Lipoxygenase inhibitors protect acute lymphoblastic leukemia cells from ferroptotic cell death
Graphical abstract
Introduction
ALL is the most frequent type of malignant neoplasm in childhood [1], [2]. The prognosis of children with very high-risk or relapsed disease is still dismal, thus calling for innovative therapeutic approaches. This includes new concepts to trigger programmed cell death, since evasion of leukemic cells to undergo cell death represents a frequent cause of treatment failure [3].
Several forms of programmed cell death have been described [4]. Ferroptosis is a recently defined mode of regulated cell death that depends on iron and is characterized by the generation of lipid-based ROS and lipid peroxidation [5]. Several ROS-generating enzymes contain iron or iron derivatives as essential co-factors for their proper function, for example LOX, nicotinamide adenine dinucleotide phosphate hydride (NADPH) oxidases (NOX), xanthine oxidase, and cytochrome P450 enzymes [6]. In addition, redox-active labile iron pools can directly catalyze free radical formation via Fenton chemistry [7].
LOX are key enzymes that catalyze the oxygenation of polyunsaturated fatty acyl groups to lipid hydroperoxides [8], while GPX family members are responsible for the reduction of hydrogen and lipid peroxides [9]. Among them, only GPX4 can directly reduce lipid hydroperoxides within biological membranes and therefore plays a crucial role in regulating the redox state of the cell membrane [9]. In addition, non-enzymatic lipophilic antioxidants such as α-Tocopherol (α-Toc) can scavenge membrane peroxyl radicals [10]. The membrane redox state is governed on the one side by the generation of lipid peroxides and on the other side by membrane-associated enzymatic and non-enzymatic peroxide scavengers. Disturbance of this homeostasis can lead to permeabilization of membranes such as the cell membrane and subsequently to cell death.
Ferroptosis has been implicated as a cell death process in a number of human diseases [5]. In cancer, the induction of ferroptosis may represent a therapeutic option, especially in those types of cancer that are refractory to other forms of programmed cell death, as ferroptosis proceeds independently of caspases. However, little is yet known about the signaling pathways that control ferroptosis in cancer cells. Therefore, in the present study we investigated the role of LOX in the regulation of ferroptotic cell death in ALL cells.
Section snippets
Cell culture and chemicals
ALL cell lines were obtained from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 or Dulbecco's Modified Eagle Medium (DMEM) medium (Life Technologies, Inc., Eggenstein, Germany), supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), 1 mM glutamine (Invitrogen, Karlsruhe, Germany), 1% penicillin/streptomycin (Invitrogen) and 25 mM HEPES (Biochrom). FADD-deficient Jurkat cells were kindly provided by Dr. J. Blenis [11], RSL3 was kindly provided by Dr. B. Stockwell.
RSL3 induces lipid peroxidation and ferroptotic cell death independently of FADD
We used RSL3, a small-molecule inhibitor of GPX4 [18], as a prototypic stimulus to trigger ferroptosis and employed the human T-cell ALL cell lines Jurkat and Molt-4 as cellular models. Treatment with RSL3 induced cell death in both ALL cell lines (Fig. 1A). This RSL3-stimulated cell death was associated with an increase in lipid peroxidation, as determined by the membrane-targeted lipid ROS sensor BODIPY-C11, a fluorescent dye that detects lipid peroxides (Fig. 1B). The addition of Fer-1, a
Discussion
In the present study, we report that LOX are involved in the regulation of RSL3-induced ferroptotic cell death in ALL cells. This conclusion is based on data showing that different LOX inhibitors protect ALL cells against lipid peroxidation, ROS generation and cell death upon treatment with RSL3, a prototypic ferroptosis inducer. The kinetics of RSL3-stimulated events with lipid peroxidation and ROS production preceding the occurrence of cell death underscore that lipid peroxidation and ROS
Conflict of interest
None to declare.
Source of funding
This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, European Community, IUAP VII, Wilhelm Sander-Stiftung and BMBF (to S.F.).
Acknowledgements
We thank B. Stockwell (Columbia University, New York, NY, USA) for kindly providing RSL3 and C. Hugenberg for expert secretarial assistance.
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