ML385 promotes ferroptosis and radiotherapy sensitivity by inhibiting the NRF2-SLC7A11 pathway in esophageal squamous cell carcinoma

Radiotherapy is important in treating esophageal squamous cell carcinoma (ESCC) comprehensively. Resistance to radiotherapy is a prominent factor contributing to treatment failure in patients with ESCC. The objective of this study was to investigate the impact of ML385, an inhibitor of nuclear factor erythroid 2-related factor 2 (NRF2), on the radiosensitivity of ESCC and elucidate its underlying mechanism. We treated KYSE150 and KYSE510 cells with ML385 and ionising radiation separately or simultaneously, and observed the proliferation, apoptosis, cell cycle and ferroptosis of different conditions by colony formation assay and flow cytometry. Our findings reveal that NRF2 was activated by radiation and translocated from the cytoplasm to the nucleus after radiation. However, ML385 inhibited the expression and cytoplasm-to-nucleus translocation of NRF2. Compared with radiation, ML385 combined with radiation exhibited a significant inhibition on the clone formation ability of ESCC cells, induced apoptosis and promoted G2/M phase arrest. The treatment of ML385 combined with radiation markedly increased ROS and lipid peroxidation levels and decreased glutathione levels compared with the control, thus promoting the occurrence of ferroptosis. In addition, the expression trend of NRF2 was the same as that of proteins related ferroptosis, such as SLC7A11 and GPX4. After overexpression of SLC7A11, we found that significantly restored glutathione levels and alleviated ML385 combined with radiation-induced lipid peroxidation, indicating that ML385 plays a key role in radiotherapy sensitization by inhibiting the NRF2-SLC7A11 pathway. In vivo, ML385 also promoted the killing effect of radiation on xenografted tumours in nude mice. This study identifies NRF2 inhibitor ML385 as a radiosensitizer of ESCC, which highlights the therapeutic potential of the NRF2-SLC7A11 pathway and provides a deeper understanding of the mechanism of ferroptosis in esophageal squamous cell carcinoma.