Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells

• Published in: Experimental and therapeutic medicine Vol. 10; no. 5; pp. 1789 - 1795
• Main Authors: SOTO-NUÑEZ, MARIBEL, DÍAZ-MORALES, KAREN AZUCENA, CUAUTLE-RODRÍGUEZ, PATRICIA, TORRES-FLORES, VÍCTOR, LÓPEZ-GONZÁLEZ, JOSÉ SULLIVAN, MANDOKI-WEITZNER, JUAN JOSÉ, MOLINA-GUARNEROS, JUAN ARCADIO
• Format: Journal Article
• Language: English
• Published: Greece D.A. Spandidos 01.11.2015; Spandidos Publications; Spandidos Publications UK Ltd
• Subjects: 7-hydroxycoumarin; A549 cells; Apoptosis; Cancer cells; Cancer therapies; caspase-3; Cell growth; Coumarins; Health aspects; Kinases; microinjection; Physiological aspects; Studies; United States > US; apoptosis; microinjection; A549 cells; 7-hydroxycoumarin; caspase-3
• ISSN: 1792-0981; 1792-1015
• DOI: 10.3892/etm.2015.2765

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.