Rivak Punchoo

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

Cholecalciferol induces apoptosis via autocrine metabolism in epidermoid cervical cancer cells

The anti-cancer effects of vitamin D are of fundamental interest. Cholecalciferol is sequentially hydroxylated endogenously to calcidiol and calcitriol. Here, SiHa epidermoid cervical cancer cells were treated with cholecalciferol (10–2600 nmol/L). Cell count and viability were assayed using Crystal Violet and Trypan Blue, respectively. Apoptosis was assessed using flow cytometry for early and late biomarkers along with brightfield microscopy and transmission electron microscopy. Autocrine vitamin D metabolism was analysed by reverse transcription-quantitative PCR and immunoblotting for activating enzymes: 25-hydroxylases (CYP2R1 and CYP27A1) and 1α-hydroxylase (CYP27B1), the catabolic 24-hydroxylase (CYP24A1), and the vitamin D receptor (VDR). Data were analysed using one-way ANOVA and Bonferroni post-hoc test, and p < 0.05 was considered significant. After cholecalciferol, cell count ( p = 0.011) and viability ( p < 0.0001) decreased, apoptotic biomarkers were positive, mitochondrial membrane potential decreased ( p = 0.0145), and phosphatidylserine externalisation ( p = 0.0439), terminal caspase activity ( p = 0.0025), and nuclear damage ( p = 0.004) increased. Microscopy showed classical features of apoptosis. Gene and protein expression were concordant. Immunoblots revealed increased CYP2R1 ( p = 0.021), VDR ( p = 0.04), and CYP24A1 ( p = 0.0274) and decreased CYP27B1 ( p = 0.031). The authors conclude that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of growth inhibition and apoptosis in SiHa cells.