit, 48201, MI, USA.
authors.forename: Aamir
authors.initials: A
authors.lastname: Ahmad
authors.affiliation: Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, 4100 John R. Street, Detroit, 48201, MI, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Sethi
authors.affiliation: Department of Molecular Structural Biology, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Resch
authors.affiliation: Department of Molecular Structural Biology, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
authors.affiliation: Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, 4100 John R. Street, Detroit, 48201, MI, USA.
authors.forename: Fazlul H
authors.initials: FH
authors.lastname: Sarkar
authors.affiliation: Organic Chemistry Laboratory, University Bayreuth, Universitaetsstrasse 30, 95440, Bayreuth, Germany. Rainer.Schobert@uni-bayreuth.de.
authors.forename: Rainer
authors.initials: R
authors.lastname: Schobert
authors.affiliation: Organic Chemistry Laboratory, University Bayreuth, Universitaetsstrasse 30, 95440, Bayreuth, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Biersack
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001632:Bibenzyls / Q000494:pharmacology*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D042783:Endothelial Cells / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D016328:NF-kappa B / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000473:pathology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000814:Aniline Compounds; D000972:Antineoplastic Agents, Phytogenic; D001632:Bibenzyls; D007093:Imidazoles; D016328:NF-kappa B; C000610583:brimamin; C040105:combretastatin; D048670:MAP Kinase Kinase 4
keywords: Brimamin; Combretastatin A-4; JNK; NF-κB; Vascular-disrupting agents