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.