Liquiritigenin targets transferrin receptor to potentiate ferroptosis sensitivity in colorectal cancer cells.

[BACKGROUND] Ferroptosis, a type of regulated cell death driven by iron-dependent lipid peroxidation, has emerged as a promising strategy for cancer therapy. However, the efficacy of conventional ferroptosis inducers is often limited by poor tumor responsiveness. High-throughput screening offers a promising approach for the rapid discovery of safe and efficient ferroptosis sensitizers.

[PURPOSE] This study aimed to systematically identify a natural compound capable of sensitizing colorectal cancer (CRC) cells to ferroptosis via high-throughput screening and delineate the underlying molecular mechanism.

[METHODS] A high-throughput screen of a natural compound library was performed using the CellTiter-Lumi™ viability assay. The synergistic potential of the hit compound with ferroptosis inducers was assessed via combination matrix assays. Ferroptosis sensitivity was assessed through cellular morphology, labile iron pool (using FerroOrange), lipid peroxidation (via C11-BODIPY and MDA assays), and reactive oxygen species (using DCFH-DA). Mechanistic studies integrated target engagement assays (CETSA and DARTS), computational simulations (molecular docking and dynamics), functional validation (genetic manipulation, CHX-chase, co-immunoprecipitation and ubiquitination assays), and public single-cell RNA sequencing data analysis. Ferroptosis sensitization was verified in a subcutaneous xenograft model in vivo and in a human organoid model ex vivo.

[RESULTS] Through screening, we identified liquiritigenin (LIQ), an active component derived from Glycyrrhiza glabra l., as a potent ferroptosis sensitizer in CRC cells. LIQ treatment predisposed CRC cells to a ferroptosis phenotype, characterized by elevated lipid peroxidation and increased labile iron pool levels, effects reversed by a ferroptosis inhibitor. Mechanistically, LIQ directly bound to and stabilized transferrin receptor (TFRC) by inhibiting β-TrCP-mediated ubiquitination and degradation, which resulted in intracellular iron accumulation, exacerbated lipid peroxidation, and ultimately sensitized cells to ferroptosis. Of note, LIQ acted synergistically with the ferroptosis inducer sulfasalazine or RSL3 to suppress tumor growth in vivo and ex vivo, with the combination demonstrating a favorable safety profile.

[CONCLUSION] Our findings reveal a previously unrecognized role for LIQ as a ferroptosis sensitizer and highlight its translational potential as a dietary intervention in CRC therapy.