Huayu Wan induces ferroptosis and enhances anti-PD-1 efficacy in cisplatin-resistant esophageal squamous cell carcinoma

Cisplatin resistance remains a major therapeutic challenge in esophageal squamous cell carcinoma (ESCC). Here, we investigated the antitumor effects and underlying mechanisms of Huayu Wan (HYW) in cisplatin-resistant ESCC. HYW markedly inhibited the growth of cisplatin-resistant human KYSE150 and murine mEC25 cells and induced a ferroptotic phenotype characterized by reactive oxygen species accumulation, increased intracellular iron, glutathione depletion, lipid peroxidation, and reduced GPX4 expression. Ferrostatin-1 partially reversed HYW-induced cytotoxicity. HYW also promoted immunogenic cell death-associated changes, including extracellular ATP and HMGB1 release and cell-surface calreticulin exposure. Mechanistically, HYW increased p53 expression and impaired the SLC7A11/GCLC-dependent cystine–glutathione antioxidant system, whereas GCLC overexpression and P53 inhibition partially restored redox homeostasis and cell survival. In parallel, HYW suppressed AKT–mTOR signaling and promoted NCOA4-associated ferritinophagy, as evidenced by increased NCOA4 and LC3-II and decreased FTH expression. Pharmacological inhibition of autophagy and NCOA4 knockdown attenuated iron accumulation and HYW-induced cytotoxicity. In cisplatin-resistant syngeneic tumors, HYW suppressed tumor growth with generally acceptable systemic tolerability, enhanced ferroptosis- and immunogenic stress-associated changes, reduced PD-L1 expression, and increased CD8 + T-cell infiltration. Moreover, HYW combined with anti-PD-1 treatment produced greater antitumor effects than either monotherapy. These findings suggest that HYW promotes ferroptosis through coordinated disruption of glutathione metabolism and NCOA4-associated ferritinophagy while enhancing antitumor immune responses in cisplatin-resistant ESCC.