Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death

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.

Potential Applications of NRF2 Modulators in Cancer Therapy

“…Moreover, due to patients' individualities and tumors heterogeneity, variable response rates are often observed making the identification of more effective drugs very urgent [272,273]. In this regard, the concomitant inhibition of antioxidant circuits and metabolic pathways that support the redox balance of malignant cells, delineates a promising anti-cancer strategy [274][275][276][277][278][279][280][281][282][283]. It is known that most of the metabolic changes promoting cancer cells proliferation and tumor growth induce also an increased ROS generation, counterbalanced by an antioxidant response that prevents cell death [234,284,285].…”

Targeting ferroptosis in rhabdomyosarcoma cells

“…ROS production and lipid peroxidation were analyzed at early time points before cells succumb to cell death to monitor events prior to the induction of cell death as previously described. 19 To analyze ROS production cells were pelleted and resuspended with phosphate-buffered saline containing 5 μM of the fluorescent dye CM-H 2 DCFDA (Invitrogen), which has been reported to detect ROS such as hydrogen peroxides, hydroxyl radicals or peroxyl radicals, for 30 min at 37 C and immediately analyzed by flow cytometry using fluorescein isothiocyanate channel. For determination of lipid peroxidation, cells were pelleted and resuspended with phosphate-buffered saline containing 5 μM of the fluorescent dye BODIPY-C11 (Invitrogen) for 30 min at 37 C and immediately analyzed by flow cytometry using fluorescein isothiocyanate channel.…”

Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells

“…The sensitivity of gastric cancer cells to auranofin could be increased by the silencing of genes involved in autophagy [ 38 ], and BRCA1 deficiency has further been implicated to increase sensitivity of ovarian cancer cells to auranofin [ 39 ]. Synergistic activity on cancer cell growth in preclinical models is reported for the combination of auranofin with many anticancer agents and is in line with the use of auranofin as combination therapy in current clinical trials [ 24 , 25 , 36 , 40 , 41 ]. The sensitizing activity of auranofin is further supported by studies showing an enhancement of radiation response in breast cancer cells [ 42 , 43 ].…”

“…There is strong evidence that the cytostatic activity of auranofin and other TrxR inhibitors on various cancer cells is mediated via an increase in cellular ROS, subsequently leading to apoptosis [ 17 , 19 , 20 , 21 , 22 , 26 ]. Other mechanisms discussed are inhibition of the PI3K/AKT pathway in lung cancer cells [ 23 , 36 ], inhibition of the proteasome in rhabdomyosarcoma and hepatoma cells [ 25 , 37 ], and adenosine triphosphate (ATP) depletion by inhibition of glycolysis in stem-like cancer cells [ 24 ]. The sensitivity of gastric cancer cells to auranofin could be increased by the silencing of genes involved in autophagy [ 38 ], and BRCA1 deficiency has further been implicated to increase sensitivity of ovarian cancer cells to auranofin [ 39 ].…”

“…Auranofin is an organic gold complex clinically used as antiarthritic drug. In various cancer cells, auranofin has been shown to elicit strong cytotoxicity through diverse mechanisms such as overproduction of ROS, inhibition of glycolysis and the proteasome, and activation of apoptotic pathways [ 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. Furthermore, auranofin treatment has been described to overcome cisplatin resistance in ovarian cancer cells [ 27 ].…”