Calycosin Promotes Ferroptosis in Cervical Cancer via the AMPK/PI3K‐TFEB‐Autophagy‐Lysosome Axis

Cervical cancer ranks among the most common malignancies in women globally, and chemoresistance is a primary cause of treatment failure and patient mortality. Ferroptosis, an iron‐dependent form of programmed cell death driven by lipid peroxidation, has been shown to reverse chemoresistance in tumor cells. Calycosin, a major active component of the traditional Chinese medicine Astragalus membranaceus, exhibits broad anti‐tumor activity; however, whether it exerts anti‐tumor effects in cervical cancer by inducing ferroptosis remains unclear. This study aimed to investigate the anti‐tumor effects of calycosin on human cervical cancer cell lines and the molecular mechanisms underlying its induction of ferroptosis, with a specific focus on the regulatory roles of autophagy and lysosomal biogenesis and the associated signaling pathways. Cell viability was assessed using the CCK‐8 assay, and cell proliferation was observed via EdU staining. Ultrastructural changes were examined by transmission electron microscopy (TEM). Levels of malondialdehyde (MDA), glutathione (GSH), and Fe2+ were measured using commercial kits. Protein expression related to ferroptosis, autophagy, lysosomes, and signaling pathways was detected by Western blotting. Mechanistic validation was performed using the ferroptosis inhibitor Fer‐1, the autophagy inhibitor 3‐MA, the lysosomal inhibitor chloroquine, and the PI3K inhibitor LY294002. An in vivo xenograft tumor model was established in nude mice. Calycosin significantly inhibited the proliferation of cervical cancer cells and induced ferroptosis, characterized by mitochondrial shrinkage, increased MDA and Fe2+ levels, decreased GSH levels, and downregulated expression of SLC7A11, GPX4, and FTH1 proteins. Concurrently, calycosin induced autophagy and lysosomal biogenesis, which enhanced the ferroptosis process. Mechanistically, calycosin promoted Beclin1‐dependent autophagy by activating the AMPK/ULK1 pathway and inhibiting the PI3K/AKT/mTOR pathway; furthermore, it promoted lysosomal biogenesis via the ERK/TFEB pathway, ultimately enhancing ferroptosis. In conclusion, calycosin induces ferroptosis in human cervical cancer cells through multi‐pathway synergistic effects. The specific mechanisms include: (1) direct disruption of the System Xc−/GSH/GPX4 axis, triggering lipid peroxidation; (2) induction of protective autophagy via AMPK/ULK1 activation and PI3K/AKT/mTOR inhibition, thereby promoting ferroptosis; and (3) regulation of the ERK/PI3K‐TFEB axis to promote lysosomal biogenesis, enhancing iron metabolic dysregulation and oxidative stress. This study is the first to systematically elucidate the molecular mechanism by which calycosin exerts its anti‐cervical cancer effect through an “autophagy‐lysosome‐ferroptosis” cascade network, providing a theoretical basis and experimental support for its development as a potential anti‐cancer agent.