title: Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study.
abstract: OBJECTIVE
  Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent.
  
  
  RESULTS
  DC altered cell proliferation, decreased viability and increased apoptosis in Vero and MCF-7 cell lines but did not show any toxic effect on mouse ovarian tissues and developing oocytes in vitro and in vivo.
  
  
  BACKGROUND
  DC suppresses cell proliferation and increases apoptosis in various cancer cells such as breast, urogenital and melanoma. DC has also been reported to alter the anticancer effects of several chemotherapeutics, including cisplatin, gemcitabine and doxorubicin in prostate, liver and uroepithelial cancer cells, respectively.
  
  
  METHODS
  Vero (African green monkey kidney epithelial cells) and MCF-7 (human cancerous breast epithelial cells) cell lines and mouse granulosa cells isolated from 21-day-old female BALB/c mice (n = 21) were used to assess the effects of DC (10, 50, 100 and 200 µm) for 24 and 48 h on cell proliferation, viability and apoptotic cell death. In vivo experiments were performed with a single i.p. injection of 32 mg/kg DC in 21-day-old female BALB/c mice (n = 12). Following 48 h, animals were sacrificed by cervical dislocation and histological sections of isolated ovaries were evaluated for apoptosis. Viability assays were based on the trypan blue dye exclusion method and an automated cell counter device was used. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and Annexin-V immunofluorescence were assessed by 3D confocal microscopy to address apoptotic cell death. We also assessed whether DC inhibits cell proliferation and viability through NQO1 [NAD(P)H Quinone Oxidoreductase 1], an intracellular inhibitor of reactive oxygen species (ROS). The meiotic spindle and chromosomes were studied in mouse oocytes by α-β-tubulin and 7-aminoactinomycine D (7-AAD) immunostaining in vitro and in vivo.
  
  
  RESULTS
  DC does not block oocyte maturation and no significant alteration was noted in meiotic spindle or chromosome morphology in metaphase-II (M-II) stage oocytes following DC treatment in vitro or in vivo. In contrast, exposure to DC for 24 h suppressed cell proliferation (P = 0.026 at 200 µm), decreased viability (P = 0.002 at 200 µm) and increased apoptosis (P = 0.048 at 100 µm) in Vero and MCF-7 cell lines, compared with controls. These changes were not related to intracellular NQO1 levels. Mouse granulosa cells were unaffected by 50 or 100 µm DC treatment for 24 and 48 h in vitro. DC treatment in vivo did not alter the number of primordial follicles or the ratio of apoptosis in primordial, primary and secondary follicles, as well as in antral follicles, compared with the controls.
  
  
  CONCLUSIONS
  DC was tested for ovarian toxicity only in isolated mouse oocytes/ovaries and healthy BALB/c mice. No cancer formation was used as an in vivo test model. The possibility that DC may potentiate ovarian toxicity when combined with traditional chemotherapeutic agents, such as mitomycin-C, cisplatin, gemcitabine and doxorubicin, must be taken into account, as DC is known to alter their effects in some cancer cells.
  
  
  CONCLUSIONS
  The present study evaluated, for the first time, the effect of DC on ovarian tissue. The results suggested that DC is not toxic to ovarian tissues and developing oocytes; therefore, DC should be assessed further as a potential anticancer agent when female fertility preservation is a concern.
  
  
  METHODS
  N/A.
  
  
  BACKGROUND
  This work includes data from dissertation thesis entitled 'Effects of dicoumarol on mitotic and meiotic cells as an anticancer agent' by DA, 2014 and was partly supported by The National Scientific and Technological Research Council of Turkey (SBAG-109S415) to AC, OC and SO. The authors confirm that this article content presents no conflicts of interest.
authors.affiliation: Ankara University Biotechnology Institute, Tandogan, Ankara 06500, Turkey.
authors.forename: Duru
authors.initials: D
authors.lastname: Aras
authors.affiliation: Department of Histology and Embryology, Laboratory for Stem Cells and Reproductive Biology, Ankara University School of Medicine, Sihhiye, Ankara 06100, Turkey.
authors.forename: Ozgur
authors.initials: O
authors.lastname: Cinar
authors.affiliation: Department of Histology and Embryology, Laboratory for Stem Cells and Reproductive Biology, Ankara University School of Medicine, Sihhiye, Ankara 06100, Turkey.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Cakar
authors.affiliation: Department of Obstetric and Gynaecology, Centre for Assisted Reproduction, Ankara University School of Medicine, Cebeci, Ankara 06590, Turkey.
authors.forename: Sinan
authors.initials: S
authors.lastname: Ozkavukcu
authors.affiliation: Department of Histology and Embryology, Laboratory for Stem Cells and Reproductive Biology, Ankara University School of Medicine, Sihhiye, Ankara 06100, Turkey alpcan@medicine.ankara.edu.tr.
authors.forename: Alp
authors.initials: A
authors.lastname: Can
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D002522:Chlorocebus aethiops; D001728:Dicumarol / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008940:Mitotic Index; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D009865:Oocytes / Q000187:drug effects; D059351:Organ Sparing Treatments; D010053:Ovary / Q000187:drug effects; D008941:Spindle Apparatus / Q000187:drug effects / Q000648:ultrastructure; D014709:Vero Cells
chemical_list: D000970:Antineoplastic Agents; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); C491732:Nqo1 protein, mouse
keywords: apoptosis; dicoumarol; fertility preservation; meiosis; oocyte