Distinct Ca2+ pools regulate NADPH oxidase 2 activation driving Ca2+-independent mitochondrial ROS formation and mitochondrial permeability transition in arsenic trioxide-treated NB4 cells

Abstract Clinically relevant concentrations of arsenic trioxide (ATO) induce apoptosis in NB4 cells through a complex, yet poorly defined interplay between endoplasmic reticulum—derived Ca 2+ signalling and mitochondrial oxidative stress. This study enhances our understanding of these mechanisms by demonstrating that exposure to 1 µM ATO initiates a biphasic Ca 2+ release: an initial flux from inositol 1,4,5-trisphosphate receptors (IP₃Rs), followed by a secondary release via ryanodine receptors (RyRs). Unlike IP 3 R-derived Ca 2+ , the fraction of the cation released through RyRs is subsequently taken up by mitochondria. Notably, IP 3 R-derived Ca 2+ uniquely activates NADPH oxidase 2 (NOX 2), a key event leading to the downstream generation of mitochondrial superoxide (mitoO 2 .− ). Importantly, mitochondrial Ca 2+ accumulation itself is not required for mitoO 2 .− emission. ATO-induced genomic DNA strand breaks are mediated by NOX 2-derived reactive oxygen species (ROS), both directly and indirectly, through the subsequent induction of mitochondrial ROS formation. Furthermore, mitochondrial uptake of RyR-derived Ca 2+ is essential for triggering the mitochondrial permeability transition and the ensuing apoptotic cell death. Although sodium arsenite elicited comparable effects on Ca 2+ homeostasis, it promoted mitoO 2 .− generation via a distinct, NOX 2-independent pathway that relied on RyR-mediated mitochondrial Ca 2+ accumulation. Thus, in NB4 cells, ATO exposure orchestrates a functional crosstalk between discrete Ca 2+ sources to regulate a cascade of events culminating in NOX 2 activation, mitoO 2 .− production, and initiation of the mitochondrial apoptotic pathway.