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Osteosarcoma cells promote intracellular iron detoxification to mitigate GPX4-mediated ferroptosis

Cancer Gene Therapy (2026)Cite this article

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Abstract

Osteosarcoma is an aggressive and highly metastatic cancer that arises in bones. Ferroptosis, an iron-dependent form of cell death, is critically controlled by glutathione peroxidase 4 (GPX4). In this study, we characterized GPX4 and its upstream regulator xCT across five osteosarcoma cell lines (U2OS, MG-63, HOS, Saos2, and 143B). We also demonstrated that chemical inhibition of these proteins using RSL3 (GPX4 inhibitor) and erastin (xCT inhibitor) significantly suppressed osteosarcoma cell growth. In U2OS and MG-63 cells, GPX4 inhibition triggered lipid peroxidation, NRF2 activation, and upregulated antioxidant genes including, AKR1C1, HO-1, and NQO1, indicating a compensatory response to oxidative stress. Furthermore, GPX4 inhibition, through RSL3 treatment or transient GPX4 knockdown, increased ferritin heavy chain 1 and ferroportin expression, promoting intracellular iron depletion and conferring resistance to ferroptosis. RSL3 exposure also elevated post-translational regulators such as HIF-1α, c-MET and MSPR/RON, associated with cell survival pathways. Pharmacological inhibition of these signaling molecules synergistically enhanced RSL3-induced cytotoxicity. Collectively, our findings reveal that GPX4 inhibition initiates ferroptosis while simultaneously activating NRF2-driven antioxidant defenses, iron homeostasis mechanisms, and adaptive cell survival signaling. The results highlight potential therapeutic strategies that combine GPX4 inhibition with targeted disruption of compensatory pathways to overcome ferroptosis resistance in osteosarcoma.

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Fig. 1: Detection of ferroptosis in osteosarcoma cells.
Fig. 2: Upregulation of NRF2 and lipid peroxidation in osteosarcoma cells in response to GPX4-mediated ferroptosis.
Fig. 3: Antioxidant response to GPX4-mediated ferroptosis in osteosarcoma cells.
Fig. 4: Effect of GPX4 inhibition on iron regulation in osteosarcoma cells.
Fig. 5: Identification of signaling molecules associated with ferroptosis in osteosarcoma cells.
Fig. 6: Schematic illustration of the molecular response in osteosarcoma cells to GPX4-mediated ferroptosis.

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Data availability

All data generated or analyzed during this study are included in the main manuscript or supplementary materials. The RNA-seq data analyzed in this study were obtained from the National Cancer Institute Genomic Data Commons Data Portal, specifically from the TARGET-OS project (dbGap accession: phs000218). These data are publicly available at https://portal.gdc.cancer.gov/. Other data relevant to this study are available upon request from the corresponding author.

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Acknowledgements

We acknowledge the UF ICBR Gene Expression & Genotyping Core (RRID: SCR_019145) for RNA-seq and the Cytometry Core (RRID: SCR_019119) for flow cytometry. Figure 6 was crafted using Biorender (biorender.com). We also acknowledge the UF HiPerGator Research Computing for computational resources and technical support. This work was partly supported by the UF AI Initiative.

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Authors and Affiliations

  1. Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

    Md Abdullah, Donghee Lee, Eslenur Nipa, Jessy Castellanos-Gell, Ann M. Chan, Oscar Hernandez Maldonado, Marc E. Salute, Rebecca Nance-Richey, Rowan J. Milner & Jong Hyuk Kim

  2. Department of Comparative, Diagnostic, and Population Medicine, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

    Jessy Castellanos-Gell

  3. UF Health Cancer Institute, University of Florida, Gainesville, FL, USA

    Jong Hyuk Kim

  4. Artificial Intelligence Academic Initiative (AI2) Center, University of Florida, Gainesville, FL, USA

    Jong Hyuk Kim

  5. Intelligent Critical Care Center (IC3), University of Florida, Gainesville, FL, USA

    Jong Hyuk Kim

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Contributions

Conception and design: MA and JHK; Development of methodology: MA, DHL, and JHK; Acquisition of data: MA, DHL, EN, JCG, and JHK; Analysis and interpretation of data: MA, DHL, and JHK; Writing, review, and/or revision of the manuscript: MA, DHL, EN, JCG, AMC, OHM, MES, RNR, RJM, and JHK; Administrative, technical, or material support: MA, EN, and JHK; Study supervision: JHK.

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Correspondence to Jong Hyuk Kim.

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This study did not involve any live animals, human participants, or human specimens. Therefore, ethics approval and informed consent were not required. All methods were performed in accordance with the relevant guidelines and regulations.

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Abdullah, M., Lee, D., Nipa, E. et al. Osteosarcoma cells promote intracellular iron detoxification to mitigate GPX4-mediated ferroptosis. Cancer Gene Ther (2026). https://doi.org/10.1038/s41417-026-01021-y

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