SAT-306 Calcitriol and Calcifediol Mediate Oxidative Stress-induced Apoptosis in HeLa Cervical Cancer Cells

Abstract Disclosure: H.C. Chan: None. S. Bhoora: None. E. Zhou: None. S. Marais: None. T. Pillay: None. R. Punchoo: None. The Vitamin D metabolome shows anti-neoplastic activity in many pre-clinical studies. High concentrations of reactive oxygen species (ROS) can mediate ROS-induced cell death in cancer cells. The anti-cancer induction of ROS-mediated apoptosis in uterine cervical cancer by activated and partially activated vitamin D hormones is unknown. This study assessed cell growth and oxidative stress-induced apoptosis in HeLa cervical cell line treated with calcifediol (25-hydroxycholecalciferol, 25(OH)D₃) and a calcitriol analogue, (1.25 dihydroxycholecalciferol, calcipotriol hydrate, calcipotriol). HeLa cells were incubated under standard cell culture conditions and all experiments controlled using triplicated biological and technical replicates, and solvent and medium cultures. Treatments spanned physiological and supraphysiological doses based on human physiological reference intervals and growth inhibition studies, respectively. Flow cytometry analysed cell viability, mitochondrial membrane potential, total ROS, Ki67, Caspase 3/7 and Annexin V. Cell count was measured by the Crystal Violet assay and morphological apoptosis assessed by light microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC). Data were expressed as mean(%)±SEM and analysed by one-way ANOVA with Bonferroni post-analysis (significance = p<0.05). A 50% growth inhibition measured by the Crystal Violet assay was achieved at concentrations >9000 nM for 25(OH)D₃ and >250 μM for calcipotriol. Elevated levels of ROS-positive cells were detected at supraphysiological treatments (10 000 nM of 25(OH)D₃, p=0.0326; vs 250 μM of calcipotriol, p<0.0001). Supraphysiological treatments also disrupted the mitochondrial membrane potential, (25(OH)D₃, p=0.0047 vs calcipotriol, p=0.0002) and reduced the cell count (25(OH)D₃, p=0.0013 vs calcipotriol p < 0.0001). These findings were consistent with flow cytometric viability results (25(OH)D₃, p=0.0007; and calcipotriol, p<0.0001); however, ROS induced insignificant cell proliferation measured by Ki67 nuclear antigen (25(OH)D₃, p=0.6268 vs calcipotriol, p=0.2754). Biochemical biomarkers of apoptosis were activated evidenced by increased caspase 3/7 expression, (25(OH)D₃, p=0.0009 vs calcipotriol, p<0.0001) and increased externalisation of phosphatidyl serine residues (25(OH)D₃, p<0.0001 vs calcipotriol, p<0.0001). Morphological analysis showed the presence of significant cell rounding, membrane blebbing, karyorrhexis and apoptotic bodies at supraphysiological treatments of 25(OH)D₃ and calcipotriol. These findings support ROS-mediated induction of intrinsic apoptosis at supraphysiological doses of 25(OH)D₃ and calcipotriol in HeLa. Current work is underway to analyse the antioxidant defence system and regulation of ROS-activated intracellular cascades in HeLa. Presentation: Saturday, July 12, 2025