Luteolin triggers autophagy-dependent ferroptosis via suppressing SLC40A1-related Fe2+ efflux in hepatocellular carcinoma (HCC)

Background Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal malignancies globally, with limited therapeutic options and a poor prognosis. Luteolin, a naturally occurring flavonoid widely distributed in various plants, has been documented to exert anti-tumor activities against multiple cancer types, including HCC. Ferroptosis, a novel form of regulated cell death characterized by iron-dependent lipid peroxidation, and autophagy, a conserved cellular catabolic process, are closely implicated in tumor progression and therapeutic responsiveness. SLC40A1, a key iron exporter responsible for Fe 2+ efflux, plays a crucial role in maintaining cellular iron homeostasis, which is essential for tumor cell survival. However, the underlying mechanism by which luteolin modulates autophagy and ferroptosis in HCC, particularly its association with SLC40A1-mediated Fe 2+ transport, remains unclear. Methods In this study, HCC cell lines (Hepa1-6, Huh7 and Hep3B) were treated with varying concentrations of luteolin. Cell viability was assessed using the CCK-8 assay and crystal staining. In vivo experiments using HCC xenograft and spleen injection mouse models were conducted to validate the in vitro findings. Ferroptosis was evaluated by measuring intracellular Fe 2+ levels, lipid reactive oxygen species (ROS) production, and glutathione (GSH) content. Autophagy was determined by analyzing the LC3B-II/LC3B-I ratio, p62 expression and some autophagy-related genes via Western blot, as well as LC3 puncta formation using immunofluorescence. The expression of SLC40A1 was detected by qPCR. SLC40A1 overexpression and knockdown models were established to verify its role in luteolin-induced effects in vivo and in vitro. Results Luteolin significantly inhibited HCC cell proliferation in a dose-dependent manner. Furthermore, luteolin treatment led to increased intracellular Fe 2+ accumulation, enhanced lipid ROS generation, and reduced GSH levels, indicating the induction of ferroptosis. Concurrently, luteolin promoted autophagy in HCC cells, as evidenced by an elevated LC3B-II/LC3B-I ratio, decreased p62 expression, and increased LC3 puncta formation. Importantly, inhibition of autophagy by knocking out ATG7 reversed luteolin-induced ferroptosis, suggesting that luteolin-induced ferroptosis is autophagy-dependent. Additionally, luteolin downregulated SLC40A1 expression in HCC cells. Overexpression of SLC40A1 attenuated luteolin-induced Fe 2+ accumulation and ferroptosis, while SLC40A1 knockdown mimicked the effects of luteolin. In vivo, luteolin suppressed xenograft tumor growth, enhanced autophagy, induced ferroptosis, and downregulated SLC40A1 expression in tumor tissues. Conclusion Our findings demonstrate that luteolin induces autophagy-dependent ferroptosis in HCC by suppressing SLC40A1-dependent Fe 2+ export. This study uncovers a novel anti-tumor mechanism of luteolin, providing a potential therapeutic strategy for HCC treatment by targeting SLC40A1-mediated iron homeostasis and the crosstalk between autophagy and ferroptosis.