xCT Expression Research Articles

Paeonol improves angiotensin II-induced cardiac hypertrophy by suppressing ferroptosis

The aim of this study was to investigate the protective effect of paeonol (pae) on an angiotensin II (AngII)-induced cardiac hypertrophy mouse model. First, AngII mouse models were constructed and randomly grouped into the control (con), AngII, and AngII + Pae groups. Compared with that in the blank group, the surface area of myocardial cells in the AngII group increased significantly. In contrast to that in the AngII group, the cardiomyocyte surface area in the Pae group was significantly reduced. Ultrasound results showed that the myocardial function of mice in the AngII group was decreased compared with that in the Con group, while the myocardial function of mice in the Pae treatment was significantly improved. Moreover, the Fe2+ and lipid peroxide levels of primary cardiomyocytes were significantly increased after treatment with AngII and were significantly decreased after the addition of Pae. Compared with those in the Con group, cristae were reduced and the outer membrane was lost in the myocardial tissues of the AngII group, and myocardial MDA, ROS, and Fe2+ levels were increased. However, myocardial damage was significantly alleviated after Pae treatment, and myocardial MDA, ROS, and Fe2+ levels were reduced. Moreover, in myocardial tissue, AngII reduced the protein levels of xCT and GPX4, while the levels of both xCT and GPX4 were increased after Pae treatment. In conclusion, Pae protected the hearts of AngII mice by upregulating the protein expression of xCT and GPX4 and resisting AngII-induced ferroptosis in cardiomyocytes.

Defining the ferroptotic phenotype of beta cells in type 1 diabetes and its inhibition as a potential antidiabetic strategy.

Recently, the involvement of ferroptotic cell death in the reduction of β-cell mass in diabetes has been demonstrated. To elucidate the mechanisms of β-cell ferroptosis and potential antidiabetic effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in vivo, a mouse model of type 1 diabetes (T1D) was used. Animals were divided into three groups: control (vehicle-treated), diabetic (streptozotocin-treated, 40 mg/kg, from days 1-5), and diabetic treated with Fer-1 (1 mg/kg, from days 1-21). On day 22, glycemia and insulinemia were measured and pancreases were isolated for microscopic analyses. Diabetes disturbed general parameters of β-cell mass (islet size, β-cell abundance and distribution) and health (insulin and PDX-1 expression), increased lipid peroxidation in islet cells, and phagocytic removal of iron-containing material. It also downregulated the main players of the antiferroptotic pathway - Nrf2, GPX4, and xCT. In contrast, Fer-1 ameliorated the signs of deterioration of β-cell/islets, decreased lipid peroxidation, and reduced phagocytic activity, while upregulated expression of Nrf2 (and its nuclear translocation), GPX4, and xCT in β-cell/islets. Overall, our study confirms ferroptosis as an important mode of β-cell death in T1D and suggests antiferroptotic agents as a promising strategy for the prevention and treatment of diabetes.

Galangin Attenuates Myocardial Ischemic Reperfusion-Induced Ferroptosis by Targeting Nrf2/Gpx4 Signaling Pathway.

Myocardial ischemic reperfusion injury (MIRI) is a crucial clinical problem globally. The molecular mechanisms of MIRI need to be fully explored to develop new therapeutic methods. Galangin (Gal), which is a natural flavonoid extracted from Alpinia Officinarum Hance and Propolis, possesses a wide range of pharmacological activities, but its effects on MIRI remain unclear. This study aimed to determine the pharmacological effects of Gal on MIRI. C57BL/6 mice underwent reperfusion for 3 h after 45 min of ischemia, and neonatal rat cardiomyocytes (NRCs) subjected to hypoxia and reoxygenation (HR) were cultured as in vivo and in vitro models. Echocardiography and TTC-Evans Blue staining were performed to evaluate the myocardial injury. Transmission electron microscope and JC-1 staining were used to validate the mitochondrial function. Additionally, Western blot detected ferroptosis markers, including Gpx4, FTH, and xCT. Gal treatment alleviated cardiac myofibril damage, reduced infarction size, improved cardiac function, and prevented mitochondrial injury in mice with MIRI. Gal significantly alleviated HR-induced cell death and mitigated mitochondrial membrane potential reduction in NRCs. Furthermore, Gal significantly inhibited ferroptosis by preventing iron overload and lipid peroxidation, as well as regulating Gpx4, FTH, and xCT expression levels. Moreover, Gal up-regulated nuclear transcriptive factor Nrf2 in HR-treated NRCs. Nrf2 inhibition by Brusatol abolished the protective effect of Gal against ferroptosis. This study revealed that Gal alleviates myocardial ischemic reperfusion-induced ferroptosis by targeting Nrf2/Gpx4 signaling pathway.

Butyrate reverses ferroptosis resistance in colorectal cancer by inducing c-Fos-dependent xCT suppression

Ferroptosis has emerged to be a promising approach in cancer therapies; however, colorectal cancer (CRC) is relatively insensitive to ferroptosis. Exactly how the gut microenvironment impacts the ferroptotic sensitivity of CRC remains unknown. Herein, by performing metabolomics, we discovered that butyrate concentrations were significantly decreased in CRC patients. Butyrate supplementation sensitized CRC mice to ferroptosis induction, showing great in vivo translatability. Particularly, butyrate treatment reduced ferroptotic resistance of cancer stem cells. Mechanistically, butyrate inhibited xCT expression and xCT-dependent glutathione synthesis. Moreover, we identified c-Fos as a novel xCT suppressor, and further elucidated that butyrate induced c-Fos expression via disrupting class I HDAC activity. In CRC patients, butyrate negatively correlated with tumor xCT expression and positively correlated with c-Fos expression. Finally, butyrate was found to boost the pro-ferroptotic function of oxaliplatin (OXA). Immunohistochemistry data showed that OXA non-responders exhibited higher xCT expression compared to OXA responders. Hence, butyrate supplementation is a promising approach to break the ferroptosis resistance in CRC.

Inefficacy of N-acetylcysteine in mitigating cue-induced amphetamine-seeking

Glutamatergic imbalances are characteristic of SUDs. Astrocytic and neuronal transporters help regulate glutamate homeostasis and disruptions in this homeostasis engender SUD. The cysteine-glutamate exchanger (xCT) is primarily localized on astrocytes and maintains glutamate concentrations. This process is disrupted by cocaine use, and the therapeutic N-acetylcysteine (NAC) lowers cue-induced relapse to cocaine by restoring xCT function. However, little research has shown how these effects extend to other psychostimulants, such as amphetamine (AMP). Here, we assessed xCT expression following relapse to AMP cues, and if NAC can attenuate relapse via changes to astrocyte and xCT expression. We administered NAC (100 mg/kg ip) daily during a 14-day abstinence period following AMP (0.1 mg/kg/infusion; 2 h sessions) self-administration. Relapse was tested following one (WD 1) or 14 days (WD 14) of withdrawal. The overall number of astrocytes was also quantified within the medial prefrontal cortex (mPFC) and nucleus accumbens (ACb). NAC failed to lower cue-induced AMP craving via cue-induced relapse and reinstatement testing. Cue-induced craving did not increase from WD 1 to WD 14. AMP-exposed rats had greater astrocyte counts in the mPFC and ACb when compared AMP-naïve rats. Repeated injection with NAC decreased xCT expression within the mPFC and ACb. Overall, these results suggest that NAC may be an ineffective treatment option for lowering cue-induced relapse to AMP. Further, the results suggest that stimulating xCT via NAC may not be an effective therapeutic approach for decreasing cue-seeking for AMP.

Inhibition of xCT by sulfasalazine alleviates the depression-like behavior of adult male mice subjected to maternal separation stress

Maternal separation (MS) can induce emotional disorders. Our previous study reported that MS resulted in depression-like behavior. In this study, we aimed to explore the role of xCT in depression-like behavior in adult mice subjected to MS stress. Pups were divided into the control group, the control + sulfasalazine (SSZ, 75 mg/kg/day, i.p.) group, the MS group, and the MS+SSZ group. After MS, all pups were raised until PD60. Then, the depression-like behavior was detected by the novelty suppressed feeding (NSF) test, the forced swimming test (FST), and the tail suspension test (TST). The synaptic plasticity was examined by electrophysiological recordings and molecular biotechnology. The data showed that, compared with the control group, the mice in the MS group presented depression-like behavior, impairment of long-term potentiation (LTP), a reduction in the number of astrocytes, and activation of the microglia. Moreover, the expression of xCT was increased in the prefrontal cortex of MS mice, the EAAT2 and the Group Ⅱ metabotropic glutamate receptors (mGluR2/3) were decreased, and the level of pro-inflammatory factors was increased in the prefrontal cortex. After the administration with SSZ, the depression-like behavior and the impairment of LTP were alleviated, the number of astrocytes was increased, and the microglial activation was inhibited. Moreover, the levels of EAAT2 and mGluR2/3 were ameliorated, the over-activation of the microglia was mitigated, and the levels of glutamate and pro-inflammatory factors were decreased. In conclusion, the inhibition of xCT by SSZ could alleviate depression-like behavior partly via modulating the homeostasis of the glutamate system and dampening neuroinflammation.

Sulfasalazine inhibits esophageal cancer cell proliferation by mediating ferroptosis.

This study aimed to explore the potential mechanism by which sulfasalazine (SAS) inhibits esophageal cancer cell proliferation. A cell counting kit-8 (CCK-8) assay was used to detect the effect of SAS (0, 1, 2, and 4 mM) on the proliferation of TE-1 cells. Subsequently, TE-1 cells were divided into control group, SAS group, SAS + ferrostatin-1 (ferroptosis inhibitor) group, and SAS + Z-VAD (OH)-FMK (apoptosis inhibitor) group, and cell proliferation was measured using a CCK-8 assay. Real-time quantitative polymerase chain reaction and western blotting were used to determine the expression of solute carrier family member 7 11 (SLC7A11, also called xCT), glutathione peroxidase 4 (GPX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) in TE-1 cells. Measurement of ferroptosis in TE-1 cells was achieved by flow cytometry. Compared with the control group (0 mM SAS), the proliferation of TE-1 cells was significantly inhibited by different concentrations of SAS for different time lengths, and 4 mM SAS treatment for 48 h could obtain the maximum inhibition rate (53.9%). In addition, SAS treatment caused a significant decrease in the mRNA and protein expression of xCT and GPX4, and a significant increase in ACSL4 expression in TE-1 cells treated with SAS. Flow cytometry results showed that the ferroptosis level was significantly increased after SAS treatment. However, the activation of ferroptosis by SAS was partially eliminated by treatment with ferrostatin-1 or Z-VAD (OH)-FMK. In conclusion, SAS inhibits the proliferation of esophageal carcinoma cells by activating the ferroptosis pathway.

Sculponeatin A promotes the ETS1-SYVN1 interaction to induce SLC7A11/xCT-dependent ferroptosis in breast cancer

BackgroundE26 transformation specificity-1 (ETS1) is a transcription factor that is overexpressed in breast cancer (BC) and promotes tumor progression. Sculponeatin A (stA), a new diterpenoid extracted from Isodon sculponeatus, has no reported antitumor mechanism. PurposeHere, we explored the antitumor activity of stA in BC and further clarified its mechanism. MethodsFerroptosis was detected by flow cytometric, glutathione, malondialdehyde, and iron determination assays. The effect of stA on the upstream signaling pathway of ferroptosis was detected by Western blot, gene expression, gene alterations and other approaches. The binding of stA and ETS1 was examined through a microscale thermophoresis assay and a drug affinity responsive target stability assay. An in vivo mouse model experiment was performed to evaluate the therapeutic and potential mechanism of stA. ResultsstA exhibits therapeutic potential in BC by inducing SLC7A11/xCT-dependent ferroptosis. stA decreases the expression of ETS1, which is responsible for xCT-dependent ferroptosis in BC. stA inhibits the transcriptional expression of xCT by directly binding to the ETS domain of the ETS1 protein. In addition, stA promotes proteasomal degradation of ETS1 by triggering ubiquitin ligase synoviolin 1 (SYVN1)-mediated ubiquitination. The K318 site of ETS1 mediates ubiquitination of ETS1 by SYVN1. In a mouse model, stA inhibits tumor growth without causing obvious toxicity. ConclusionTaken together, the results confirm that stA promotes the ETS1-SYVN1 interaction to induce ferroptosis in BC mediated by ETS1 degradation. stA is expected to be used in research of candidate drugs for BC and drug design based on ETS1 degradation.

Growth differentiation factor 15 induces cisplatin resistance through upregulation of xCT expression and glutathione synthesis in gastric cancer.

Gastric cancer is a common cancer worldwide, particularly in East Asia. Chemotherapy is used in adjuvant or palliative therapies for gastric cancer. However, subsequent chemoresistance often develops. Growth differentiation factor 15 (GDF15) links to several cancers, but its effect on chemoresistance in gastric cancer remains unclear. Here, we analyzed clinical samples from genetic databases and included patients with gastric cancer. We dissected the regulatory mechanism underlying GDF15-mediated resistance of cisplatin in human gastric cancer cells. We showed that GDF15 serum levels might be a valuable biomarker for predicting prognosis in gastric cancer. The expressions of GDF15 and its receptor glial cell-derived neurotrophic factor family receptor a-like (GFRAL) in gastric tumors are important for malignant progression. Moreover, GDF15 expression is increased in gastric cancer cells with cisplatin resistance, resulting from elevated intracellular glutathione (GSH) and antioxidant activities. Upregulated GDF15 could increase intracellular GSH content by activating the GFRAL-GCN2-eIF2α-ATF4 signaling, enhancing cystine-uptake transporter xCT expression, and contributing biosynthesis of GSH in human gastric cancer cells. In conclusion, our results indicate that GDF15 could induce chemoresistance by upregulating xCT expression and GSH biosynthesis in human gastric cancer cells. Targeting GDF15 could be a promising treatment method for gastric cancer progression.

XCT-mediated glutamate excretion in white adipocytes stimulates interferon-γ production by natural killer cells in obesity

Obesity-mediated hypoxic stress underlies inflammation, including interferon (IFN)-γ production by natural killer (NK) cells in white adipose tissue. However, the effects of obesity on NK cell IFN-γ production remain obscure. Here, we show that hypoxia promotes xCT-mediated glutamate excretion and C-X-C motif chemokine ligand 12 (CXCL12) expression in white adipocytes, resulting in CXCR4+ NK cell recruitment. Interestingly, this spatial proximity between adipocytes and NK cells induces IFN-γ production in NK cells by stimulating metabotropic glutamate receptor 5 (mGluR5). IFN-γ then triggers inflammatory activation of macrophages and augments xCT and CXCL12 expression in adipocytes, forming a bidirectional pathway. Genetic or pharmacological inhibition of xCT, mGluR5, or IFN-γ receptor in adipocytes or NK cells alleviates obesity-related metabolic disorders in mice. Consistently, patients with obesity showed elevated levels of glutamate/mGluR5 and CXCL12/CXCR4 axes, suggesting that a bidirectional pathway between adipocytes and NK cells could be a viable therapeutic target in obesity-related metabolic disorders.

Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis

BackgroundOsteosarcomas (OS) is a kind of malignant bone tumor which occurs primarily in children and adolescents, and the clinical therapeutics remain disappointing. As a new programmed cell death, ferroptosis is characterized by iron dependent and intracellular oxidative accumulation, which provides a potential alternative intervene for the OS treatment. Baicalin, a major bioactive flavone derived from traditional Chinese medicine Scutellaria baicalensis, has been proved to have anti-tumor properties in OS. Whether ferroptosis participated in the baicalin mediated anti-OS activity is an interesting project. PurposeTo explore the pro-ferroptosis effect and mechanisms of baicalin in OS. Methods/study designPro-ferroptosis effect of baicalin on cell death, cell proliferation, iron accumulation, lipid peroxidation production was determined in MG63 and 143B cells. The levels of glutathione (GSH), oxidized (GSSG) glutathione and malondialdehyde (MDA) were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione peroxidase 4 (GPX4) and xCT were detected by western blot in baicalin-mediated ferroptosis regulation. In vivo, a xenograft mice model was adopted to explore the anticancer effect of baicalin. ResultsIn the present study, it was found that baicalin significantly suppress tumor cell growth in vitro and in vivo. By promoting the Fe accumulation, ROS formation, MDA production and suppressing the ratio of GSH/GSSG, baicalin was found to trigger ferroptosis in OS and ferroptosis inhibitor ferrostatin-1 (Fer-1) successfully reversed these suppressive effects, indicating that ferroptosis participated in the baicalin mediated anti-OS activity. Mechanistically, baicalin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced its stability via inducing ubiquitin degradation, which suppressed the Nrf2 downstream targets GPX4 and xCT expression, and led to stimulating ferroptosis. ConclusionsOur findings for the first time indicated that baicalin exerted anti-OS activity through a novel Nrf2/xCT/GPX4-dependent ferroptosis regulatory axis, which hopefully provides a promising candidate for OS treatment.

Enhancing vulnerability of Afatinib using Erastin via xCT-mediated ROS/P38MAPK signaling feedback loop in gastric cancer cells

Ferroptosis, being classified as a form of regulated cell death, was driven by the oxidative injury induced by lipid peroxidation (LPO). Recently, ferroptosis has been confirmed to exert a critical effect in the pathogenesis and treatment of various tumors, including gastric cancer (GC). Erastin, as a frequently used ferroptosis inducer, caused ferroptosis by downregulating the xCT expression resulting in increasing reactive oxygen species (ROS) and aggravating the LPO. However, the mechanisms of Erastin in ferroptosis regulation, especially in GC, remain largely elusive. This work firstly demonstrated that Erastin inhibited cell growth and promoted apoptosis and ferroptosis in AGS and BGC823 cells. Then, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of Erastin-related targets screened by using PharmMapper Web, the P38MAPK signaling was explored and validated in AGS and BGC-823 cells. Besides, the Fer-1 and P38 inhibitor were performed to investigate the mechanisms of ferroptosis induced by Erastin in depth. This work revealed a feedback mode among xCT, ROS and the P38MAPK pathway, which affected each other. It meant that Erastin regulated ferroptosis through the xCT-mediated ROS/P38MAPK signaling feedback loop. In addition, it was noticed that in co-operation with Erastin, the cytotoxic effects of Afatinib on cells were aggravated by further strengthening ferroptosis with activation of the P38MAPK pathway. In summary, those works provided evidence that Erastin plays an important role in increasing the cytotoxic effect on GC cells treated with Afitinib. Furthermore, the Erastin-induced ferroptosis via the xCT-mediated ROS/P38MAPK pathway feedback loop provides new strategies for GC comprehensive treatment.

Abietic acid inhibits acetaminophen-induced liver injury by alleviating inflammation and ferroptosis through regulating Nrf2/HO-1 axis.

Abietic acid has been known to exhibit anti-inflammatory activity. This study was designed to investigate the protective effects of abietic acid on acetaminophen (APAP)-induced liver injury. The data demonstrated that abietic acid significantly ameliorated APAP-induced liver pathological changes, TNF-α and IL-1β production. APAP could increase malondialdehyde (MDA) and Fe2+ levels, and decrease ATP and glutathione (GSH) levels, as well as glutathione peroxidase 4 (GPX4) and xCT expression. However, these changes induced by APAP were prevented by abietic acid, indicating abietic acid could inhibit APAP-induced ferroptosis. Furthermore, abietic acid inhibited APAP-induced NF-κB activation and increased the expression of Nrf2 and HO-1. Additionally, the inhibitory effects of abietic acid on APAP-induced liver injury were prevented in Nrf2-/- mice. In vitro, the inhibition of abietic acid on APAP-induced inflammation and ferroptosis were reversed when Nrf2 was knockdown. In summary, abietic acidexhibited a therapeutic effectagainst liver injury by attenuating inflammation and ferroptosis.

Dithiolethiones D3T and ACDT Protect Against Iron Overload-Induced Cytotoxicity and Serve as Ferroptosis Inhibitors in U-87 MG Cells.

Iron overload-induced oxidative stress is implicated in various neurodegenerative disorders. Given the numerous adverse effects associated with current iron chelators, natural antioxidants are being explored as alternative therapeutic options. Dithiolethiones found in cruciferous vegetables have emerged as promising candidates against a wide range of toxicants owing to their lipophilic and cytoprotective properties. Here, we test the dithiolethiones 3H-1,2-dithiole-3-thione (D3T) and 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) against ferric ammonium citrate (FAC)-induced toxicity in U-87 MG astrocytoma cells. Exposure to 15mM FAC for 24h resulted in 54% cell death. A 24-h pretreatment with 50μM D3T and ACDT prevented this cytotoxicity. Both dithiolethiones exhibited antioxidant effects by activating the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor and upregulating levels of intracellular glutathione (GSH). This resulted in the successful inhibition of FAC-induced reactive oxygen species, lipid peroxidation, and cell death. Additionally, D3T and ACDT upregulated expression of the Nrf2-mediated iron storage protein ferritin which consequently reduced the total labile iron pool. A 24-h pretreatment with D3T and ACDT also prevented cell death induced by the ferroptosis inducer erastin by upregulating the transmembrane cystine/glutamate antiporter (xCT) expression. The resulting increase in intracellular GSH and alleviation of lipid peroxidation was comparable to that caused by ferrostatin-1, a specific ferroptosis inhibitor. Collectively, our findings demonstrate that dithiolethiones may show promise as potential therapeutic options for the treatment of iron overload disorders.

Inhibition of lung adenocarcinoma by combinations of sulfasalazine (SAS) and disulfiram-copper (DSF-Cu) in cell line models and mice.

Sulfasalazine (SAS) is a repurposed antitumor drug which inhibits the proliferation and survival of cancer cells by inhibiting the xCT cellular antioxidant system. Recent clinical studies have shown that, due to poor bioavailability, the antitumor effects of SAS monotherapy are minimal. Therefore, we hypothesized that DSF, another repurposed drug that has demonstrated anticancer effects, or its complex with copper (DSF-copper, DSF-Cu) could potentiate the antilung cancer effects of SAS. Exposure of non-small cell lung cancer cells to therapeutically achievable concentrations of SAS-induced low-to-moderate cytotoxic effects (20-40% reduction in cell viability) and, unexpectedly, induced the antioxidant protein NRF2 and its downstream effectors xCT and ALDH1A1. However, combinations of SAS and DSF-Cu, but not SAS and DSF, induced a significantly higher cytotoxic effect (64-88% reduction in cell viability), apoptosis and generation of mitochondrial reactive oxygen species as compared with SAS or DSF-Cu alone. Moreover, DSF-Cu abrogated SAS-induced NRF2, xCT and ALDH1A1 expression. In a mouse model of lung tumor, SAS + DSF-Cu showed a higher efficacy than the individual drugs in reducing the number and size of tumors as well as the incidence and multiplicity of lung adenocarcinoma. Taken together, our findings indicate that the observed antilung cancer effects of SAS plus DSF-Cu are mediated, at least in part, via impairment of reactive oxygen species defense and -enhancement of oxidative stress and provide evidence for the preventive/therapeutic potential of this combinatorial approach against lung cancer.

Abstract 4776: NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis

Abstract Background: Esophageal adenocarcinoma (EAC) is the predominant type of esophageal cancer in the United States, with the 5-year survival rate is below 20%. EAC develops through Barrett’s esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease (GERD), where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator, involving in many cancer hallmarks. Methods and Results: Using western blotting and immunohistochemistry technologies, we detected high NRF2 protein levels in EAC cell lines and primary EAC tissues, as compared with normal esophagus and non-neoplastic Barrett’s esophagus samples. The knockdown of NRF2 using NRF2 specific siRNAs significantly increased oxidative stress in response to cellular stress stimuli by bile salts or cisplatin. This was associated with an increase in DNA damage and inhibition of EAC cell growth. Brusatol, a NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with CDDP induced significant lipid peroxidation and ferroptosis as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. Moreover, the combination of Brusatol and CDDP significantly inhibited EAC tumor xenografts growth in vivo. We confirmed the in vitro data showing ferroptosis as an important mechanism in the xenografted tumors treated with Brusatol or Brusatol and CDDP combination using IHC staining. Conclusion: Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC. Citation Format: Farah Ballout, Heng Lu, Zheng Chen, Tianling Hu, Lei Chen, Wael El-Rifai, Dunfa Peng. NRF2 is overexpressed in esophageal adenocarcinoma and its targeting sensitizes tumor cells to cisplatin through induction of ferroptosis and apoptosis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4776.

Levosimendan Reverses Cardiac Malfunction and Cardiomyocyte Ferroptosis During Heart Failure with Preserved Ejection Fraction via Connexin 43 Signaling Activation.

In recent decades, the occurrence of heart failure with preserved ejection fraction (HFpEF) has outweighed that of heart failure with reduced ejection fraction by degrees, but few drugs have been demonstrated to improve long-term clinical outcomes in patients with HFpEF. Levosimendan, a calcium-sensitizing cardiotonic agent, improves decompensated heart failure clinically. However, the anti-HFpEF activities of levosimendan and underlying molecular mechanisms are unclear. In this study, a double-hit HFpEF C57BL/6N mouse model was established, and levosimendan (3 mg/kg/week) was administered to HFpEF mice aged 13 to 17 weeks. Different biological experimental techniques were used to verify the protective effects of levosimendan against HFpEF. After four weeks of drug treatment, left ventricular diastolic dysfunction, cardiac hypertrophy, pulmonary congestion, and exercise exhaustion were significantly alleviated. Junction proteins in the endothelial barrier and between cardiomyocytes were also improved by levosimendan. Among the gap junction channel proteins, connexin 43, which was especially highly expressed in cardiomyocytes, mediated mitochondrial protection. Furthermore, levosimendan reversed mitochondrial malfunction in HFpEF mice, as evidenced by increased mitofilin and decreased ROS, superoxide anion, NOX4, and cytochrome C levels. Interestingly, after levosimendan administration, myocardial tissue from HFpEF mice showed restricted ferroptosis, indicated by an increased GSH/GSSG ratio; upregulated GPX4, xCT, and FSP-1 expression; and reduced intracellular ferrous ion, MDA, and 4-HNE levels. Regular long-term levosimendan administration can benefit cardiac function in a mouse model of HFpEF with metabolic syndromes (namely, obesity and hypertension) by activating connexin 43-mediated mitochondrial protection and sequential ferroptosis inhibition in cardiomyocytes.

CircOMA1 modulates cabergoline resistance by downregulating ferroptosis in prolactinoma.

Prolactinomas are one of the most common pituitary neuroendocrine tumors (PitNETs), accounting for approximately 50% of all pituitary tumors. Dopamine agonists are the main treatment for prolactinoma, but a small number of patients are still resistant to pharmacotherapy. Recent discoveries have revealed that ferroptosis is involved in regulating tumor drug resistance. However, the role of ferroptosis in prolactinoma has not been reported. In this study, we aimed to explore the mechanism of a circRNA in ferroptosis in prolactinoma. The expression of circOMA1 in prolactinoma tissues was examined by quantitative reverse transcription PCR (qRT-PCR). The biological function of circOMA1 was evaluated in vitro and in vivo. To explore the role of ferroptosis in prolactinoma, we used qRT-PCR and western blotting. Glutamate-cysteine ligase, modifier subunit (GCLM) was predicted to be a direct target gene of miR-145-5p by bioinformatics analysis, which was confirmed by luciferase reporter assays. circOMA1 was overexpressed in drug-resistant prolactinoma tissues compared with sensitive prolactinoma samples. We further found that circOMA1 promoted MMQ cells growth in vivo and in vitro. In addition, GCLM was directly targeted by miR-145-5p and indirectly regulated by circOMA1. Importantly, circOMA1 induced ferroptosis resistance through the increased expression of Nrf2, GPX4, and xCT, and circOMA1 attenuated CAB-induced ferroptosis in MMQ cells in vivo and in vitro. The present study demonstrates that circOMA1 attenuates CAB efficacy through ferroptosis resistance and may be a new therapeutic target for the individualized treatment of DA-resistant prolactinoma patients.

Maltol inhibits oxygen glucose deprivation‑induced chromatinolysis in SH‑SY5Y cells by maintaining pyruvate level.

Maltol, a chemical isolated from ginseng root, has shown treatment effects on several pathological processes including osteoarthritis, diabetic peripheral neuropathy and liver fibrosis. Nevertheless, its effect on ischemia‑induced neuron death remains elusive. In the present study, the treatment effect of maltol on ischemia‑induced neuron damage was investigated by using oxygen and glucose deprivation (OGD) model in SH‑SY5Y cells. Invitro studies revealed that maltol protected SH‑SY5Y cells against OGD‑induced chromatinolysis by inhibiting two reactive oxygen species (ROS)‑regulated pathways. One was DNA double‑strand breaks and the other was nuclear translocation of apoptosis inducing factor. Mechanistically, maltol not only inhibited OGD‑induced depletion of glutathione and cysteine by maintaining cystine/glutamate antiporter (xCT) level, but also abrogated OGD‑induced catalase downregulation. Meanwhile, maltol also alleviated OGD‑induced inactivation of mTOR by attenuating OGD‑induced depletion of adenosine triphosphate and pyruvate and downregulation of pyruvate kinase M2, indicating that maltol inhibited the glycolysis dysfunction caused by OGD. Considering that activated mammalian target of the rapamycin (mTOR) could lead to enhanced xCT expression and decreased catalase degradation by autophagy, these findings indicated that maltol attenuated OGD‑induced ROS via inhibition of mTOR inactivation by maintaining pyruvate level. Taken together, it was demonstrated that maltol prevented OGD‑induced chromatinolysis in SH‑SY5Y cells via inhibiting pyruvate depletion.

Genetic Disruption of System xc-Mediated Glutamate Release from Astrocytes Increases Negative-Outcome Behaviors While Preserving Basic Brain Function in Rat.

The importance of neuronal glutamate to synaptic transmission throughout the brain illustrates the immense therapeutic potential and safety risks of targeting this system. Astrocytes also release glutamate, the clinical relevance of which is unknown as the range of brain functions reliant on signaling from these cells hasn't been fully established. Here, we investigated system xc- (Sxc), which is a glutamate release mechanism with an in vivo rodent expression pattern that is restricted to astrocytes. As most animals do not express Sxc, we first compared the expression and sequence of the obligatory Sxc subunit xCT among major classes of vertebrate species. We found xCT to be ubiquitously expressed and under significant negative selective pressure. Hence, Sxc likely confers important advantages to vertebrate brain function that may promote biological fitness. Next, we assessed brain function in male genetically modified rats (MSxc) created to eliminate Sxc activity. Unlike other glutamatergic mechanisms, eliminating Sxc activity was not lethal and didn't alter growth patterns, telemetry measures of basic health, locomotor activity, or behaviors reliant on simple learning. However, MSxc rats exhibited deficits in tasks used to assess cognitive behavioral control. In a pavlovian conditioned approach, MSxc rats approached a food-predicted cue more frequently than WT rats, even when this response was punished. In attentional set shifting, MSxc rats displayed cognitive inflexibility because of an increased frequency of perseverative errors. MSxc rats also displayed heightened cocaine-primed drug seeking. Hence, a loss of Sxc-activity appears to weaken control over nonreinforced or negative-outcome behaviors without altering basic brain function.SIGNIFICANCE STATEMENT Glutamate is essential to synaptic activity throughout the brain, which illustrates immense therapeutic potential and risk. Notably, glutamatergic mechanisms are expressed by most types of brain cells. Hence, glutamate likely encodes multiple forms of intercellular signaling. Here, we hypothesized that the selective manipulation of astrocyte to neuron signaling would alter cognition without producing widespread brain impairments. First, we eliminated activity of the astrocytic glutamate release mechanism, Sxc, in rat. This impaired cognitive flexibility and increased expression of perseverative, maladaptive behaviors. Notably, eliminating Sxc activity did not alter metrics of health or noncognitive brain function. These data add to recent evidence that the brain expresses cognition-specific molecular mechanisms that could lead to highly precise, safe medications for impaired cognition.