Strong Protection by 4-Hydroxyestrone against Erastin-Induced Ferroptotic Cell Death in Estrogen Receptor-Negative Human Breast Cancer Cells: Evidence for Protein Disulfide Isomerase as a Mechanistic Target for Protection

Ferroptosis is a recently identified form of regulated cell death, characterized by excessive iron-dependent lipid peroxidation. Recent studies have demonstrated that protein disulfide isomerase (PDI) is an important mediator of chemically induced Ferroptosis and also a new target for protection against ferroptosis-associated cell death. In the present study, we identified that 4-hydroxyestrone (4-OH-E₁), a metabolic derivative of endogenous estrogen, is a potent small-molecule inhibitor of PDI, and can strongly protect against chemically induced ferroptotic cell death in the estrogen receptor-negative MDA-MB-231 human breast Cancer cells. Pull-down and CETSA assays demonstrated that 4-OH-E₁ can directly bind to PDI both in vitro and in intact cells. Computational modeling analysis revealed that 4-OH-E₁ forms two hydrogen bonds with PDI His256, which is essential for its binding interaction and thus inhibition of PDI's catalytic activity. Additionally, PDI knockdown attenuates the protective effect of 4-OH-E₁ as well as cystamine (a known PDI Inhibitor) against chemically induced Ferroptosis in human breast Cancer cells. Importantly, inhibition of PDI by 4-OH-E₁ and cystamine or PDI knockdown by siRNAs each markedly reduces iNOS activity and NO accumulation, which has recently been demonstrated to play an important role in erastin-induced Ferroptosis. In conclusion, this study demonstrates that 4-OH-E₁ is a novel inhibitor of PDI and can strongly inhibit Ferroptosis in human breast Cancer cells in an estrogen receptor-independent manner. The mechanistic understanding gained from the present study may also aid in understanding the estrogen receptor-independent cytoprotective actions of endogenous estrogen metabolites in many noncancer cell types.