Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells
Por:
Soto-Nuñez M., Díaz-Morales K.A., Cuautle-Rodríguez P., Torres-Flores V., López-González J.S., Mandoki-Weitzner J.J., Molina-Guarneros J.A.
Publicada:
1 nov 2015
Resumen:
Coumarins have attracted intense interest in recent years due to their apoptogenic effects. The aim of the present study was to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C-3) activity in A549 human lung carcinoma cells. A range of analytical techniques, including colorimetric and fluorometric assays, western blotting, single-cell microinjection, fluorescence microscopy and image analysis were conducted to elucidate the effects of 7-HC. A 24-h exposure to 1.85 mM 7-HC induced a 65% increase in C-3 activity, and a notable conversion of procaspase-3 to C-3, in addition to poly(ADP-ribose)polymerase cleavage. Furthermore, morphological changes associated with apoptosis were observed. Exposure of the cells to 7-HC for 3 or 6 h increased calcium conductance by 27%. By performing the single-cell microinjection of a specific fluorescent substrate of C-3 into previously 7-HC-exposed cells, a typical enzymatic kinetic profile of C-3 activation was identified a number of hours prior to the morphological and biochemical changes associated with apoptosis being observed. These results suggest that the rapid in vivo activation of C-3 is induced by 7-HC, the most relevant biotransformation product of coumarin in humans. © 2015, Spandidos Publications. All rights reserved.
Filiaciones:
Soto-Nuñez M.:
Univ Nacl Autonoma Mexico, Sch Med, Dept Pharmacol, Mexico City 04510, DF, Mexico
Díaz-Morales K.A.:
Univ Nacl Autonoma Mexico, Sch Med, Dept Pharmacol, Mexico City 04510, DF, Mexico
Cuautle-Rodríguez P.:
Univ Nacl Autonoma Mexico, Sch Med, Div Res, Mexico City 04510, DF, Mexico
Torres-Flores V.:
Univ Nacl Autonoma Mexico, Sch Med, Dept Pharmacol, Mexico City 04510, DF, Mexico
López-González J.S.:
Department of Chronic-Degenerative Diseases, National Institute of Respiratory Diseases ‘Ismael Cosio Villegas’, Mexican Ministry of Health, Mexico City, 14080, Mexico
Mandoki-Weitzner J.J.:
Univ Nacl Autonoma Mexico, Sch Med, Dept Pharmacol, Mexico City 04510, DF, Mexico
Molina-Guarneros J.A.:
Univ Nacl Autonoma Mexico, Sch Med, Dept Pharmacol, Mexico City 04510, DF, Mexico
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