Hesperetin induces mitochondria independent apoptosis-like death in Candida albicans

Hesperetin, a flavonoid abundantly found in citrus fruits, exhibits broad-spectrum antimicrobial activity and has been proposed as a potential alternative to conventional antifungal agents. However, its precise mode of action(s), particularly in fungal systems, remains poorly understood. This study investigated the molecular mechanism by which hesperetin exerts its antifungal effects on Candida albicans. Considering the reported redox-modulating properties of flavonoids, oxidative stress was evaluated by monitoring intracellular reactive oxygen species (ROS) generation. Hesperetin treatment markedly increased intracellular ROS levels, and ROS production was significantly reduced by diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase (NOX), indicating that ROS generation originated from a NOX-dependent. Despite the elevated ROS, mitochondrial ROS release, fission and cytochrome c release were not observed, suggesting a mitochondria-independent pathway. Instead, cytosolic metacaspase activation was detected, accompanied by phosphatidylserine (PS) externalization, membrane disruption, and DNA fragmentation-hallmarks of apoptosis-like cell death. Collectively, these findings indicate that hesperetin induces NOX-mediated ROS production, leading to mitochondria-independent apoptosis-like death in Candida albicans.