Yunnan Baiyao inhibits EBV-positive nasopharyngeal carcinoma via ferritinophagy-dependent ferroptosis

YB overcomes chemoresistance in EBV-positive NPC. • YB induces ferroptosis rather than necroptosis in EBV-positive NPC. • YB activates ferritinophagy-dependent ferroptosis in EBV-positive NPC. • NCOA4 knockdown rescues YB-treated EBV-positive NPC. Background EBV-positive nasopharyngeal carcinoma frequently exhibits significant chemoresistance, posing a major challenge in clinical treatment. Yunnan Baiyao, a traditional Chinese patent medicine, has shown promise in antitumor research beyond its conventional hemostatic applications. This study investigated the efficacy of Yunnan Baiyao against EBV-positive nasopharyngeal carcinoma and explored the underlying molecular mechanisms of cell death. Methods Yunnan Baiyao-treated EBV-positive C666–1 and EBV-negative HK1/SUNE1 cells were subjected to Sulforhodamine B assay, colony formation assay, and C11-BODIPY/Fe²⁺ flow cytometry to verify antitumor activity and ferroptosis induction. Ferroptosis, autophagy, and ferritinophagy were confirmed via live-cell imaging, Transmission Electron Microscopy, and Western blotting. Specific inhibitors (Q-VD-Oph, Nec-1, etc.) validated the roles of apoptosis, necroptosis, and other pathways. Lentiviral NCOA4 knockdown verified its core function. Nude mouse xenograft models (with Liproxstatin-1 rescue and NCOA4-knockdown tumors) evaluated in vivo efficacy and dependence on ferroptosis/NCOA4. Results C666–1 cells (EBV-positive) are more resistant to cisplatin compared with SUNE1 and HK1 cells (both EBV-negative). Yunnan Baiyao exerted concentration- and time-dependent cytotoxicity on C666–1 cells. Notably, C666–1 cells were more sensitive to Yunnan Baiyao than SUNE1 cells. Yunnan Baiyao induced ferroptosis (not apoptosis/necroptosis/pyroptosis) with elevated lipid peroxidation, intracellular Fe²⁺ accumulation, and mitochondrial shrinkage. Yunnan Baiyao activated autophagic flux, and BAF (autophagy inhibitor) partially reversed ferroptosis. Yunnan Baiyao-induced cell death relied on NCOA4-mediated ferritinophagy; NCOA4 knockdown abrogated its effects in vitro. In vivo, Yunnan Baiyao significantly inhibited tumor growth, reversed by Lip-1 treatment or NCOA4 knockdown. Conclusions Yunnan Baiyao overcomes chemoresistance in EBV-positive nasopharyngeal carcinoma by triggering NCOA4-mediated ferritinophagy-dependent ferroptosis. These findings provide a mechanistic foundation for further preclinical evaluation of Yunnan Baiyao as a candidate therapeutic strategy for chemoresistant nasopharyngeal carcinoma. Download: Download high-res image (146KB) Download: Download full-size image