Identification of CLIC1 as a direct molecular target of luteolin associated with ferroptosis induction in hepatocellular carcinoma

Ferroptosis is an iron-dependent form of regulated cell death with important tumor-suppressive functions in hepatocellular carcinoma (HCC). Luteolin, a naturally occurring flavonoid, exhibits anti-tumor activity, but its molecular mechanism in HCC remains unclear. This study investigated whether luteolin inhibits HCC growth and identified its potential molecular target involved in ferroptotic responses. Cysteine-reactive competitive activity-based protein profiling (CC-ABPP), surface plasmon resonance (SPR), molecular docking, co-immunoprecipitation, immunofluorescence, and Pearson correlation analyses were performed to characterize luteolin-associated targets and their relationships with ferroptosis-related alterations. Antitumor and ferroptosis-related effects were evaluated using CCK-8, colony formation, RT-qPCR, western blotting, fluorescence imaging, and a xenograft mouse model. Luteolin significantly inhibited HCC cell proliferation and induced ferroptosis-associated alterations, as demonstrated by decreased GPX4 and SLC7A11 expression, increased ACSL4 expression, elevated Fe 2+ , reactive oxygen species, and lipid peroxidation, together with altered ferroptosis-related gene expression. These effects were partially reversed by ferroptosis inhibitors. CC-ABPP and SPR identified chloride intracellular channel 1 (CLIC1) as a previously unrecognized direct binding target of luteolin, whereas co-immunoprecipitation and fluorescence spatial correlation analyses supported an association between CLIC1 and GPX4. Pearson correlation analyses further revealed relationships between CLIC1/GPX4 expression patterns and ferroptosis-related parameters. High CLIC1 expression was associated with poor prognosis in HCC. In vivo , luteolin suppressed xenograft tumor growth and induced ferroptosis-related molecular changes. Collectively, these findings indicate that luteolin inhibits HCC growth by promoting ferroptosis-associated response and identifying CLIC1 as a novel molecular target of luteolin potentially involved in these processes.