SAT-293 - Vitamin D Metabolites Promote Oxidative Stress-Induced Apoptosis in HPV16-Positive Siha Cervical Cancer Cells

Vitamin D metabolites promote oxidative stress–mediated apoptosis in HeLa adenocarcinoma cervical cells following 25-hydroxycholecalciferol (25(OH)D₃) and calcipotriol treatment (1). This study extends these findings to HPV16-positive SiHa squamous cervical cancer cells to assess whether comparable redox-dependent effects occur and to examine accompanying antioxidant responses and oxidative signalling pathways.

SiHa cells were cultured under standard conditions, with triplicate assays across three independent experiments. Treatments included physiological and supraphysiological 25-hydroxycholecalciferol (25(OH)D₃) and calcipotriol doses. Flow cytometry assessed viability, mitochondrial membrane potential, oxidative stress, Annexin V binding, and caspase 3/7 activity; and PlasDIC microscopy assessed apoptotic morphology. Antioxidant catalase and SOD enzyme induction by ELISA, and MAPK, PI3K/AKT, and NF-κB signalling pathways by Western blots were analysed. One-way ANOVA with Bonferroni correction was applied (significance = p< 0.05).

Growth inhibition was observed (25% at 7000 nM 25(OH)D₃; 75% at 100 μM calcipotriol). Supraphysiological exposure caused mitochondrial depolarisation (25(OH)D₃ p< 0.0001; calcipotriol p< 0.0001) and ROS accumulation (25(OH)D₃ 7.58%; calcipotriol 25.86%). Total SOD and catalase concentrations showed dose-response changes and levels significantly decreased at higher treatment doses. Reduced relative viable cell counts occurred (25(OH)D₃ p=0.0014; calcipotriol p< 0.0001). Apoptotic morphology was significant: cell rounding, membrane blebbing, karyorrhexis, and apoptotic bodies were significantly increased in all experimental cultures. Phosphatidylserine externalisation was significant (25(OH)D₃ p=0.0144; calcipotriol p< 0.0001). Caspase 3/7 activation was highest with calcipotriol treatment (p=0.0002). A significant down-regulation of key signalling proteins was identified in MAPK, PI3K/AKT, and NF-κB pathways (p < 0.05).

Supraphysiological vitamin D metabolite exposure induces ROS-driven mitochondrial dysfunction and apoptosis in SiHa cells, consistent with previous HeLa findings (1). Collectively, the attenuated antioxidant enzyme activity together with suppression of survival signalling suggests dysregulated redox homeostasis which promotes mitochondrial collapse and apoptosis execution. Validation studies in primary cervical cells and HPV-negative C33A cervical cells are currently being investigated.

References
1. Cherry Chan HC, Bhoora S, Zhou E, Marais S, Pillay T, Punchoo R. SAT-306 Calcitriol and Calcifediol Mediate Oxidative Stress-induced Apoptosis in HeLa Cervical Cancer Cells. J Endocr Soc [Internet]. 2025 Oct 22 [cited 2025 Dec 31];9(Suppl 1):bvaf149.2448. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC12545022/

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