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

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 D₃ 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 D₃ 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.