title: Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine.
abstract: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer.
  
  
  
  Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs.
  
  
  
  Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762.
  
  
  
  AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Makoto
authors.initials: M
authors.lastname: Isono
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Michèle J
authors.initials: MJ
authors.lastname: Hoffmann
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Pinkerneil
authors.affiliation: Department of Urology, National Defense Medical College, Namiki 3-2, 359-8513, Tokorozawa, Japan.
authors.forename: Akinori
authors.initials: A
authors.lastname: Sato
authors.affiliation: Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Michaelis
authors.affiliation: Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Paul-Ehrlich‑Str. 40, 60596, Frankfurt am Main, Germany.
authors.forename: Jindrich
authors.initials: J
authors.lastname: Cinatl
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Niegisch
authors.affiliation: Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany. Wolfgang.Schulz@uni-duesseldorf.de.
authors.forename: Wolfgang A
authors.initials: WA
authors.lastname: Schulz
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1 / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering; D013876:Thiophenes / Q000494:pharmacology*; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D000093542:Gemcitabine
chemical_list: C532363:3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide; D000964:Antimetabolites, Antineoplastic; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D013876:Thiophenes; D003841:Deoxycytidine; D014508:Urea; D011065:Poly(ADP-ribose) Polymerases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D020169:Caspases; D000093542:Gemcitabine
keywords: AZD7762; Bladder cancer; Checkpoint kinase; Gemcitabine; Urothelial carcinoma