Luteolin Induces GPX4-dependent Ferroptosis and Enhances Immune Activation in Colon Cancer

Abstract

Background: Ferroptosis is pivotal in colon cancer progression and immunity. While the natural flavonoid luteolin has anticancer properties, its ferroptosis targets and immunomodulatory effects in colon cancer are unclear.

Purpose: This study aims to validate luteolin's anticancer efficacy in colon cancer and elucidate its mechanism involving ferroptosis induction and antitumor immune activation.

Methods: Integrated WGCNA, machine learning, and functional enrichment techniques to analyze luteolin's potential targets. Comprehensive assays including CCK-8, colony formation, flow cytometry, western blotting, and reactive oxygen species (ROS)/malondialdehyde (MDA)/glutathione (GSH) /FerroOrange profiling, combined with MC38 syngeneic models, were used to investigate luteolin's antiproliferative effects, glutathione peroxidase 4 (GPX4) - dependent ferroptosis induction, and modulation of tumor-infiltrating immune cells. Molecular docking, Cellular Thermal Shift Assay (CETSA), Drug Affinity Responsive Target Stability (DARTS), and surface plasmon resonance (SPR) validated GPX4-luteolin interactions. Anti-CD8 antibody and clodronate liposomes (CLD) immune depletion studies demonstrate that CD8⁺ T cells and macrophages play pivotal roles in activating antitumor immunity.

Results: Bioinformatics indicated luteolin modulates oxidative stress signaling. Luteolin dose-dependently inhibited colon cancer cell proliferation, reversed by ferroptosis inhibitor Ferrostatin-1. Luteolin triggered ROS/Fe²⁺ accumulation, lipid peroxidation, MDA elevation, GSH depletion, and GPX4 downregulation. GPX4 overexpression conferred ferroptosis resistance, reversed by luteolin. Besides, luteolin directly bound GPX4, enhancing its thermal stability and suppressing pronase E-mediated degradation. In vivo, luteolin exhibited antitumor efficacy through GPX4 downregulation and ferroptosis induction, synergistically promoting intratumoral M1 macrophage polarization and CD8⁺ T lymphocyte activation.

Conclusion: Luteolin induces ferroptosis by directly targeting GPX4, and promotes antitumor immune responses. These findings reveal a novel mechanism underlying luteolin-induced ferroptosis and provide theoretical support for its immunotherapeutic application in colon cancer.

Keywords: Cancer immunity; Colon cancer; Ferroptosis; GPX4; Luteolin.