Inhibition of GLRX5 promotes ferroptotic cell death of head and neck cancer (HNC) cells. (A-C) Cell viability of HNC cells exposed to cyst(e)ine (CC) deprivation and erastin or sulfasalazine (SAS) treatment in HNC cells. The cell viability was relative to control not treated with CC deprivation, erastin, or SAS. HN3R and HN4R were the cisplatin-resistant HNC cell lines developed from the parental cisplatin-sensitive cell lines of HN3 and HN4, respectively. * P<0.05. (D-F) Ferroptosis in HNC cells induced by SAS treatment. Propidium iodide (PI)-positive cells were stained and counted using fluorescent microscopy and flow cytometry after 1 mM SAS treatment for 48 h. NT indicates control treated with DMSO only. Scale bar, 50 μm. ** P < 0.01. NS indicates statistically not significant. (G, H) mRNA and protein expression of GLRX5 in the HN3R and HN4R cells transfected with siRNA control (siCtr) or siGLRX5. (I) Changes of cellular lipid reactive oxygen species (ROS) levels of HN4R cells exposed to 1 mM SAS for 8 h. The cells were also pretreated with deferoxamine (DFO, 100 μM) or co-treated with ferrostatin-1 (Fer-1, 2 μM), or α-tocopherol (αTP, 200 μM). The error bars represent standard errors from three replicates. ** P < 0.01 between siCtr and siGLRX5.