Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Oxidative stress in conjunction with glutathione depletion has been linked with various acute and chronic degenerative disorders, yet the molecular mechanisms have remained unclear. In contrast to the belief that oxygen radicals are detrimental to cells and tissues by unspecific oxidation of essential biomolecules, we now demonstrate that oxidative stress is sensed and transduced by glutathione peroxidase 4 (GPx4) into a-yet-unrecognized cell-death pathway. Inducible GPx4 inactivation in mice and cells revealed 12/15-lipoxygenase-derived lipid peroxidation as specific downstream event, triggering apoptosis-inducing factor (AIF)-mediated cell death. Cell death could be entirely prevented either by alpha-tocopherol (alpha-Toc), 12/15-lipoxygenase inhibitors, or siRNA-mediated AIF silencing. Accordingly, 12/15-lipoxygenase-deficient cells were highly resistant to glutathione depletion. Neuron-specific GPx4 depletion caused neurodegeneration in vivo and ex vivo, highlighting the importance of this pathway in neuronal cells. Since oxidative stress is common in the etiology of many human disorders, the identified pathway reveals promising targets for future therapies.

Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/ reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in ␥-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.

Tumor cells generate substantial amounts of reactive oxygen species (ROS), engendering the need to maintain high levels of antioxidants such as thioredoxin (Trx)-and glutathione (GSH)-dependent enzymes. Exacerbating oxidative stress by specifically inhibiting these types of ROS-scavenging enzymes has emerged as a promising chemotherapeutic strategy to kill tumor cells. However, potential redundancies among the various antioxidant systems may constrain this simple approach. Trx1 and thioredoxin reductase 1 (Txnrd1) are upregulated in numerous cancers, and Txnrd1 has been reported to be indispensable for tumorigenesis. However, we report here that genetic ablation of Txnrd1 has no apparent effect on tumor cell behavior based on similar proliferative, clonogenic, and tumorigenic potential. This finding reflects widespread redundancies between the Trx-and GSH-dependent systems based on evidence of a bypass to Txnrd1 deficiency by compensatory upregulation of GSH-metabolizing enzymes. Because the survival and growth of Txnrd1-deficient tumors were strictly dependent on a functional GSH system, Txnrd1 −/− tumors were highly susceptible to experimental GSH depletion in vitro and in vivo. Thus, our findings establish for the first time that a concomitant inhibition of the two major antioxidant systems is highly effective in killing tumor, highlighting a promising strategy to combat cancer. Cancer Res; 70(22); 9505-14. ©2010 AACR.