OBJECTIVE: Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: N/A.
UNASSIGNED: 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.

BACKGROUND: Enormous interest had been paid to the coordination chemistry of alkali and alkaline metal ions because of their role inside body viz; their Li(+)/Na(+) exchange inside the cell lead to different diseases like neuropathy, hypertension, microalbuminuria, cardiac and vascular hypertrophy, obesity, and insulin resistance. It has been presumed that alkali metal ions (whether Na(+) or K(+)) coordinated to chelating ligands can cross the hydrophobic cell membrane easily and can function effectively for depolarizing the ion difference. This unique function was utilized for bacterial cell death in which K(+) has been found coordinated valinomycin (antibiotic).
RESULTS: Distinct sodium adduct (1) with dicoumarol ligand, 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-chromen-3-yl)-phenyl-methyl]-chromen-2-one (L) is isolated from the saturated solution of sodium methoxide. Single crystal X-ray diffraction studies of the adduct reveals that sodium is in the form of cation attached to a methoxide, methanol and a dicoumarol ligand where carbonyl functional groups of the coumarin derivative are acting as bridges. The sodium compound (1) is also characterized by IR, (1)H-NMR, and (13)C{(1)H}-NMR spectroscopic techniques. The composition is confirmed by elemental analysis. DFT study for 1 has been carried out using B3LYP/6-13G calculations which shown the theoretical confirmation of the various bond lengths and bond angles. Both the compounds were studied subsequently for the U2OS tumoricidal activity and it was found that L has LD50 value of 200 μM whereas the sodium analog cytotoxicity did not drop down below 60%.
CONCLUSIONS: A sodium analogue (1) with medicinally important dicoumarol ligand (L) has been reported. The crystal structure and DFT study confirm the formation of cationic sodium compound with dicoumarol. The ligand was found more active than the sodium analog attributed to the instability of 1 in solution state. Coumarin compound with sodium was observed to be less cytotoxic than the ligand, its LD50 value never dropped below 60%.