Abstract
Rhabdomyosarcoma (RMS) cells have recently been reported to be sensitive to oxidative stress. Therefore, we investigated whether concomitant inhibition of the two main antioxidant defense pathways, that is, the thioredoxin (TRX) and the glutathione (GSH) systems, presents a new strategy to trigger cell death in RMS. In this study, we discover that GSH-depleting agents, i.e. γ-glutamylcysteine synthetase inhibitor, buthionine sulfoximine (BSO) or the cystine/glutamate antiporter inhibitor erastin (ERA), synergize with thioredoxin reductase (TrxR) inhibitor auranofin (AUR) to induce cell death in RMS cells. Interestingly, AUR causes accumulation of ubiquitinated proteins when combined with BSO or ERA, in line with recent reports showing that AUR inhibits the proteasome besides TrxR. Consistently, AUR/BSO or AUR/ERA cotreatment increases ubiquitination and expression of the short-lived proteins NOXA and MCL-1, accompanied by increased binding of NOXA to MCL-1. Notably, NOXA knockdown significantly rescues RMS cells from AUR/BSO- or AUR/ERA-induced cell death. In addition, AUR acts together with BSO or ERA to stimulate BAX/BAK and caspase activation. Of note, BSO or ERA abolish the AUR-stimulated increase in GSH levels, leading to reduced GSH levels upon cotreatment. Although AUR/BSO or AUR/ERA cotreatment enhances reactive oxygen species (ROS) production, only thiol-containing antioxidants (i.e., N-acetylcysteine (NAC), GSH), but not the non-thiol-containing ROS scavenger α-Tocopherol consistently suppress AUR/BSO- and AUR/ERA-stimulated cell death in both cell lines. Importantly, re-supply of GSH or its precursor NAC completely prevents AUR/ERA- and AUR/BSO-induced accumulation of ubiquitinated proteins, NOXA upregulation and cell death, indicating that GSH depletion rather than ROS production is critical for AUR/BSO- or AUR/ERA-mediated cell death. Thus, by demonstrating that GSH-depleting agents enhance the antitumor activity of AUR, we highlight new treatment options for RMS by targeting the redox homeostasis.
Highlights
RMS is the most common soft-tissue sarcoma found in children[1] and comprises two major histological subtypes, that is, alveolar RMS (ARMS) and embryonal RMS (ERMS).[2,3,4]
We investigated in this study whether targeting the cellular redox homeostasis represents a suitable approach to induce cell death in RMS
As inhibition of the proteasome has been shown to result in upregulation of short-lived proteins, we investigated expression levels and the ubiquitination status of NOXA and MCL-1, two proteins of the BCL-2 family with a rapid turnover.[33,34]
Summary
RMS is the most common soft-tissue sarcoma found in children[1] and comprises two major histological subtypes, that is, alveolar RMS (ARMS) and embryonal RMS (ERMS).[2,3,4] As the prognosis especially after metastasis or relapse still remains poor,[5] there is a high medical need for new therapies. ROS exert important functions in many biological and biochemical processes, whereas at high levels ROS can lead to cell death.[15] Cancer cells often exhibit increased basal ROS levels, for example, because of their elevated metabolic activity, oncogene activation or mitochondrial dysfunction that are compensated by concomitant upregulation of antioxidant pathways to cope with higher levels of oxidative stress.[16] These changes render cancer cells susceptible to treatment regimens targeting their redox homeostasis.[17]. The glutathione (GSH) and the thioredoxin (TRX) system are two important ROS scavenging pathways that are often upregulated in cancer cells.[18,19] As the reduced form of GSH is the most abundant non-protein thiol in the cell,[20] changes in intracellular GSH levels have an important role in regulating redox homeostasis. BSO inhibits γ-glutamylcysteine synthetase,[26] the rate-limiting enzyme of the GSH synthesis,[27] leading to GSH depletion.[26,27] ERA blocks the Received 20.5.17; revised 14.7.17; accepted 18.7.17; Edited by G Raschellà
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