Inhibition Of System Xc Research Articles

Dual-Site Chemosensor for Visualizing •OH-GSH Redox and Tracking Ferroptosis-Inducing Pathways In Vivo.

Oxidative stress, characterized by an imbalance between oxidative and antioxidant processes, results in excessive accumulation of intracellular reactive oxygen species. Among these responses, the regulation of intracellular hydroxyl radicals (•OH) and glutathione (GSH) is vital for physiological processes. Real-time in situ monitoring these two opposing bioactive species and their redox interactions is essential for understanding physiological balance and imbalance. In this study, we developed a dual-site fluorescence chemosensor OG-3, which can independently image both exogenous and endogenous •OH and GSH in separate channels both within cells and in vivo, eliminating issues of spatiotemporal inhomogeneous distribution and cross-interference. With its imaging capabilities of monitoring •OH-GSH redox, OG-3 elucidated two different pathways for ferroptosis induction: (i) inhibition of system xc- to block cystine uptake (extrinsic pathway) and (ii) GPX4 inactivation, leading to the loss of antioxidant defense (intrinsic pathway). Moreover, we assessed the antiferroptotic function and effects of ferroptosis inhibitors by monitoring •OH and GSH fluctuations during ferroptosis. This method provides a reliable platform for identifying potential ferroptosis inhibitors, contributing to our understanding of relevant metabolic and physiological mechanisms. It shows potential for elucidating the regulation of ferroptosis mechanisms and investigating further strategies for therapeutic applications.

NIR-promoted ferrous ion regeneration enhances ferroptosis for glioblastoma treatment

Ferroptosis, a unique iron-dependent mode of cell death characterized by lipid peroxide accumulation, holds significant potential for the treatment of glioblastoma (GBM). However, the effectiveness of ferroptosis is hindered by the limited intracellular ferrous ions (Fe2+) and hydrogen peroxide (H2O2). In this study, a novel near-infrared (NIR)-light-responsive nanoplatform (ApoE-UMSNs-GOx/SRF) based on upconversion nanoparticles (UCNPs) was developed. A layer of mesoporous silica and a lipid bilayer were coated on UCNPs sequentially and loaded with glucose oxidase (GOx) and sorafenib, respectively. Further attachment of the ApoE peptide endowed the nanoplatform with BBB penetration and GBM targeting capabilities. Our results revealed that ApoE-UMSNs-GOx/SRF could efficiently accumulated in the orthotopic GBM and induce amplified ferroptosis when combining with NIR irradiation. The UCNPs mediated the photoreduction of Fe3+ to Fe2+ by converting NIR to UV light, and excess H2O2 was produced by the reaction of glucose with the loaded GOx. These processes greatly promoted the production of ROS, which together with inhibition of system Xc− by the loaded sorafenib, leading to enhanced accumulation of lipid peroxides and significantly improved the antiglioma effect both in vitro and in vivo. Our strategy has the potential to enhance the effectiveness of ferroptosis as a therapeutic approach for GBM.

Application of ferroptosis strategy to overcome tumor therapy resistance in breast and different cancer cells.

This literature review emphasizes the innovative role of ferroptosis in cancer treatment. Ferroptosis is a kind of deliberate cell death that is characterized by the generation of lipid peroxides and needs the presence of iron. Ferroptosis is a controlled cell death process that adheres to certain rules and regulations. The inhibition of System Xc- and the involvement of GPX4 are two of the primary areas of exploration that are engaged in the process of ferroptosis. This review explores the treatments that are used to treat ferroptosis in a range of malignancies, with a particular focus on breast carcinoma. Attention is paid to certain pathways, such as the FSP1-independent regulation of glutathione, involvement of cholesterol, and the prominin 2-MVB/exosome-ferritin pathway. Ferroptosis plays a key role in resistance to tumor therapy.

Induction of ferroptosis by photodynamic therapy and enhancement of antitumor effect with ferroptosis inducers.

Photodynamic therapy (PDT) is an effective tumor treatment that involves the administration of a photosensitizer to generate cytotoxic 1O2 [reactive oxygen species (ROS)] from molecular oxygen that is produced from energy absorption following tumor irradiation at specific wavelengths. Ferroptosis is induced by the disruption of the glutathione peroxidase 4 (GPX4) antioxidant system, leading to lipid peroxidation. We hypothesized that talaporfin sodium-photodynamic therapy (TS-PDT)-generated ROS would lead to ferroptosis via accumulation of lipid peroxidation. Cell viability assay in TS-PDT-treated cells in combination with a ferroptosis inhibitor (ferrostatin-1: Fer-1) or ferroptosis inducers (imidazole ketone erastin: IKE, Ras-selective lethal 3: RSL3) was performed. Accumulation of lipid peroxidation, GPX4 antioxidant system and cystine/glutamate antiporter (system xc-) activity in TS-PDT-treated cells was investigated. In xenograft mice, the antitumor effect of TS-PDT in combination with ferroptosis inducers (IKE or sorafenib) was examined. TS-PDT-induced cell death was partly suppressed by Fer-1 and accompanied by lipid peroxidation. TS-PDT combined with IKE or RSL3 enhanced the induction of cell death. TS-PDT inhibited cystine uptake activity via system xc-. In vivo, the combination of TS-PDT and ferroptosis inducers (IKE or sorafenib) reduced tumor volume. This study found that the mechanism underlying TS-PDT-induced ferroptosis constitutes direct lipid peroxidation by the generated ROS, and the inhibition of system xc-, and that the combination of a ferroptosis inducer with TS-PDT enhances the antitumor effect of TS-PDT. Our findings suggest that ferroptosis-inducing therapies combined with PDT may benefit cancer patients.

Interferon-γ induces salivary gland epithelial cell ferroptosis in Sjogren's syndrome via JAK/STAT1-mediated inhibition of system Xc-

The elevated level of interferon-γ (IFN-γ) in Sjogren's syndrome (SS) triggers salivary gland epithelial cells (SGEC) death. However, the underlying mechanisms of IFN-γ-induced SGEC death modes are still not fully elucidated. We found that IFN-γ triggers SGEC ferroptosis via Janus kinase/signal transducer and activator of transcription 1 (JAK/STAT1)-mediated inhibition of cystine-glutamate exchanger (System Xc−). Transcriptome analysis revealed that ferroptosis-related markers are differentially expressed in SS human and mouse salivary glands with distinct upregulation of IFN-γ and downregulation of glutathione peroxidase 4 (GPX4) and aquaporin 5 (AQP5). Inducing ferroptosis or IFN-γ treatment in the Institute of cancer research (ICR) mice aggravated and inhibition of ferroptosis or IFN-γ signaling in SS model non-obese diabetic (NOD) mice alleviated ferroptosis in the salivary gland and SS symptoms. IFN-γ activated STAT1 phosphorylation and downregulated system Xc− components solute carrier family 3 member 2 (SLC3A2), glutathione, and GPX4 thereby triggering ferroptosis in SGEC. JAK or STAT1 inhibition in SGEC rescued IFN-γ-downregulated SLC3A2 and GPX4 as well as IFN-γ-induced cell death. Our results indicate the role of ferroptosis in SS-related death of SGEC and SS pathogenicity.

Research Models to Study Ferroptosis's Impact in Neurodegenerative Diseases.

Ferroptosis is a type of regulated cell death promoted by the appearance of oxidative perturbations in the intracellular microenvironment constitutively controlled by glutathione peroxidase 4 (GPX4). It is characterized by increased production of reactive oxygen species, intracellular iron accumulation, lipid peroxidation, inhibition of system Xc-, glutathione depletion, and decreased GPX4 activity. Several pieces of evidence support the involvement of ferroptosis in distinct neurodegenerative diseases. In vitro and in vivo models allow a reliable transition to clinical studies. Several in vitro models, including differentiated SH-SY5Y and PC12 cells, among others, have been used to investigate the pathophysiological mechanisms of distinct neurodegenerative diseases, including ferroptosis. In addition, they can be useful in the development of potential ferroptosis inhibitors that can be used as disease-modifying drugs for the treatment of such diseases. On the other hand, in vivo models based on the manipulation of rodents and invertebrate animals, such as Drosophila melanogaster, Caenorhabditis elegans, and zebrafish, have been increasingly used for research in neurodegeneration. This work provides an up-to-date review of the main in vitro and in vivo models that can be used to evaluate ferroptosis in the most prevalent neurodegenerative diseases, and to explore potential new drug targets and novel drug candidates for effective disease-modifying therapies.

Patulin disrupts SLC7A11-cystine-cysteine-GSH antioxidant system and promotes renal cell ferroptosis both in vitro and in vivo.

Patulin (PAT) is a common food-borne mycotoxin with diverse toxic effects including nephrotoxicity. The induction of oxidative stress is suggested to be a key mechanism contributed to toxicities of PAT. Reduced glutathione (GSH), a sulfhydryl-containing tripeptide, is a key reason for PAT-mediated oxidative stress. Cystine/glutamate antiporter (system xc-)-mediated cystine uptake plays a critical role in maintaining redox balance via promoting GSH biosynthesis. In this study, we addressed if GSH reduction by PAT was associated with inhibition of system xc--mediated GSH biosynthesis. Results showed that PAT significantly decreased activity of SLC7A11, a core subunit of system xc-, through activating AMPK-mediated formation of beclin1-SLC7A11 complex. Furthermore, PAT promoted ferroptosis induced by a known ferroptosis inducer RSL3 in normal renal cells, and exacerbated folic acid-induced nephrotoxicity in a mouse model of acute kidney injury. The findings of the present study provide new insights into PAT-induced kidney toxicity, and implicate that patients with ferroptosis-associated diseases maybe more susceptible to PAT.

System Xc− inhibition blocks bone marrow-multiple myeloma exosomal crosstalk, thereby countering bortezomib resistance

Multiple myeloma (MM) cells derive proliferative signals from the bone marrow (BM) microenvironment via exosomal crosstalk. Therapeutic strategies targeting this crosstalk are still lacking. Bortezomib resistance in MM cells is linked to elevated expression of xCT (the subunit of system Xc−). Extracellular glutamate released by system Xc− can bind to glutamate metabotropic receptor (GRM) 3, thereby upregulating Rab27-dependent vesicular trafficking. Since Rab27 is also involved in exosome biogenesis, we aimed to investigate the role of system Xc− in exosomal communication between BM stromal cells (BMSCs) and MM cells. We observed that expression of xCT and GRMs was increased after bortezomib treatment in both BMSCs and MM cells. Secretion of glutamate and exosomes was simultaneously enhanced which could be countered by inhibition of system Xc− or GRMs. Moreover, glutamate supplementation increased exosome secretion by increasing expression of Alix, TSG101, Rab27a/b and VAMP7. Importantly, the system Xc− inhibitor sulfasalazine reduced BMSC-induced resistance to bortezomib in MM cells in vitro and enhanced its anti-MM effects in vivo. These findings suggest that system Xc− plays an important role within the BM and could be a potential target in MM.

Chronic Sulfasalazine Treatment in Mice Induces System xc - - Independent Adverse Effects.

Despite ample evidence for the therapeutic potential of inhibition of the cystine/glutamate antiporter system xc − in neurological disorders and in cancer, none of the proposed inhibitors is selective. In this context, a lot of research has been performed using the EMA- and FDA-approved drug sulfasalazine (SAS). Even though this molecule is already on the market for decades as an anti-inflammatory drug, serious side effects due to its use have been reported. Whereas for the treatment of the main indications, SAS needs to be cleaved in the intestine into the anti-inflammatory compound mesalazine, it needs to reach the systemic circulation in its intact form to allow inhibition of system xc −. The higher plasma levels of intact SAS (or its metabolites) might induce adverse effects, independent of its action on system xc −. Some of these effects have however been attributed to system xc − inhibition, calling into question the safety of targeting system xc −. In this study we chronically treated system xc − - deficient mice and their wildtype littermates with two different doses of SAS (160 mg/kg twice daily or 320 mg/kg once daily, i.p.) and studied some of the adverse effects that were previously reported. SAS had a negative impact on the survival rate, the body weight, the thermoregulation and/or stress reaction of mice of both genotypes, and thus independent of its inhibitory action on system xc −. While SAS decreased the total distance travelled in the open-field test the first time the mice encountered the test, it did not influence this parameter on the long-term and it did not induce other behavioral changes such as anxiety- or depressive-like behavior. Finally, no major histological abnormalities were observed in the spinal cord. To conclude, we were unable to identify any undesirable system xc −-dependent effect of chronic administration of SAS.

Targeting SLC3A2 subunit of system XC− is essential for m6A reader YTHDC2 to be an endogenous ferroptosis inducer in lung adenocarcinoma

The m6A reader YT521-B homology containing 2 (YTHDC2) has been identified to inhibit lung adenocarcinoma (LUAD) tumorigenesis by suppressing solute carrier 7A11 (SLC7A11)-dependent antioxidant function. SLC7A11 is a major functional subunit of system XC−. Inhibition of system XC− can induce ferroptosis. However, whether suppressing SLC7A11 is sufficient for YTHDC2 to be an endogenous ferroptosis inducer in LUAD is unknown. Here, we found that induction of YTHDC2 to a high level can induce ferroptosis in LUAD cells but not in lung and bronchus epithelial cells. In addition to SLC7A11, solute carrier 3A2 (SLC3A2), another subunit of system XC− was equally important for YTHDC2-induced ferroptosis. YTHDC2 m6A-dependently destabilized Homeo box A13 (HOXA13) mRNA because a potential m6A recognition site was identified within its 3′ untranslated region (3′UTR). Interestingly, HOXA13 acted as a transcription factor to stimulate SLC3A2 expression. Thereby, YTHDC2 suppressed SLC3A2 via inhibiting HOXA13 in an m6A-indirect manner. Mouse experiments further confirmed the associations among YTHDC2, SLC3A2 and HOXA13, and demonstrated that SLC3A2 and SLC7A11 were both important for YTHDC2-impaired tumor growth and –induced lipid peroxidation in vivo. Moreover, higher expression of SLC7A11, SLC3A2 and HOXA13 indicate poorer clinical outcome in YTHDC2-suppressed LUAD patients. In conclusion, YTHDC2 is believed to be a powerful endogenous ferroptosis inducer and targeting SLC3A2 subunit of system XC− is essential for this process. Increasing YTHDC2 is an alternative ferroptosis-based therapy to treat LUAD.

Endogenous glutamate determines ferroptosis sensitivity via ADCY10-dependent YAP suppression in lung adenocarcinoma.

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system XC- because of the impaired uptake of cystine. However, the outcome following the accumulation of endogenous glutamate in lung adenocarcinoma (LUAD) has not yet been determined. Yes-associated protein (YAP) is sustained by the hexosamine biosynthesis pathway (HBP)-dependent O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation), and glutamine-fructose-6-phosphate transaminase (GFPT1), the rate-limiting enzyme of the HBP, can be phosphorylated and inhibited by adenylyl cyclase (ADCY)-mediated activation of protein kinase A (PKA). However, whether accumulated endogenous glutamate determines ferroptosis sensitivity by influencing the ADCY/PKA/HBP/YAP axis in LUAD cells is not understood.Methods: Cell viability, cell death and the generation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the responses to the induction of ferroptosis following the inhibition of system XC-. Tandem mass tags (TMTs) were employed to explore potential factors critical for the ferroptosis sensitivity of LUAD cells. Immunoblotting (IB) and quantitative RT-PCR (qPCR) were used to analyze protein and mRNA expression. Co-immunoprecipitation (co-IP) assays were performed to identify protein-protein interactions and posttranslational modifications. Metabolite levels were measured using the appropriate kits. Transcriptional regulation was evaluated using a luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). Drug administration and limiting dilution cell transplantation were performed with cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. The associations among clinical outcome, drug efficacy and ADCY10 expression were determined based on data from patients who underwent curative surgery and evaluated with patient-derived primary LUAD cells and tissues.Results: The accumulation of endogenous glutamate following system XC- inhibition has been shown to determine ferroptosis sensitivity by suppressing YAP in LUAD cells. YAP O-GlcNAcylation and expression cannot be sustained in LUAD cells upon impairment of GFPT1. Thus, Hippo pathway-like phosphorylation and ubiquitination of YAP are enhanced. ADCY10 acts as a key downstream target and diversifies the effects of glutamate on the PKA-dependent suppression of GFPT1. We also discovered that the protumorigenic and proferroptotic effects of ADCY10 are mediated separately. Advanced-stage LUADs with high ADCY10 expression are sensitive to ferroptosis. Moreover, LUAD cells with acquired therapy resistance are also prone to higher ADCY10 expression and are more likely to respond to ferroptosis. Finally, a varying degree of secondary labile iron increase is caused by the failure to sustain YAP-stimulated transcriptional compensation for ferritin at later stages further explains why ferroptosis sensitivity varies among LUAD cells.Conclusions: Endogenous glutamate is critical for ferroptosis sensitivity following the inhibition of system XC- in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors.

ATF3 promotes erastin-induced ferroptosis by suppressing system Xc\u2013

The amino acid antiporter system Xc− is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc− often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc− activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc− is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc−, depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc− and promote ferroptosis repressed by this antiporter.

Abstract 278: System xc- inhibition shares features of necroptosis and ferroptosis in hepatocellular carcinoma cells

Abstract Ferroptosis is characterized by iron-dependent non-apoptotic cell death involving the accumulation of lipid peroxides. Inhibition of system xc- which uptake L-cystine with the exchange of L-glutamate decreases glutathione synthesis, leading to ferroptosis. However, molecular interplay between ferroptosis and necroptosis has not been clearly identified. In this study, we investigated molecular process for cell death by SLC7A11 inhibition in hepatocellular carcinoma cell lines. The sensitivity for SLC7A11 inhibition by sulfasalazine was in the order of HepG2, Huh7 and Huh6, and then PLC/PRF/5. The decrease of cell viability by sulfasalazine and erastin was prevented by co-treatment of β-mercaptoethanol in all kinds of cell lines, indicating that the limited supply of L-cystine may contribute to cell death. Both ferrostatin and necrostatin, an inhibitor of ferroptosis and necroptosis, respectively, rescued sulfasalazine-induced growth inhibition by 60% in Huh7 and Huh6 and by 20% in HepG2. However, they failed to restore cell survival in PLC/PRF/5 cells. Sulfasalazine at the dose inducing equivalent level of cell death significantly increased lipid peroxidation in Huh7 and Huh6, but not in HepG2 and PLC/PRF/5 cells, as determined by C11-BODIPY dye. RIPK activation in necroptosis pathway by sulfasalazine was monitored in these cell lines. These results suggest that SLC7A11 inhibition triggers mixed-type of cell death via ferroptosis and necroptosis in a context-dependent manner in hepatocellular carcinoma cells (MD Abdullah and DH Kim are equally contributed to this work). Citation Format: Md Abdullah, Do Hyung Kim, Seung Jin Lee. System xc- inhibition shares features of necroptosis and ferroptosis in hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 278.

Augmenter of liver regeneration protects the kidney from ischaemia-reperfusion injury in ferroptosis.

Acute kidney injury (AKI) is a common and severe clinical condition with high morbidity and mortality. Ischaemia‐reperfusion (I/R) injury remains the major cause of AKI in the clinic. Ferroptosis is a recently discovered form of programmed cell death (PCD) that is characterized by iron‐dependent accumulation of reactive oxygen species (ROS). Compelling evidence has shown that renal tubular cell death involves ferroptosis, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR) is a widely distributed multifunctional protein that is expressed in many tissues. Our previous study demonstrated that ALR possesses an anti‐oxidant function. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. Here, to elucidate the role of ALR in ferroptosis, ALR expression was inhibited using short hairpin RNA lentivirals (shRNA) in vitro model of I/R‐induced AKI. The results suggest that the level of ferroptosis is increased, particularly in the shRNA/ALR group, accompanied by increased ROS and mitochondrial damage. Furthermore, inhibition of system xc‐ with erastin aggravates ferroptosis, particularly silencing of the expression of ALR. Unexpectedly, we demonstrate a novel signalling pathway of ferroptosis. In summary, we show for the first time that silencing ALR aggravates ferroptosis in an in vitro model of I/R. Notably, we show that I/R induced kidney ferroptosis is mediated by ALR, which is linked to the glutathione‐glutathione peroxidase (GSH‐GPx) system.

Astrocytic cystine/glutamate antiporter is a key regulator of erythropoietin expression in the ischemic retina.

Ischemic retinopathies and optic neuropathies are important causes of vision loss. The neuroprotective effect of erythropoietin (EPO) in ischemic neuronal injury and the expression of EPO and its receptor in retinal tissue have been well documented. However, the exact regulatory mechanism of EPO expression in retinal ischemia still remains to be elucidated. In this study, we investigated the role of cystine/glutamate antiporter (system xc-) in the regulation of astrocytic EPO expression by using both in vitro and in vivo models. Under hypoxia, the expression of astrocytic system xc- is up-regulated both in vitro and in vivo. Inhibition of system xc- resulted in depletion of intracellular glutathione (GSH) and decrement of GSH disulfide ratios in human brain astrocytes (HBAs). In HBAs, hypoxia-induced stabilization of hypoxia-inducible factor (Hif)-2α is nearly completely abolished by inhibition of system xc-. Hypoxia-induced up-regulation of astrocytic EPO expression is suppressed by both pharmacological inhibition and siRNA-mediated knockdown of system xc-. In contrast, basal EPO expression under normoxia is not affected by system xc- modulation. In summary, under hypoxia, increased system xc- acts as the major source of intracellular GSH, which helps in stabilizing Hif-2α and subsequent up-regulation of EPO in astrocytes.-Lee, B. J., Jun, H. O., Kim, J. H., Kim, J. H. Astrocytic cystine/glutamate antiporter is a key regulator of erythropoietin expression in the ischemic retina.

Ferroptosis is a lysosomal cell death process

Ferroptosis is a form of regulated cell death resulting from iron accumulation and lipid peroxidation. While impaired ferroptosis is tightly linked to human diseases and conditions, the mechanism and regulation of ferroptosis remain largely unknown. Here, we demonstrate that STAT3 is a positive regulator of ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Activation of the MAPK/ERK pathway, but not inhibition of system Xc−, was required for STAT3 activation during erastin-induced ferroptosis. Importantly, pharmacological inhibition and genetic silencing of STAT3 through small molecules (e.g., cryptotanshinone and S3I-201) or siRNA blocked erastin-induced ferroptosis in PDAC cells. Mechanically, STAT3-mediated cathepsin B expression was required for ferroptosis. Consequently, inhibition of lysosome-dependent cell death by pharmacological blockade of cathepsin activity (using CA-074Me) or vacuolar type H+-ATPase (using bafilomycin A1) limited erastin-induced ferroptosis. These studies indicate that ferroptosis is a lysosomal cell death process.

System xc- and neuro-inflammaging

Event Abstract Back to Event System xc- and neuro-inflammaging Lise Verbruggen1*, Eduard Bentea1, Lauren Deneyer1, Giulia Albertini2, Ilse J. Smolders2, Hideyo Sato3 and Ann Massie1 1 Vrije Universiteit Brussel, Pharmaceutical Biotechnology and Molecular Biology, Belgium 2 Pharmaceutical Chemistry and Drug Analysis, Belgium 3 Department of Medical Technology, Niigata University, Japan, Japan System xc- is a mainly glial cystine/glutamate antiporter (specific subunit xCT) that imports cystine in exchange for glutamate. Cystine is intracellularly reduced to cysteine, a building block of the major brain antioxidant glutathione. Cystine uptake is obligatory coupled to glutamate release which can, given the extrasynaptic localization of system xc-, activate both extrasynaptic metabotropic glutamate receptors (leading to modulation of synaptic glutamatergic transmission) and extrasynaptic NMDA receptors (possibly leading to excitotoxicity). Recently, system xc- was shown to also modulate neuroinflammation, with the absence of system xc- favoring the anti-inflammatory (M2) over the pro-inflammatory (M1) microglial phenotype [1]. As system xc- can provide glutathione to the cell, the expression of xCT is increased in conditions of oxidative stress and neuroinflammation [2] . The involvement of system xc- in the process of brain aging has never been investigated. Although it was reported that the plasma of xCT-/- mice (mice lacking functional system xc-) is in a more oxidized state [3], possibly leading to accelerated aging, we could observe a significantly increased life-span in xCT-/- vs xCT+/+ mice and we previously reported absence of any signs of accelerated brain aging after genetic loss of xCT [4]. Since increased levels of reactive oxygen species and inflammation are hallmarks of normal brain aging, we hypothesized that in the aging brain enhancement of system xc- could modulate the threshold for glutamate toxicity and drive neuroinflammation. As such, inhibition of system xc- might delay age-related diseases by reducing the build-up of several types of damage and thus making cells more resistant to a range of typical age-related stressors. To gain more insight into the role that system xc- fulfills in the process of brain aging, we investigated both the effect of aging on system xc- and the effect of loss of system xc- on age-induced neuroinflammation. xCT protein expression was unaltered in hippocampus, cortex and striatum of adult (3-5 months) versus aged (20-25 months) xCT+/+ mice. Absence of system xc- did not affect the age-related changes in number or shape of astrocytes in dentate gyrus as evidenced by immunohistochemistry for GFAP on sections of adult and aged xCT+/+ as well as xCT-/- mice. Astrocyte number decreased in the dentate gyrus of aged mice compared to adult mice, independent of genotype. Convex closure of astrocytic processes, cell body area and diameter remained stable between different groups. As for microglia, we observed absence of age-induced microglial proliferation in the dentate gyrus of xCT-/- mice compared to their xCT+/+ littermates, as evidenced by Iba1 immunohistochemistry. Also, the age-induced increase in microglial cell body area that was seen in xCT+/+ mice, was absent in xCT-/- mice. Finally, the genotype-effects that were seen on microglial cells in adult and aged dentate gyrus, were absent in substantia nigra. Yet, age-induced microglial priming might still be affected in substantia nigra of xCT-/- mice, as we observed reduced susceptibility to lactacystin-induced neurodegeneration accompanied by lack of nigral microglial activation in aged xCT-/- mice compared to age-matched xCT+/+ littermates. Taken together, the current (preliminary) findings suggest that the brain of xCT-/- mice ages healthier with reduced levels of hippocampal neuroinflammation and protection against toxin-induced neurodegeneration.

PET Imaging with [(18)F]FSPG Evidences the Role of System xc(-) on Brain Inflammation Following Cerebral Ischemia in Rats.

In vivo Positron Emission Tomography (PET) imaging of the cystine-glutamate antiporter (system xc-) activity with [18F]FSPG is meant to be an attractive tool for the diagnosis and therapy evaluation of brain diseases. However, the role of system xc- in cerebral ischemia and its involvement in inflammatory reaction has been scarcely explored. In this work, we report the longitudinal investigation of the neuroinflammatory process following transient middle cerebral artery occlusion (MCAO) in rats using PET with [18F]FSPG and the translocator protein (TSPO) ligand [18F]DPA-714. In the ischemic territory, [18F]FSPG showed a progressive binding increase that peaked at days 3 to 7 and was followed by a progressive decrease from days 14 to 28 after reperfusion. In contrast, [18F]DPA-714 evidenced maximum binding uptake values over day 7 after reperfusion. Ex vivo immnunohistochemistry confirmed the up-regulation of system xc- in microglial cells and marginally in astrocytes. Inhibition of system xc- with sulfasalazine and S-4-CPG resulted in increased arginase (anti-inflammatory M2 marker) expression at day 7 after ischemia, together with a decrease in TSPO and microglial M1 proinflammatory markers (CCL2, TNF and iNOS) expression. Taken together, these results suggest that system xc- plays a key role in the inflammatory reaction underlying experimental stroke.

Sulfasalazine attenuates ACL transection and medial menisectomy-induced cartilage destruction by inhibition of cystine/glutamate antiporter.

We had previously demonstrated that excitatory amino acid glutamate plays a role in the progression and severity of knee osteoarthritis (OA), and early hyaluronic acid injection attenuates the OA progression by attenuation of knee joint glutamate level, which was also related to the cystine/glutamate antiporter system X (system XC-) expression. System XC- uptakes cystine into chondrocytes for glutathione (GSH) synthesis, but the role of system XC- in OA is rarely addressed. Sulfasalazine (SSZ) is a system XC- inhibitor; SSZ was applied intra-articularly to study the function of system XC- in the development of OA in rats subjected to anterior cruciate ligament transection and medial meniscectomy (ACLT + MMx). Moerover, the system XC- activator N-acetylcysteine (NAC) was also applied to verify the role of system XC-. The intra-articular injection of SSZ significantly attenuated knee swelling and cartilage destruction in the knees of ACLT + MMx rats and this effect was blocked by NAC. The results showed that inhibition of system XC- function can attenuate ACLT + MMx-induced cartilage destruction. In the present study, system XC- inhibitor SSZ was shown to reduce glutamate content in synovial fluid and GSH in chondrocytes. It was also showed SSZ could attenuate ACLT + MMx-induced cartilage destruction, and treatment of NAC reversed the protective effect of SSZ.

Abstract 4315: System Xc- in glioma alters redox regulation, energetics and morphology

Abstract Glioblastoma multiforme is the most common malignant primary brain tumor with a median life expectancy of 12 months from diagnosis. Due to the metabolic demands of rapidly proliferating cancer cells and higher free radical generation, we show that human glioma cells, compared to primary normal human astrocytes, express higher levels of system XC-, a sodium-independent electroneutral transporter composed of a catalytic subunit, xCT, and a heavy chain subunit, 4F2hc. System XC- is responsible for maintaining redox homeostasis by importing cystine, where it is then reduced to cysteine and used to synthesize the major antioxidant glutathione (GSH). We demonstrate that inhibition of system XC-, accomplished using sulfasalazine (SAS), increased intracellular reactive oxygen species (ROS) and decreased survival of U251 glioma cells in a dose-dependent manner. To further delineate the role of system XC- in glioma survival and minimize the off-target effects of SAS, we generated stable xCT-over-expressing and xCT-knockdown U251 glioma cell lines. The xCT-over-expressing glioma cells were more resistant to oxidative stress and chemotherapy treatment. Interestingly, the xCT-over-expressing U251 cells exhibit increased basal ATP generation compared to xCT-knockdown and control cells, despite no difference in viability or proliferation. The higher ATP levels correlated with increased basal levels of ROS in the xCT-over-expressing cells. Since mitochondria generate much of the cellular energy and produce most of the cellular ROS, we performed transmission electron microscopy (TEM) for morphological examination of the mitochondria. TEM revealed no alteration in mitochondrial morphology but there was a significant increase in the number of mitochondria in the xCT-over-expressing cells compared to xCT-knockdown and control cells. This indicates that system XC- may not only play a role in redox regulation but also in altered energetics. Further studies to assess the mitochondrial biogenesis and metabolism are currently under investigation. Additionally, the xCT-over-expressing U251 cells exhibited an altered morphology compared to the typical elongated morphology of parental and empty vector controls. This cuboidal morphology was associated with increased cortical F-actin staining compared to the elongated F-actin stress fibers in control cells. Since actin stress fibers promote cell migration, we assessed the migratory and invasive characteristics of the xCT-modified cell lines. The xCT-over-expressing U251 cells exhibited decreased migration and invasion while the xCT-knock-down U251 cells exhibited increased invasion. These findings indicate that system XC- over-expression in glioma not only promotes survival under oxidative stress and chemotherapy treatment, but may also modulate mitochondrial function and the migratory/ invasive properties to promote integration into host tissue. Citation Format: Monika D. Polewski, Rosyli F. Reveron, Karen S. Aboody. System Xc- in glioma alters redox regulation, energetics and morphology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4315. doi:10.1158/1538-7445.AM2014-4315