Cell–cell contacts protect against t-BuOOH-induced cellular damage and ferroptosis in vitro

Ferroptosis is a recently discovered pathway of regulated necrosis dependent on iron and lipid peroxidation. It has gained broad attention since it is a promising approach to overcome resistance to apoptosis in cancer chemotherapy. We have recently identified tertiary-butyl hydroperoxide (t-BuOOH) as a novel inducer of ferroptosis. t-BuOOH is a widely used compound to induce oxidative stress in vitro. t-BuOOH induces lipid peroxidation and consequently ferroptosis in murine and human cell lines. t-BuOOH additionally results in a loss of mitochondrial membrane potential, formation of DNA double-strand breaks, and replication block. Here, we specifically address the question whether cell-cell contacts regulate t-BuOOH-induced ferroptosis and cellular damage. To this end, murine NIH3T3 or human HaCaT cells were seeded to confluence, but below their saturation density to allow the establishment of cell-cell contacts without inducing quiescence. Cells were then treated with t-BuOOH (50 or 200 µM, respectively). We revealed that cell-cell contacts reduce basal and t-BuOOH-triggered lipid peroxidation and consequently block ferroptosis. Similar results were obtained with the specific ferroptosis inducer erastin. Cell-cell contacts further protect against t-BuOOH-induced loss of mitochondrial membrane potential, and formation of DNA double-strand breaks. Interestingly, cell-cell contacts failed to prevent t-BuOOH-mediated replication block or formation of the oxidative base lesion 8-oxo-dG. Since evidence of protection against cell death was both (i) observed after treatment with hydrogen peroxide, methyl methanesulfonate or UV-C, and (ii) seen in several cell lines, we conclude that protection by cell-cell contacts is a widespread phenomenon. The impact of cell-cell contacts on toxicity might have important implications in cancer chemotherapy.