Protoapigenone, a natural derivative of the
flavonoid apigenin, has been shown to exhibit potent antitumor activity in vitro and in vivo; the precise mechanism of action, however, is not fully elucidated. In this study, we investigated and compared the mechanisms by which
protoapigenone and
apigenin caused cell death in the human
breast cancer MDA-MB-231 cells. Flow cytometry analysis revealed that
protoapigenone induced apoptosis with 10-fold greater potency than
apigenin.
Cancer cells treated with
protoapigenone resulted in persistent activation of
mitogen-activated protein kinase (MAPK) ERK, JNK, and p38, hyperphosphorylation of Bcl-2 and Bcl-xL, and loss of mitochondrial membrane potential (
MMP). The MAPK inhibitors effectively prevented the loss of
MMP and apoptosis induced by
protoapigenone. Treatment of cells with
protoapigenone led to increased levels of
reactive oxygen species (ROS) and decreased levels of intracellular
glutathione. The
thiol-
antioxidant N-acetylcysteine abolished
protoapigenone-induced MAPK activation,
mitochondrial dysfunction, and apoptosis. These results suggest that the induction of oxidative stress preceding the activation of MAPK is required to initiate the mitochondria-mediated apoptosis induced by
protoapigenone. Additionally,
protoapigenone-induced JNK activation was linked to
thiol modification of
glutathione S-transferase π (GSTpi), which impeded GSTpi inhibition of JNK. In contrast to
protoapigenone,
apigenin-induced apoptosis was neither dependent on ROS nor on MAPK. Structure-activity relationship studies suggested that the
thiol reacting effect of
protoapigenone might be associated with an α, β-unsaturated
ketone moiety in the structure of ring B.