Non‐Apoptotic Inhibition of Colorectal Cancer Proliferation by Sclareol via Ferroptosis Involving <scp>SLC7A11</scp>/<scp>GPX4</scp> Modulation

Colorectal cancer (CRC) is a prevalent and lethal malignancy necessitating new treatments. Sclareol, the primary active component of Salvia sclarea L., exhibits diverse pharmacological properties, making it a promising anti-cancer drug warranting further investigation. To evaluate Sclareol's action in CRC cell death, various methods were performed, including cell viability and colony formation assays, EdU assay, flow cytometry for cell cycle analysis and apoptosis, as well as TUNEL staining, transmission electron microscopy (TEM), reactive oxygen species (ROS) detection, and Fe²⁺ fluorometric assay. RNA sequencing and pathway analysis, combined with in silico molecular docking, and immunoblotting, were conducted to determine the mechanism of action of Sclareol. A CRC xenograft model in immunocompetent mice was used to evaluate Sclareol's in vivo efficacy and toxicity, with immunohistochemistry confirming its mechanism of action. Sclareol significantly suppressed CRC cell proliferation and induced cell cycle arrest in vitro without significantly inducing apoptosis. Ferroptosis as a promising mechanism of action was validated through TEM, ROS detection, and Fe²⁺ staining. RNA sequencing analysis revealed enrichment in metal ion-transporter activity. Molecular docking analysis showed direct binding of Sclareol to the key ferroptosis-related target solute carrier family 7 member 11 (SLC7A11). Additionally, Sclareol downregulated the expression of SLC7A11, nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4) proteins, indicating ferroptosis induction in vitro. Significant tumor growth inhibition was observed without hepatic or renal toxicities, with immunohistochemistry confirming the suppression of ferroptosis- and proliferation-related markers in vivo. Sclareol inhibits CRC growth by modulating the ferroptosis pathway through the SLC7A11/GPX4 axis. Sclareol shows promise as a therapeutic agent in CRC, warranting further pre-clinical studies to confirm its efficacy and safety.