ATM inhibition enhances Auranofin-induced oxidative stress and cell death in lung cell lines.

Ataxia-Telangiectasia (A-T), a pleiotropic chromosomal breakage syndrome, is caused by the loss of the kinase Ataxia-telangiectasia mutated (ATM). ATM is not only involved in the response to DNA damage, but also in sensing and counteracting oxidative stress. Since a disturbed redox balance has been implicated in the pathophysiology of A-T lung disease, we aimed to further explore the interplay between ATM and oxidative stress in lung cells. Using a kinetic trapping approach, we could demonstrate an interaction between the trapping mutant TRX1-CS and ATM upon oxidative stress. We could further show that combined inhibition of thioredoxin reductase (TrxR) and ATM kinase activity, using Auranofin and KU55933 respectively, induced an increase in cellular reactive oxygen species (ROS) levels and protein oxidation in lung cells. Furthermore, ATM inhibition sensitized lung cells to Auranofin-induced cell death that could be rescued by ROS scavengers. As a consequence, targeted reduction of ATM by TRX1 could serve as a regulator of oxidative ATM activation and contribute to the maintenance of the cellular redox homeostasis. These results highlight the importance of the redox-active function of ATM in preventing ROS accumulation and cell death in lung cells.

Thioredoxin inhibitor PX-12 induces mitochondria-mediated apoptosis in acute lymphoblastic leukemia cells.

Imbalances in redox homeostasis have been described to be involved in the development, progression and relapse of leukemia. As the thioredoxin (Trx) system, one of the major cellular antioxidant networks, has been implicated in acute lymphoblastic leukemia (ALL), we investigated the therapeutic potential of Trx inhibition in ALL. Here, we show that the Trx inhibitor PX-12 reduced cell viability and induced cell death in a dose- and time-dependent manner in different ALL cell lines. This antileukemic activity was accompanied by an increase in reactive oxygen species (ROS) levels and enhanced PRDX3 dimerization. Pre-treatment with the thiol-containing ROS scavenger N-acetylcysteine (NAC), but not with non-thiol-containing scavengers α-tocopherol (α-Toc) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP), significantly rescued PX-12-induced cell death. Furthermore, PX-12 triggered activation of BAK. Importantly, knockdown of BAK reduced PX-12-stimulated ROS production and cell death. Similarly, silencing of NOXA provided significant protection from PX-12-mediated cell death. The relevance of mitochondria-mediated, caspase-dependent apoptosis was further supported by data showing that PX-12 triggered cleavage of caspase-3 and that addition of the broad-range caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD.fmk) potently blocked cell death upon PX-12 treatment. This study provides novel insights into the mechanisms of PX-12-induced cell death in ALL and further highlights the therapeutic potential of redox-active compounds in ALL.

Targeting ferroptosis in rhabdomyosarcoma cells.

Recent data suggest that rhabdomyosarcoma (RMS) cells might be vulnerable to oxidative stress-induced cell death. Here, we show that RMS are susceptible to cell death induced by Erastin, an inhibitor of the glutamate/cystine antiporter xc- that can increase reactive oxygen species (ROS) production via glutathione (GSH) depletion. Prior to cell death, Erastin caused GSH depletion, ROS production and lipid peroxidation. Importantly, pharmacological inhibitors of lipid peroxidation (i.e., Ferrostatin-1, Liproxstatin-1), ROS scavengers (i.e., α-Tocopherol, GSH) and the iron chelator Deferoxamine inhibited ROS accumulation, lipid peroxidation and cell death, consistent with ferroptosis. Interestingly, the broad-spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I as well as the PKCα- and ß-selective inhibitor Gö6976 significantly reduced Erastin-induced cell death. Similarly, genetic knockdown of PKCα significantly protected RMS cells from Erastin-induced cell death. Furthermore, the broad-spectrum nicotinamide adenine dinucleotide phosphate-oxidase (NOX) inhibitor Diphenyleneiodonium and the selective NOX1/4 isoform inhibitor GKT137831 significantly decreased Erastin-stimulated ROS, lipid ROS and cell death. These data provide new insights into the molecular mechanisms of ferroptosis in RMS, contributing to the development of new redox-based treatment strategies.