High-dose ionizing radiation-induced ferroptosis leads to radiotherapy sensitization of nasopharyngeal carcinoma

Abstract

Background and purpose

Radiotherapy (RT) is the most commonly-used treatment for nasopharyngeal carcinoma (NPC). Ferroptosis is a type of regulated cell death that is associated with cancer development. This study aims to investigate the relationship between RT and iron metabolism in NPC patients, as well as the effects of different doses of ionizing radiation (IR) on NPC cells and the role played by ferroptosis

Methods and Materials

A retrospective analysis was conducted on changes in iron metabolism markers—serum iron, hemoglobin, TIBC, UIBC, and transferrin saturation—before and after radiotherapy in 73 participants. CCK-8 assay was used to assess the effects of varying radiation doses on the viability of 5-8F cells, determining the radiation dose applied in this study. Real-time quantitative PCR (RT-qPCR) was performed to examine how different radiation doses affect the expression of ferroptosis-related genes (FRGs), including ACSL4, ATP5G3, PTGS2, and IREB2. Western blot analysis was employed to measure the protein expression levels of GPX4 under varying radiation doses. The contents of lactate dehydrogenase (LDH), reactive oxygen species (ROS), malonaldehyde (MDA), [Fe2+], adenosine triphosphate (ATP), and glutathione (GSH) were assessed to evaluate the induction of ferroptosis. In addition, flow cytometry was used to analyze cell cycle distribution in NPC cells following various doses of IR. FRGs expression was assessed in NPC cell lines with varying radiosensitivity

Results

Compared to pre-radiotherapy levels, serum iron and transferrin saturation significantly increased after treatment, while hemoglobin and UIBC showed marked reductions. The CCK8 results indicated that after exposure to 2–18 Gy of radiation, the viability of 5-8F cells was lowest at a dose of 10 Gy. In irradiated NPC cells, mRNA expression of pro-ferroptosis factors such as ACSL4, PTGS2, and IREB2 was generally upregulated, whereas protein levels of the anti-ferroptosis factor GPX4 were downregulated. As the IR dose increased, the contents of LDH, ROS, and MDA were upregulated, while ATP and GSH levels were downregulated. In addition, high-dose IR exposure can block NPC cells in the G₂/M phase. Moreover, we found that the expression levels of three ferroptosis driver genes were lower in radioresistant NPC cell line

Conclusions

High dose IR can induce ferroptosis in NPC cells, highlighting the potential for developing novel ferroptosis-based targets and overcoming radioresistance in clinical NPC treatments.