Esther Zhou

Analysis of Reactive Oxygen Species–Induced Cellular Damage in Cervical Cancer

Abstract 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

Induction of Cell Death and Regulation of Autocrine Vitamin D Metabolism in Cervical Cancer by Physiological and GI20 Doses of 25-Hydroxycholecalciferol

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)2D3, 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.