Stabilization of Keap1 by alkannin triggers ferroptotic cell death in colorectal cancer via suppression of the Nrf2/GPX4 signaling

Colorectal Cancer (CRC) is one of the most common and deadly types of Cancer globally. Ferroptosis, a type of regulated cell death that relies on iron, has become a promising target for treating CRC. Alkannin, a natural compound from Lithospermum erythrorhizon, exhibits anti-tumor activity, yet its mechanism in CRC is unclear. This study investigated alkannin's role in regulating Ferroptosis via the Keap1/Nrf2/GPX4 axis. Using network pharmacology and experimental validation in HCT116 and SW480 cells and a xenograft mouse model, we found that alkannin markedly inhibited the viability, proliferation, and migratory capacity of CRC cells, demonstrating significant anti-tumor activity. Network pharmacology revealed a primary association between alkannin's therapeutic effects and the induction of Ferroptosis, along with the regulation of oxidative stress pathways, with a notable focus on the Keap1/Nrf2 axis. Experimental evidence confirmed that alkannin induced Ferroptosis, as reflected by increased intracellular Fe²⁺ levels and lipid peroxidation, along with reduced glutathione (GSH) content. These effects were reversed by Ferroptosis inhibitors, which also attenuated alkannin-induced cytotoxicity. Mechanistically, alkannin enhanced Keap1 protein stability by suppressing its ubiquitination. It promoted the interaction between Keap1 and Nrf2, leading to decreased Nrf2 expression and inhibition of its nuclear translocation, thereby downregulating the expression of Glutathione Peroxidase 4 (GPX4), a key suppressor of Ferroptosis. Genetic silencing of Keap1 significantly diminished alkannin-induced ferroptotic cell death. In vivo, alkannin effectively inhibited tumor growth in xenografted nude mice. Furthermore, it induced Ferroptosis in tumor tissues, as evidenced by similar biochemical changes, which were counteracted by co-administration of a Ferroptosis inhibitor. Consistently, alkannin upregulated Keap1 expression while reducing the protein levels of Nrf2 and GPX4 in tumor tissues. In conclusion, alkannin induces Ferroptosis in CRC by stabilizing Keap1 to inhibit the Nrf2/GPX4 pathway, supporting its potential as a CRC therapeutic agent.