Supraphysiological 25-Hydroxyvitamin D₃ and Calcipotriol Induce Reactive Oxygen Species, Mitochondrial Dysfunction...

Cervical cancer remains a major global health challenge, necessitating continued advances in therapeutic strategies. Emerging evidence suggests that vitamin D derivatives may exert anti-cancer effects, including induction of apoptosis, potentially through redox-dependent mechanisms. This study investigated whether vitamin D exposure is associated with reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and apoptosis in cervical cancer cells. HeLa cells were exposed to physiological, intermediate, and supraphysiological concentrations of 25-hydroxyvitamin D₃, as well as calcipotriol, a 1,25-dihydroxyvitamin D analogue. Cell viability assays (crystal violet, Alamar Blue, and Muse cell viability) demonstrated dose-dependent reductions in viability. Flow cytometric analyses demonstrated increased apoptotic markers, evidenced by Annexin V binding and caspase-3/7 activation. The oxidative stress assay showed increased ROS-positive cell populations, accompanied by mitochondrial membrane depolarization. Catalase and total superoxide dismutase activities decreased at the highest effective concentrations. In parallel, decreased total expression of proteins within the MAPK, PI3K/AKT, and NF-κB pro-survival pathways was observed. These cellular changes were accompanied by apoptotic morphological features visualized by PlasDIC and brightfield microscopy, including membrane blebbing, karyorrhexis, and apoptotic body formation. Collectively, these findings demonstrate concurrent redox disturbance, mitochondrial dysfunction, and apoptosis following supraphysiological vitamin D exposure. NAC co-treatment produced partial attenuation of the ROS-positive population and improved mean viability following calcipotriol treatment, supporting a possible contribution of ROS to calcipotriol-associated cytotoxicity; however, the present experiments do not establish ROS as a necessary mediator of apoptosis.