Vitamin D is a steroid hormone crucial for bone mineral metabolism. In addition, vitamin D has pleiotropic actions in the body, including anti-cancer actions. These anti-cancer properties observed within in vitro studies frequently report the reduction of cell proliferation by interruption of the cell cycle by the direct alteration of cell cycle regulators which induce cell cycle arrest. The most recurrent reported mode of cell cycle arrest by vitamin D is at the G1/G0 phase of the cell cycle. This arrest is mediated by p21 and p27 upregulation, which results in suppression of cyclin D and E activity which leads to G1/G0 arrest. In addition, vitamin D treatments within in vitro cell lines have observed a reduced C-MYC expression and increased retinoblastoma protein levels that also result in G1/G0 arrest. In contrast, G2/M arrest is reported rarely within in vitro studies, and the mechanisms of this arrest are poorly described. Although the relationship of epigenetics on vitamin D metabolism is acknowledged, studies exploring a direct relationship to cell cycle perturbation is limited. In this review, we examine in vitro evidence of vitamin D and vitamin D metabolites directly influencing cell cycle regulators and inducing cell cycle arrest in cancer cell lines.

Vitamin D has displayed anti-cancer actions in numerous in vitro studies. Here, we investigated the anti-cancer actions of cholecalciferol, a vitamin D precursor, on a metastatic cervical cancer cell line, namely, CaSki. Experimental cultures were incubated for 72 h and treated with cholecalciferol (10–1000 ng/mL). In the present study, cell count, viability, proliferation and cell cycle were analyzed by a crystal violet assay, trypan blue assay, Ki67 proliferation, and a cell cycle assay, respectively. Biomarkers of apoptosis, necrosis, and autophagic cell death were measured by the Caspase 3/7 and Annexin V/7-AAD Muse™ assays, a LC3-II assay, and a lactate dehydrogenase release assay, respectively. The ultrastructural features of cell death were assessed by transmission electron microscopy. A statistical analysis was performed using a one-way ANOVA and Bonferroni’s post-hoc analysis test, and p < 0.05 is considered statistically significant here. The results identify statistical decreases in cell count and viability at high-dose treatments (100 and 1000 ng/mL). In addition, significant increases in apoptotic biochemical markers and apoptotic ultrastructure are shown to be present at high-dose treatments. In conclusion, high-dose cholecalciferol treatments inhibit cell count and viability, which are both mediated by apoptotic induction in the CaSki cell line.

Serological tests based on the enzyme immunoassay (EIA) are the primary tool for the diagnosis of human immunodeficiency virus (HIV) in adults and have rapidly evolved to quicker, affordable and more accurate test formats to detect early HIV infection. Second- and third-generation HIV rapid tests detect the immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to the HIV and are used at the point of care and in HIV self-testing. The tests are affordable and accessible in state and private diagnostic laboratories. The present-day fourth- and fifth-generation EIAs can detect both p24 antigen and IgG and IgM HIV antibodies and thereby diagnose early HIV infection at approximately 2 weeks. The fourth- and fifth-generation EIAs also report sensitivity and specificity of more than 99%. The correct interpretation of HIV diagnosis of false-positive and false-negative EIA test results requires collaborative scrutiny of patient factors and laboratory test methodologies.

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.

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

Cervical cancer disproportionately affects low-resource countries and is a significant health burden in South Africa. Pre-clinical studies have demonstrated numerous anti-cancer actions of vitamin D metabolites. Here, the anti-cancer action of the vitamin D precursor, cholecalciferol, was investigated in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Apoptotic cell death was investigated by flow cytometry, which measured mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and the expression of DNA damage markers. Additionally, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis. Expression of the vitamin D metabolising system (VDMS) – consisting of cholecalciferol activating (CYP2R1 and CYP27A1), calcidiol activating (CYP27B1) and calcidiol inactivating (CYP24A1) enzymes, and the vitamin D receptor (VDR) – was assessed by qPCR and Western blots. Data were analysed using a one-way ANOVA and Bonferroni post-hoc tests and p < 0.05 was considered statistically significant. Significant decreases in cell count (p = 0.011) and cell viability (p < 0.0001) were identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, biochemical markers at 2600 nM treatment were significant for apoptosis, and included decreased ∆Ψm (p = 0.0145); increased PS externalisation (p = 0.0439); terminal caspase activation (p = 0.0025); and nuclear damage (p = 0.004). Moreover, biochemical apoptosis was corroborated by classical apoptotic features observed by brightfield microscopy and TEM. Additionally, a significant increase in CYP2R1 gene (p < 0.0001) and protein (p = 0.021) expression, and a converse significant decrease in CYP27B1 gene (p = 0.003) and protein expression (p = 0.031) were observed at 2600 nM cholecalciferol treatment. Furthermore, significant increases in VDR gene (p = 0.033) and protein (p = 0.04) expression, and CYP24A1 gene (p < 0.0001) and protein (p = 0.0274) expression were observed at 2600 nM cholecalciferol. In summary, high-dose cholecalciferol treatment of SiHa cervical cancer cells inhibits cell growth, induces apoptosis, and furthermore, upregulates CYP2R1 and VDR expression. Taken together, these findings suggest that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of cell growth inhibition, and apoptosis in SiHa experimental cultures.