Ferroptosis is a newly defined form of cell death induced by iron-dependent accumulation of lethal lipid peroxidation. Ferroptosis represent a therapeutic strategy to suppress therapy-resistant cancer cells with more property of epithelial-mesenchymal transition (EMT). However, epigenetic reprogramming of EMT has been rarely studied in the context of ferroptosis susceptibility. Therefore, we examined the therapeutic potentiality of EMT epigenetic reprogramming in promoting ferroptosis in head and neck cancer (HNC) cells. The effects of ferroptosis inducers and EMT inhibition or induction were tested in HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species and iron production, labile iron pool, glutathione contents, NAD/NADH levels, and mRNA/protein expression. Cell density and the expression levels of E-cadherin, vimentin, and ZEB1 were associated with the different susceptibility to ferroptosis inducers. CDH1 silencing or ZEB1 overexpression increased the susceptibility to ferroptosis, whereas CDH overexpression or ZEB1 silencing decreased the susceptibility, in vitro and in vivo . Histone deacetylase SIRT1 gene silencing or pharmacological inhibition by EX-527 suppressed EMT and consequently decreased ferroptosis, whereas SIRT inducers, resveratrol and SRT1720, increased ferroptosis. MiR-200 family inhibitors induced EMT and increased ferroptosis susceptibility. In HNC cells with low expression of E-cadherin, the treatment of 5-azacitidine diminished the hypermethylation of CDH1, resulting in increased E-cadherin expression and decreased ferroptosis susceptibility. Our data suggest that epigenetic reprogramming of EMT contributes to promoting ferroptosis in HNC cells.
Rationale: Loss of iron-sulfur cluster function predisposes cancer cells to ferroptosis by upregulating iron-starvation response, but the role of glutaredoxin 5 (GLRX5) silencing in ferroptosis remains unknown. We examined the role of GLRX5 functional loss in promoting ferroptosis in cisplatin-resistant head and neck cancer (HNC) cells. Methods: The effects of sulfasalazine treatment and GLRX5 gene silencing were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species (ROS) and mitochondrial iron production, labile iron pool, mRNA/protein expression, and malondialdehyde assays. Results: Cyst(e)ine deprivation, erastin, or sulfasalazine induced ferroptosis in HNC cells, which was relatively less sensitive in cisplatin-resistant HNC cells. Sulfasalazine or cyst(e)ine deprivation-induced ferroptosis resulted from increased lipid peroxidation and intracellular free iron, which were significantly promoted by short-interfering RNA or short hairpin RNA (shRNA) targeting GLRX5 ( P <0.05). GLRX5 silencing activated iron-starvation response and boosted up intracellular free iron through the iron-responsive element-binding activity of increased iron regulatory protein (increased transferrin receptor and decreased ferritin). These effects were rescued by resistant GLRX5 cDNA but not by catalytically inactive mutant GLRX5 K101Q. The same results were noted in an in vivo mouse model transplanted with vector or shGLRX5-transduced HNC cells and treated with sulfasalazine. Conclusion: Our data suggest that inhibition of GLRX5 predisposes therapy-resistant HNC cells to ferroptosis.
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