title: Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells.
abstract: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Maribel
authors.initials: M
authors.lastname: Soto-Nuñez
authors.affiliation: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Karen Azucena
authors.initials: KA
authors.lastname: Díaz-Morales
authors.affiliation: Research Division, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Patricia
authors.initials: P
authors.lastname: Cuautle-Rodríguez
authors.affiliation: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Víctor
authors.initials: V
authors.lastname: Torres-Flores
authors.affiliation: Department of Chronic-Degenerative Diseases, National Institute of Respiratory Diseases 'Ismael Cosio Villegas', Mexican Ministry of Health, Mexico City 14080, Mexico.
authors.forename: José Sullivan
authors.initials: JS
authors.lastname: López-González
authors.affiliation: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Mandoki-Weitzner
authors.affiliation: Department of Pharmacology, School of Medicine, National University of Mexico, Mexico City 04510, Mexico.
authors.forename: Juan Arcadio
authors.initials: JA
authors.lastname: Molina-Guarneros
keywords: 7-hydroxycoumarin; A549 cells; apoptosis; caspase-3; microinjection