Ciprofol induces ferroptosis and enhances anti-PD-1 efficacy via USP1-mediated GPX4 ubiquitination in hepatocellular carcinoma

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown promise in hepatocellular carcinoma (HCC), but resistance due to the immunosuppressive tumor microenvironment remains a major challenge. Ferroptosis, an iron-dependent form of regulated cell death, has emerged as a potential strategy to enhance anti-tumor immunity. Ciprofol, a novel propofol derivative widely used in clinical anesthesia, has not been explored for its role in cancer therapy. In this study, we evaluated the anti‑proliferative and pro‑ferroptotic effects of ciprofol in human HCC cell lines (Huh‑7, MHCC97‑H) and a mouse HCC line (H22) using CCK‑8, EdU, colony formation, Transwell, and flow cytometry assays. Ferroptosis was assessed by measuring ROS, Fe²⁺, MDA, SOD, and mitochondrial morphology via TEM. Molecular mechanisms were investigated by Western blot, qRT‑PCR, Co‑IP, ubiquitination assay, and surface plasmon resonance (SPR). In vivo efficacy was examined in xenograft (nude mice) and syngeneic (C57BL/6) models, and combination therapy with anti‑PD‑1 antibody was evaluated. Ciprofol inhibited HCC cell proliferation, migration, invasion, and epithelial‑mesenchymal transition (EMT) (IC₅₀: 43.7 µM for Huh‑7; 36.4 µM for MHCC97‑H). Mechanistically, ciprofol induced ferroptosis, evidenced by increased ROS, Fe²⁺, MDA, and typical mitochondrial shrinkage/cristae disruption, which were reversed by the ferroptosis inhibitor Fer‑1. Ciprofol did not alter GPX4 mRNA levels but promoted its ubiquitination and proteasomal degradation. Transcriptome analysis identified USP1 as a key downstream target. Ciprofol reduced USP1 expression, disrupted the USP1‑GPX4 interaction, and directly bound to USP1 (Kd = 1.5 µM). USP1 knockdown phenocopied ciprofol effects, inducing ferroptosis and suppressing HCC progression. In vivo, ciprofol (40 mg/kg) significantly reduced tumor growth, downregulated USP1/GPX4, and was well‑tolerated. Importantly, ciprofol improved tumor control when combined with anti-PD-1 antibody. In conclusion, ciprofol is a novel ferroptosis inducer that promotes GPX4 ubiquitination and degradation by suppressing USP1, exhibits potent anti‑HCC activity, and enhances the therapeutic efficacy of anti‑PD‑1 immunotherapy, representing a promising sensitizer for combined cancer treatment. Not applicable.