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![A: NADPH is a source of reducing equivalents for the glutathione system consisting of reduced glutathione (GSH), glutathione disulfide (GSSG), glutathione peroxidase (GPx), and glutathione reductase (GR) and the thioredoxin system consisting of reduced thioredoxin [Trx(SH)2], thioredoxin disulfide [Trx(S2)], peroxiredoxin (Prx), and thioredoxin reductase (TR). BSO inhibits γ-glutamylcysteine ligase (GCL), which catalyzes the reaction between cysteine and L-glutamate to form γ-glutamyl-cysteine. Glutathione synthetase (GS) converts γ-GCS into GSH. AUR inhibits TR activity. FaDu, Cal-27 and SCC-25 cells were treated with 0.5 µM AUR and/or 1 mM BSO for 24 h and analyzed for total GSH (B) and TR activity (C). Error bars represent the standard error of the mean (SEM) of N = 3 experiments *, p<0.05 versus control.](profile/Elise-Fletcher/publication/232766730/figure/fig1/AS:202819530432517@1425367352682/A-NADPH-is-a-source-of-reducing-equivalents-for-the-glutathione-system-consisting-of.png)
A: NADPH is a source of reducing equivalents for the glutathione system consisting of reduced glutathione (GSH), glutathione disulfide (GSSG), glutathione peroxidase (GPx), and glutathione reductase (GR) and the thioredoxin system consisting of reduced thioredoxin [Trx(SH)2], thioredoxin disulfide [Trx(S2)], peroxiredoxin (Prx), and thioredoxin reductase (TR). BSO inhibits γ-glutamylcysteine ligase (GCL), which catalyzes the reaction between cysteine and L-glutamate to form γ-glutamyl-cysteine. Glutathione synthetase (GS) converts γ-GCS into GSH. AUR inhibits TR activity. FaDu, Cal-27 and SCC-25 cells were treated with 0.5 µM AUR and/or 1 mM BSO for 24 h and analyzed for total GSH (B) and TR activity (C). Error bars represent the standard error of the mean (SEM) of N = 3 experiments *, p<0.05 versus control.
Source publication
Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative...
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Citations
... Furthermore, it was previously demonstrated that inhibition of antioxidant pathways in HNSCC cells, such as GSH and thioredoxin, influences the response to anticancer therapy involving oxidative stress, but to a different extent, given the intrinsic differences of HNSCC cell types. For instance, CAL27 cells, tongue squamous cell carcinoma, possess a significantly higher level of catalase activity compared to FaDu cells, a hypopharyngeal carcinoma cell line that influences the cell killing rescue by treatment with buthionine sulfoximine and auranofin, inhibitors of GSH and thioredoxin, respectively [55]. Although only a small increase in ROS generation was observed in SQD9 cells, laryngeal squamous cell carcinoma, we recorded a statistically significant PDT-induced cell death, comparable to that recorded in CAL27 cells ( Figure 6). ...
Temoporfin (mTHPC) is approved in Europe for the photodynamic treatment of head and neck squamous cell carcinoma (HNSCC). Although it has a promising profile, its lipophilic character hampers the full exploitation of its potential due to high tendency of aggregation and a reduced ROS generation that compromise photodynamic therapy (PDT) efficacy. Moreover, for its clinical administration, mTHPC requires the presence of ethanol and propylene glycol as solvents, often causing adverse effects in the site of injection. In this paper we explored the efficiency of a new mTHPC formulation that uses human serum albumin (HSA) to disperse the photosensitizer in solution (mTHPC@HSA), investigating its anticancer potential in two HNSCC cell lines. Through a comprehensive characterization, we demonstrated that mTHPC@HSA is stable in physiological environment, does not aggregate, and is extremely efficient in PDT performance, due to its high singlet oxygen generation and the high dispersion as monomolecular form in HSA. This is supported by the computational identification of the specific binding pocket of mTHPC in HSA. Moreover, mTHPC@HSA-PDT induces cytotoxicity in both HNSCC cell lines, increasing intracellular ROS generation and the number of γ-H2AX foci, a cellular event involved in the global response to cellular stress. Taken together these results highlight the promising phototoxic profile of the complex, prompting further studies to assess its clinical potential.
... In a preliminary in vivo experiment with 344SQ tumor-bearing 129-mice, we observed a significant delay in tumor growth after treatment with 10 mg/kg AF via daily i.p. injections for 14 days, compared to vehicle-treated mice. Similarly, several other in vivo studies demonstrated an inhibitory effect of AF monotherapy on tumor growth after i.p. injections in different cancer mouse models [9][10][11]13,[15][16][17][18][19][20][21]24,26,30,35,40] (Table 1). In CLL and BCP-ALL xenograft mouse models, i.p. ...
... Alternatively, well-designed combination strategies might limit the need for high AF doses. As listed in Table 1, AF in combination with vitamin C, anti-PD-L1, MK2206, adriamycin, cisplatin, buthionine sulfoximine, cold atmospheric plasma etc. showed statistically significant reduction in tumor growth and/or prolonged survival, without obvious side effects, in several in vivo cancer models [25,26,33,35,37,40,41,45]. ...
The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10–15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF’s optimal administration route and solvent in two syngeneic cancer mouse models.
... We demonstrated the interplay between both antioxidant systems by treating ALL cells with auranofin, a thioredoxin reductase, and BSO, a GSH synthesis inhibitors. The remarkable combination effect observed has been previously reported in head and neck cancer (92,93), breast cancer (94), and lymphoma and multiple myeloma (95) cells. In some cases, the thioredoxin system may be even more important than GSH to protect cells from oxidative stress. ...
Introduction
Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use.
Methods
To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX.
Results
MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested.
Conclusion
Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.
... The basic approach is an antioxidative therapy focused on decreasing ROS levels to prevent oncogene activation. An alternative, "pro-oxidative" approach has been devised based on increasing the ROS above the susceptible level for selectively killing cancer cells (Somu et al., 2021). Numerous pro-oxidative agents have been identified that can increase the ROS level above the susceptible limit. ...
... Another central target in redox systems is thioredoxin, which NADPH reduces to promote electron transfer for signal transduction and redox regulation. A thioredoxin inhibitor, auranofin, show significant effects in treating head and neck cancer cells (Sobhakumari et al., 2012). ...
Abstract
Reactive species are highly-reactive enzymatically, or non-enzymatically produced compounds with important roles in physiological and pathophysiological cellular processes. Although reactive species represent an extensively researched topic in biomedical sciences, many aspects of their roles and functions remain unclear. This review aims to systematically summarize findings regarding the biochemical characteristics of various types of reactive species and specify the localization and mechanisms of their production in cells. In addition, we discuss the specific roles of free radicals in cellular physiology, focusing on the current lines of research that aim to identify the reactive oxygen species-initiated cascades of reactions resulting in adaptive or pathological cellular responses. Finally, we present recent findings regarding the therapeutic modulations of intracellular levels of reactive oxygen species, which may have substantial significance in developing novel agents for treating several diseases.
... ROS can cause lipid peroxidation in the lipid bilayer of the cell membrane and thus induce changes in membrane potential and permeability (Gupta et al. 2009). In healthy cells there is an antioxidant enzyme complex -superoxide dismutases (SODs), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin (Trxs), and peroxiredoxin (Prxs)-that ensures cellular homeostasis by capturing excess ROS (Ray et al. 2012;Sobhakumari et al. 2012). In tumor cells, reduced expression of antioxidant genes such as SOD2, GPX1, Trxs and Prxs (Jarvis et al. 2012;Calvo et al. 2013;Cao et al. 2009) or their translational changes (e.g., SOD2 ...
... Inhibition of the Trx system can lead to an increase in GSH system activity and prevent the anti-tumor effect. Thus, in defeating cancer through redox systems tempering, dual inhibition of both GSH and Trx systems proved to be more effective than inhibition of individual systems (Sobhakumari et al., 2012;Benhar et al., 2016;Rodman et al., 2016;Roh et al., 2017a). Recent research provided evidence that there is another mechanism of resistance to Trx system inhibitors, mediated by hydrogen sulfide (H 2 S) (Mao et al., 2019;Mao et al., 2020). ...
The intracellular redox homeostasis is a dynamic balancing system between the levels of free radical species and antioxidant enzymes and small molecules at the core of cellular defense mechanisms. The thioredoxin (Trx) system is an important detoxification system regulating the redox milieu. This system is one of the key regulators of cells’ proliferative potential as well, through the reduction of key proteins. Increased oxidative stress characterizes highly proliferative, metabolically hyperactive cancer cells, which are forced to mobilize antioxidant enzymes to balance the increase in free radical concentration and prevent irreversible damage and cell death. Components of the Trx system are involved in high-rate proliferation and activation of pro-survival mechanisms in cancer cells, particularly those facing increased oxidative stress. This review addresses the importance of the targetable redox-regulating Trx system in tumor progression, as well as in detoxification and protection of cancer cells from oxidative stress and drug-induced cytotoxicity. It also discusses the cancer cells’ counteracting mechanisms to the Trx system inhibition and presents several inhibitors of the Trx system as prospective candidates for cytostatics’ adjuvants. This manuscript further emphasizes the importance of developing novel multitarget therapies encompassing the Trx system inhibition to overcome cancer treatment limitations.
... Indeed, this effect was reversed by pretreatment of the cells with NAC, a known reductant that protects cells from ROS, therefore evidencing oxidative stress. [132] More studies have demonstrated that treating ovarian cancer cells with auranofin results in cytochrome c-mediated cell death, and the generation of H 2 O 2 which again is inhibited by the activity of NAC. [133] ...
Excessive cellular oxidative stress is widely perceived as a key factor in pathophysiological conditions and cancer development. Healthy cells use several mechanisms to maintain intracellular levels of reactive oxygen species (ROS) and overall redox homeostasis to avoid damage to DNA, proteins, and lipids. Cancer cells, in contrast, exhibit elevated ROS levels and upregulated protective antioxidant pathways. Counterintuitively, such elevated oxidative stress and enhanced antioxidant defence mechanisms in cancer cells provide a therapeutic opportunity for the development of drugs with different anticancer mechanisms of action (MoA). In this review, oxidative stress and the role of ROS in cells are described. The tumour‐suppressive and tumour‐promotive functions of ROS are discussed, and these two different therapeutic strategies (increasing or decreasing ROS to fight cancer) are compared. Clinically approved drugs with demonstrated oxidative stress anticancer MoAs are highlighted followed by description of examples of metal‐based anticancer drug candidates causing oxidative stress in cancer cells via novel MoAs.
... Recently, Onodera et al.found that inhibitors of the redox system displayed preferential cytotoxicity to cancer cells in nutrient-deprived conditions. 127 AF treatment in PANC-1 cells showed preferential cytotoxicity under nutrient-deprived compared with nutrient-sufficient condition with an EC50 of 0.64 and 2.18, respectively.In literature there are only two studies concerning AF in treatment of head and neck squamous cell carcinoma (HNSCC).128,129 Both were based on the prooxidative activity of AF. ...
Auranofin is an oral gold(I) compound, initially developed for the treatment of rheumatoid arthritis. Currently, Auranofin is under investigation for oncological application within a drug repurposing plan due to the relevant antineoplastic activity observed both in vitro and in vivo tumor models. In this review, we analysed studies in which Auranofin was used as a single drug or in combination with other molecules to enhance their anticancer activity or to overcome chemoresistance. The analysis of different targets/pathways affected by this drug in different cancer types has allowed us to highlight several Med Res Rev. 2021;1-36. wileyonlinelibrary.com/journal/med | 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
... This was confirmed by treating the head and neck cancer cells with auranofin plus or minus the ROS scavenger N-acetyl cysteine (NAC), which ameliorates ROS-induced DNA damage [59,60]. Pre-treatment with NAC counteracted the cancer cell killing effects of auranofin, indicating that regulation of ROS is a primary mechanism used by head and neck cancers to multiply and spread [61]. In another study, auranofin inhibited the proliferation of mesothelioma cells in a dose-dependent manner; the anti-cancer effect was associated with caspase-independent apoptosis and necrosis, and increased ROS levels. ...
Advanced stages of cancer are highly associated with short overall survival in patients due to the lack of long-term treatment options following the standard form of care. New options for cancer therapy are needed to improve the survival of cancer patients without disease recurrence. Auranofin is a clinically approved agent against rheumatoid arthritis that is currently enrolled in clinical trials for potential repurposing against cancer. Auranofin mainly targets the anti-oxidative system catalyzed by thioredoxin reductase (TrxR), which protects the cell from oxidative stress and death in the cytoplasm and the mitochondria. TrxR is over-expressed in many cancers as an adaptive mechanism for cancer cell proliferation, rendering it an attractive target for cancer therapy, and auranofin as a potential therapeutic agent for cancer. Inhibiting TrxR dysregulates the intracellular redox state causing increased intracellular reactive oxygen species levels, and stimulates cellular demise. An alternate mechanism of action of auranofin is to mimic proteasomal inhibition by blocking the ubiquitin–proteasome system (UPS), which is critically important in cancer cells to prevent cell death when compared to non-cancer cells, because of its role on cell cycle regulation, protein degradation, gene expression, and DNA repair. This article provides new perspectives on the potential mechanisms used by auranofin alone, in combination with diverse other compounds, or in combination with platinating agents and/or immune checkpoint inhibitors to combat cancer cells, while assessing the feasibility for its repurposing in the clinical setting.
... Of note, the DKO of BAX and BAK (and, more recently, the triple knockout [TKO] of BAX, BAK, and BOK) have been extensively studied to reveal the essential role of these proteins in the activation of MOMP and hence the intrinsic pathway of apoptosis [26,[32][33][34]. Nonetheless, it turned that BAX/BAK DKO cells do succumb in response to specific cell death stimuli, as exemplified by granzyme B [35], chelethrine [36], N-(3-oxododecanoyl)homoserine lactone [37], or combined inhibition of glutathione and thioredoxin metabolism [38]. Although it can be argued that these cell death pathways do not rely on a physiological route towards MOMP, it appears that the cytoprotective action of BAX/ BAK inactivation is not absolute. ...
Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca ²⁺ stores and store-operated Ca ²⁺ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca ²⁺ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca ²⁺ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca ²⁺ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.
