Etoposide in combination with erastin synergistically altered iron homeostasis and induced ferroptotic cell death through regulating IREB2/FPN1 expression in estrogen receptor positive-breast cancer cells

Aim: Ferroptosis is an iron-dependent cell death mechanism that substantially differs from apoptosis. Since its mechanism involves increased oxidative stress and rich iron content, cancer cells are particularly vulnerable to ferroptotic death compared to healthy tissues. In the present study, the effect of etoposide in combination with a ferroptotic agent, erastin, was investigated in breast cancer.Main methods: Cell viability was assessed by the MTT assay. Oxidative stress, lipid peroxidation and glutathione peroxidase activity were detected using the relevant kits. Intracellular iron levels were measured by HPLC. Ferroptosis markers were explored by western blotting.Key findings: Results demonstrated that although etoposide didn't induce a significant cell death up to 50 mu M in MCF-7 cells, with the addition of erastin, a significant synergistic activity was achieved at a dose as low as 1 mu M (p < 0.05), contrary to normal breast epithelial cells. This cytotoxic effect was blocked by ferrostatin-1, which is a specific inhibitor of ferroptosis. The combined treatment of etoposide and erastin synergistically induced oxidative stress and lipid peroxidation, while suppressing glutathione peroxidase activity. More importantly, the combination treatment synergistically increased iron accumulation, which was associated with altered expres-sion of IREB2/FPN1. Additionally, ferroptosis-regulating proteins ACSF2 and GPX4 were altered more potently by the combination treatment, compared to untreated cells and erastin treatment alone (p < 0.05).Significance: In conclusion, this is the first study that reports enhanced cytotoxicity of etoposide, in combination with erastin, in ER-positive breast cancer cells via activation of ferroptotic pathways, and offers a new perspective for future regimens.