Astragaloside IV Suppresses Gastric Cancer by m6A-dependent FSP1 Modulation and Ferroptosis Induction

Astragaloside IV (AS-IV), a key active component derived from the traditional Chinese medicinal plant Astragali, has been reported to exhibit various biological activities, including antioxidative, anti-inflammatory, immunoregulatory, and antineoplastic properties. This study aimed to elucidate the role of AS-IV in inhibiting gastric cancer (GC) growth, focusing on its impact on cell ferroptosis and the underlying molecular mechanisms. Proliferation and migration of GC cells upon AS-IV treatment were examined using CCK-8, colony formation, and Transwell assays. Ferroptosis induction was analyzed via ELISA, flow cytometry, and transmission electron microscopy. Ferroptosis suppressor protein 1 (FSP1) mRNA stability was assessed by the ActD assay, while RNA immunoprecipitation (RIP) was employed to confirm the interaction between FSP1 mRNA, Fat mass and obesity-associated protein (FTO) demethylase, and YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2). The dual-luciferase reporter assay was used to explore FTO binding to N6-methyladenosine (m6A)-modified sites on FSP1 mRNA. Furthermore, AS-IV’s anti-tumor effects (20 mg/kg) were validated in vivo using gastric cancer xenograft and lung metastasis mouse models. AS-IV significantly suppressed the proliferation and migration of GC cells by inducing ferroptosis. Mechanistically, AS-IV down-regulated FTO, thus impairing its interaction with FSP1 mRNA and leading to increased m6A modification on FSP1 mRNA. This modification facilitated m6A recognition protein YTHDF2-mediated recognition and subsequent degradation of FSP1 mRNA. The reduction of FSP1 triggered ferroptosis, while the overexpression of FSP1 or inhibition of ferroptosis by ferrostatin-1 partially reversed AS-IV’s effects on cell viability and migration. In vivo, AS-IV effectively inhibited tumor growth and metastasis. This study highlights potent anti-GC effects of AS-IV, mediated by the suppression of FSP1 via the FTO/YTHDF2/m6A axis. The treatment of AS-IV inhibits the expression of demethylase FTO in GC cells, subsequently disrupting the binding between FTO protein and FSP1 mRNA. This disruption leads to a increase in the level of m6A modification on FSP1 mRNA, thereby enhancing the recognition and binding of m6A-recognizing protein YTHDF2, promoting the decay of FSP1 mRNA. Ultimately, the downregulation of FSP1 enhances ferroptosis in GC cells. Collectively. AS-IV holds great promise as a novel therapeutic strategy for gastric cancer treatment. This image was created using the Biorender website (https://www.biorender.com/).