Huaier-derived arnicolide D induces ferroptosis in gastric cancer via SRC/TNF-mediated GPX4 inactivation.

[BACKGROUND] While ferroptosis induction has emerged as a promising strategy against gastric cancer (GC), clinically viable natural inducers remain scarce. Huaier n-butanol extract (HBE), a crude extract derived from the traditional Chinese medicinal fungus Huaier (Trametes robiniophila Murr.), exhibits promising anticancer activity, although its bioactive constituents and underlying mechanisms remain to be fully elucidated.

[PURPOSE] This study aimed to investigate HBE's anti-GC effects and identify its bioactive components, with a focus on elucidating the ferroptosis-related mechanisms underlying their therapeutic actions.

[METHOD] WE EMPLOYED A COMPREHENSIVE APPROACH INCLUDING: 1) in vivo evaluation using an immunocompetent mouse GC model; 2) chemical characterization of HBE components via UHPLC-MS/MS; 3) network pharmacology analysis to predict potential targets; 4) in vitro cellular assays assessing ferroptosis markers; 5) molecular docking and surface plasmon resonance (SPR) for target validation; and 6) siRNA-mediated gene knockdown to verify mechanistic pathways.

[RESULTS] HBE significantly inhibited tumor growth without observable toxicity in immunocompetent mice by inducing ferroptosis, as evidenced by increased levels of the lipid peroxidation marker 4-HNE. In vitro experiments confirmed that HBE triggers ferroptosis through disruption of iron metabolism and redox homeostasis. This was further supported by elevated intracellular iron levels, increased lipid reactive oxygen species (ROS) and malondialdehyde (MDA), decreased reduced glutathione (GSH) content, and downregulation of FPN1, FSP1, and GPX4 protein levels as confirmed by Western blotting. Blood component analysis identified Arnicolide D as one of the key active components in HBE. In vitro studies demonstrated the potent antitumor activity of Arnicolide D across five GC cell lines, while in vivo experiments confirmed its efficacy in suppressing tumor growth without significant toxicity. Mechanistically, Arnicolide D was found to dually target TNF and SRC, leading to the suppression of GPX4 expression at both transcriptional and post-translational levels, thereby promoting ferroptosis.

[CONCLUSION] These findings not only elucidate the pharmacodynamic basis of HBE's anti-GC activity but also establish Arnicolide D as a promising dual-target ferroptosis inducer, providing a novel therapeutic strategy for GC treatment.