Dihydrolipoamide dehydrogenase regulates cystine deprivation-induced ferroptosis in head and neck cancer

Abstract

Ferroptosis is a new form of regulated cell death driven by iron-dependent lipid peroxidation. Glutaminolysis and tricarboxylic acid cycle are involved in ferroptosis, but the underlying metabolic process remains unclear. We examined the role of dihydrolipoamide dehydrogenase (DLD) in ferroptosis induction in head and neck cancer (HNC). The effects of cystine deprivation or sulfasalazine treatment and of DLD gene silencing/overexpression were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed with regard to cell death, lipid reactive oxygen species (ROS) and mitochondrial iron production, mitochondrial membrane potential, mRNA/protein expression, and α-ketoglutarate dehydrogenase (KGDH)/succinate/aconitase activities. Cystine deprivation induced ferroptosis via glutaminolysis. Cystine deprivation or import inhibition using sulfasalazine induced cancer cell death and increased lipid ROS and mitochondrial iron levels, which had been significantly decreased by short-interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting DLD (P < 0.01) but not by dihydrolipoyl succinyltransferase. The same results were noted in an in vivo mouse model transplanted with vector or shDLD-transduced HN9 cells. After cystine deprivation or sulfasalazine treatment, mitochondrial membrane potential, mitochondrial free iron level, KGDH activity, and succinate content significantly increased (P < 0.001), which had been blocked by DLD siRNA or shRNA and were consequently rescued by resistant DLD cDNA. Cystine deprivation caused iron starvation response and mitochondrial iron accumulation for Fenton reaction and ferroptosis. Our data suggest a close association of DLD with cystine deprivation- or import inhibition-induced ferroptosis. : Cystine deprivation induced ferroptosis via glutaminolysis in cancer cells. Dihydrolipoamide dehydrogenase (DLD) gene silencing blocked cystine deprivation- or sulfasalazine-induced ferroptosis in vitro and in vivo, which was restored by resistant DLD cDNA; this suggested a close relationship between DLD and cystine deprivation-induced ferroptosis. Keywords: Ferroptosis, Dihydrolipoamide dehydrogenase, Mitochondria, Cystine deprivation, Iron