Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

Calycosin, a natural flavonoid small-molecule compound derived from traditional Chinese medicine, has demonstrated remarkable pharmacological activity in the field of cancer therapy. This study systematically elucidates the molecular mechanisms of Calycosin in colorectal cancer (CRC) treatment through integrated in vivo and in vitro experiments. In vivo experiments revealed that Calycosin effectively inhibits subcutaneous tumor growth in CRC-bearing mice. In vitro assays and transcriptome sequencing confirmed that Calycosin effectively suppresses migration, invasion, epithelial-mesenchymal transition (EMT), and induces ferroptosis in human CRC cells, thereby inhibiting malignant tumor behaviors. Cellular Thermal Shift Assay (CETSA) and site-directed mutagenesis experiments first identified cytochrome P450 1B1 (CYP1B1) and Gly-329 as critical binding targets and sites for Calycosin. Functional studies showed that CYP1B1 knockdown in vitro and in vivo suppresses GPX4 expression and enhances ferroptosis in CRC cells. Mechanistically, CYP1B1 activates the AKT/SP-1 signaling pathway to upregulate GPX4 expression, thereby modulating colorectal carcinogenesis and progression. In summary, this study first unveils the crucial role of Calycosin and the CYP1B1-AKT/SP1-GPX4 regulatory axis in CRC ferroptosis, providing novel theoretical foundations for targeted therapy using traditional Chinese medicine-derived small molecules against colorectal cancer.