Hibicuslide C-induced cell death in Candida albicans involves apoptosis mechanism

Aim: To provide the observation that hibicuslide C-induced cell death in yeast Candida albicans involves apoptosis mechanism. Methods and Results: Hibicuslide C was isolated from Abutilon theophrasti by column chromatography. In reactive oxygen species (ROS) assay, C. albicans treated with hibicuslide C showed increase in ROS, and its accumulation induced fungal cell death. In particular, hydroxyl radicals were a large part of the ROS. Mitochondrial dysfunction including mitochondrial depolarization and release of cytochrome c, which is a pro-apoptotic factor, was detected by JC-1 assay and Western blot. CaspACE FITC-VAD-FMK staining using caspase inhibitor showed metacaspase activation. Also, the increase in intracellular Ca²⁺, which is a signal molecule of apoptosis, was detected by Fura-2AM and Rhod-2AM assays. Finally, annexin V-FITC and PI double staining and TUNEL assay confirmed that hibicuslide C induces early apoptosis followed by secondary necrosis in C. albicans. Conclusions: Hibicuslide C exerts antifungal activity against C. albicans through new mechanism inducing apoptosis. Significance and Impact of the Study: Candida albicans is the common cause of nosocomial infections with high mortality. Our findings provide that hibicuslide C can be a model molecule that induces apoptosis in C. albicans.