Esther Zhou

Gynaecological malignancies account for over 1.4 million new diagnoses, with outcomes constrained by late‐stage presentation and chemoresistance. Dysregulated redox homeostasis is a hallmark of tumourigenesis. Tumour cells maintain elevated basal reactive oxygen species (ROS), which support proliferative signalling but may also increase susceptibility to further oxidative stress. Vitamin D, acting through the vitamin D receptor (VDR), has antiproliferative and pro‐apoptotic effects across cancer models, but its ability to amplify ROS beyond an apoptotic threshold in gynaecological malignancies remains unresolved. We conducted a narrative literature search of PubMed/MEDLINE, ClinicalTrials.gov and the Surveillance, Epidemiology and End Results databases, using terms related to vitamin D and its metabolites, ROS/oxidative stress, apoptosis and gynaecological malignancies. Preclinical evidence indicates that calcitriol and related compounds can induce ROS‐mediated mitochondrial apoptosis, modulate autophagy and arrest the cell cycle. In ovarian cancer, non‐HGSOC cell data suggest that mutant TP53 may alter calcitriol responses, requiring validation in high‐grade serous ovarian carcinoma models. In cervical cancer, calcitriol radiosensitises human papillomavirus‐positive cell lines through autophagy suppression, whilst an in vivo calcitriol‐cisplatin study reported improved survival. In endometrial cancer, thioredoxin‐interacting protein induction is a candidate mechanism for selective ROS‐mediated apoptosis, although this remains context‐dependent. Clinical evidence is heterogeneous: higher 25‐hydroxyvitamin D status has shown modest inverse associations with ovarian cancer mortality; small trials report regression or reduced recurrence of cervical intraepithelial neoplasia; no randomised controlled trial evidence supports efficacy in invasive cervical cancer; and associations with endometrial cancer incidence are largely null. Current evidence does not support vitamin D supplementation as an oncology‐specific intervention. Future research should prioritise biomarker‐stratified trials incorporating VDR genotype, CYP24A1 profiling, pre‐specified 25(OH)D targets and pharmacokinetically informed dosing.

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 D ₃ , 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.

Reactive oxygen species (ROS) are highly reactive oxygen‐based molecules comprising hydrogen peroxide, hydroxyl radicals, superoxide anion, and singlet oxygen. These species are produced intracellularly and play an important role in cellular signaling and metabolism. Their high reactivity damages intracellular macromolecules such as lipids and DNA. In cancer biology, ROS display a dual role: they promote cancer cell proliferation at low to moderate levels, whereas excessive accumulation overwhelms antioxidant defenses, causing oxidative stress and apoptosis. This has resulted in therapeutic strategies that selectively increase ROS in cancer cells to induce apoptosis. Vitamin D has demonstrated anti‐cancer properties, with one proposed mechanism involving ROS‐mediated apoptosis. This article outlines a workflow to investigate ROS‐induced cellular damage by vitamin D ₃ in HeLa cervical cancer cells. The study begins with quantification of ROS levels and assessment of mitochondrial membrane potential in HeLa cultures. Transmission electron microscopy is used to examine mitochondrial ultrastructure. Lipid peroxidation quantifies downstream ROS‐mediated membrane and cellular injury. Antioxidant enzyme activities, including superoxide dismutase and catalase, measure cellular anti‐oxidative defence capacity. Lastly, the role of ROS inhibition of AKT signaling, leading to reduced cell survival and apoptosis, is quantified by immunoblotting. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Preparation of HeLa cell cultures and treatment with vitamin D ₃ Basic Protocol 2 : Quantification of ROS‐positive cells using the Muse Oxidative Stress assay Basic Protocol 3 : Measurement of mitochondrial membrane potential using the Muse MitoPotential assay Basic Protocol 4 : Ultrastructural evaluation of mitochondrial damage by transmission electron microscopy Basic Protocol 5 : Evaluation of lipid damage using a human 8‐iso prostaglandin F2α ELISA Basic Protocol 6 : Evaluation of total superoxide dismutase activity using an activity assay kit Basic Protocol 7 : Evaluation of catalase activity using an activity assay kit Basic Protocol 8 : Immunoblot analysis of AKT to assess PI3K/AKT signaling

Vitamin D and its metabolites exert anti-cancer properties in various cancers; however, their effects on cervical cancer remain largely unexplored. To investigate this gap, we exposed HeLa adenocarcinoma cervical cells to physiological and the growth inhibition 20% (GI20) concentration of 25-hydroxycholecalciferol, the precursor hormone of active 1,25-dihydroxycholecalciferol. We then assessed its impact on cell health, and the expression of the genes and proteins involved in the activation and catabolism of vitamin D at the cellular level by autocrine vitamin D metabolism via the vitamin D metabolizing system (VDMS). Cell health was evaluated by crystal violet and alamarBlue assays, while cell cycle progression and apoptotic cell death markers were assessed by flow cytometry. Gross morphology and ultrastructure were observed using brightfield microscopy and transmission electron microscopy. Gene and protein analyses of the autocrine VDMS were assessed using reverse transcription polymerase chain reaction and Western blot, respectively. Our findings reveal that 25(OH)D₃ inhibits cell growth and induces apoptosis in HeLa cervical cells in a dose-dependent manner through the autocrine upregulation of CYP27B1 and VDR. These autocrine effects most likely promote the bioactivation of 25(OH)D₃ and intracellular signaling of pro-apoptotic genomic pathways by liganded VDR. Furthermore, the upregulation of CYP24A1 at GI20 treatment likely increases the catabolism of 25(OH)D₃ and 1,25(OH)₂D_3, and therefore may mitigate the anti-cancer action of the high-treatment dose. In summary, 25(OH)D₃ holds immense potential as a complementary therapeutic treatment for cervical cancer.