7-hydroxicoumarin Exposure Induces Prompt Activation of caspase-3 in A-549 Cancer Cells through Single Cell Microinjection

Abstract

Coumarins and their apoptogenic effects have arisen conspicuous   interest in recent years.  The present chapter aims to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C-3) activity in A549 human lung carcinoma cells.

The effects of 7-HC were investigated using a variety of analytical approaches, including colorimetric and fluorometric assays, western blotting, single-cell microinjection, fluorescence microscopy, and image analysis. After being exposed to 1.85 mM 7-HC for 24 hours, C3 activity increased by 65%, procaspase-3 was significantly converted to C-3, and poly(ADP-ribose) polymerase was cleaved. Additionally, morphological modifications linked to apoptosis were noticed. The calcium conductance of the cells increased by 27% after being exposed to 7HC for 3 or 6 hours. Single-cell microinjection of a specific fluorescent substrate of C-3 into cells that had previously been exposed to 7-HC allowed for the identification of a typical enzymatic kinetic profile of C-3 activation several hours before the morphological and biochemical changes connected to the observation of apoptosis. These findings imply that 7-HC, the most important biotransformation product of coumarin in people, is what causes the rapid in vivo activation of C-3.