Arenobufagin induces ferroptosis in gastric cancer stem cells via the HCAR1-GPX4/SLC7A11 antioxidant axis.

Arenobufagin (ARBU), a steroid compound extracted from the venom of Bufo gargarizans, exhibits multi-target pharmacological activities, yet its role in regulating ferroptosis in gastric cancer stem cells (GCSCs) remains unclear. This study systematically evaluated the antitumor effects and mechanisms of ARBU using in vitro sphere culture, organoid models, and xenografts. ARBU inhibited GCSC proliferation and sphere formation in a concentration-dependent manner, reduced EdU incorporation and SOX2 expression in organoids, and markedly suppressed tumor growth in vivo while downregulating SOX2 and Nanog, with favorable biosafety. Mechanistically, ARBU induced ferroptosis, evidenced by elevated MDA, ROS, and Fe2+, decreased GSH and SOD, mitochondrial damage, COX2 upregulation, and GPX4/SLC7A11 downregulation. RNA-seq and functional studies further revealed that HCAR1 critically regulates GCSC self-renewal and antioxidant defense, and ARBU promoted ferroptosis via HCAR1 suppression. Collectively, these results demonstrate that ARBU inhibits GCSC proliferation and stemness by inducing ferroptosis through downregulation of the HCAR1 pathway, highlighting its potential as a therapeutic candidate for gastric cancer.