SLC26A4 Research Articles

Based on bioinformatics, SESN2 negatively regulates ferroptosis induced by ischemia reperfusion via the System Xc−/GPX4 pathway

IntroductionCerebral ischemia–reperfusion (IR) causes severe secondary brain injury. Previous studies have demonstrated that ferroptosis is involved in IR-induced brain injury. However, whether IR induces ferroptosis in brain microvascular endothelial cells (BMVECs) is not fully understood.Materials and methodsOxygen–glucose deprivation/reoxygenation (OGDR) was performed in bEND.3 cells to mimic IR injury in vitro, and a focal cerebral IR model was created in C57BL/6 mice. Transcriptomic sequencing of the cells was performed first, followed by bioinformatics analysis. Differentially expressed gene (DEG) enrichment analysis highlighted ferroptosis-related pathways.ResultsUsing Venn analysis, nine ferroptosis-related DEGs were identified, namely, Slc3a2, Slc7a11, Ccn2, Tfrc, Atf3, Chac1, Gch1, Lcn2, and Sesn2. Protein–protein interaction (PPI) analysis combined with molecular complex detection (MCODE) identified six hub genes, namely, Ddit3, Atf3, Sesn2, Trib3, Ppp1r15a, and Gadd45a. Spearman’s correlation analysis revealed a significant correlation between the hub genes and ferroptosis-related DEGs. After reperfusion, the levels of ferroptosis indicators were elevated, and the expression of the ferroptosis-related proteins Xc− and GPX4 decreased. SESN2 is a hub gene and key antioxidant regulator. SESN2 silencing reduced the expression of System Xc− and GPX4, whereas overexpression of SESN2 promoted the expression of System Xc− and GPX4.DiscussionThese results suggest that SESN2 is a negative regulator of ferroptosis. Enhancing the expression of SESN2 can alleviate ferroptosis through the activation of the System Xc−/GPX4 pathway. By integrating bioinformatics analysis with mechanistic exploration, this study revealed that ferroptosis plays a crucial role in IR-induced BMVECs injury, with SESN2 acting as a negative regulator via the System Xc−/GPX4 pathway.

Abstract P010: Monitoring of cancer ferroptosis with [18F]hGTS13, a system xc- specific radiotracer

Abstract Ferroptosis has emerged as a significant process in cancer biology due to its ability to counteract mechanisms of tumor survival and drug resistance. Exploiting this vulnerability is an emerging therapeutic strategy in cancer treatment. System xc- plays a critical role in ferroptosis by regulating the availability of cysteine, a crucial precursor for glutathione synthesis, thereby influencing the cellular antioxidant defense system. We recently reported the development and validation of [18F]hGTS13, a radiolabeled glutamate derivative, as a novel radiopharmaceutical specific for system xc-. We characterized the sensitivity of various cell lines to pro-ferroptotic compounds and evaluated the ability of [18F]hGTS13 to distinguish between sensitive and resistant cell lines and monitor changes in response to ferroptosis-inducing drugs. We then correlated changes in tracer uptake with cellular glutathione content. Furthermore, we performed PET/CT imaging of C6 glioma-bearing rats with [18F]hGTS13, revealing high, sustained uptake within the intracranial glioma, and demonstrated effective blocking of system xc- in vivo using imidazole ketone erastin. In summary, [18F]hGTS13 represents a promising tool for distinguishing cell types that demonstrate sensitivity or resistance to ferroptotic therapies, and monitoring of ferroptosis-inducing drug engagement. Its successful application in preclinical models make it a valuable asset for advancing our understanding of glioma biology and evaluating the efficacy of emerging ferroptosis-targeted therapies. As synergistic ferroptosis/radionuclide therapies are currently under investigation, [18F]hGTS13 represents a valuable tool for identifying patients who would benefit from combinatorial therapy, and monitoring treatment response in glioma and other cancers. Citation Format: Abraham S. Moses, Rim Malek, Mustafa T. Kendirli, Pierre Cheung, Madeleine Landry, Marco Herrera-Barrera, Abbas Khojasteh, Syed Bukhari, Jody Hooper, Melanie Hayden-Gephart, Scott J. Dixon, Lawrence D. Recht, Corinne Beinat.Monitoring of cancer ferroptosis with [18F]hGTS13, a system xc- specific radiotracer.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P010

Disulfidptosis-related gene SLC3A2: a novel prognostic biomarker in nasopharyngeal carcinoma and head and neck squamous cell carcinoma

IntroductionNasopharyngeal carcinoma (NPC), one of the most common malignancies of the head and neck, is characterised by a complex pathogenesis and an unfavourable prognosis. Recently, disulfidoptosis, a novel form of cell death, has been proposed. Several studies in recent years have extensively investigated the function of the disulfidoptosis-related SLC7A11 gene in cancer, but the role of its partner protein, SLC3A2, remains unknown unclear in NPC.MethodsGEO database analysis confirmed SLC3A2's prognostic impact on nasopharyngeal carcinoma. ROC, Kaplan-Meier analyses, and stage-specific expression studies showed a strong correlation with poor HNSC prognosis. GO and KEGG analyses pinpointed relevant signaling pathways. In vitro, SLC3A2's influence on cell proliferation, migration, and invasion was evaluated through CCK8, wound healing, colony formation, transwell assays, and cell cycle analysis.ResultsIn this study, we identified the high expression of SLC3A2 in NPC and head and neck squamous cell carcinoma (HNSC) and analyzed its potential mechanism and correlation with patient prognosis. Furthermore, a negative relationship was found between the expression level of SLC3A2 and the extent of immune cell infiltration and immune checkpoint. Differentially expressed genes (DEGs) between the high and low SLC3A2 expression groups were primarily involved in cytokine-cytokine receptor interaction and immune response. Finally, in vitro experiments demonstrated that SLC3A2 stimulates tumor cell proliferation and migration. DiscussionIn conclusion, these results indicated a strong association between SLC3A2 and progression in both NPC and HNSC, suggesting it as a promising biomarker for predicting adverse prognosis in NPC and HNSC patients.

Targeting estrogen-regulated system xc− promotes ferroptosis and endocrine sensitivity of ER+ breast cancer

Estrogen receptor positive (ER+) breast cancer accounts for approximately 70% of cases. Endocrine therapies targeting estrogen are the first line therapies for ER+ breast cancer. However, resistance to these therapies occurs in about half of patients, leading to decreased survival rates. Inducing ferroptosis is a promising therapeutic strategy for cancer treatment for refractory and malignant cancers including triple-negative breast cancer. Nevertheless, ER+ breast cancer is relatively resistant to ferroptosis inducers. Here, we uncovered that ERα suppressed ferroptosis in ER+ breast cancer. Silencing ERα triggered ferroptosis, which was attenuated by ferroptosis inhibitor Ferrostatin-1, and was enhanced by ferroptosis inducer Erastin. Mechanistically, ERα transcriptionally upregulated the expression of SLC7A11 and SLC3A2, two subunits of the system xc−, which is one key inhibitory regulator of ferroptosis. Overexpression of the exogenous SLC7A11 and SLC3A2 was able to mitigate ferroptosis induced by ERα inhibition. Moreover, SLC7A11 and SLC3A2 levels were elevated in endocrine-resistant breast cancer cells and tumors. Importantly, the system xc− inhibitor Sorafenib or Imidazole ketone erastin effectively inhibited the growth of tamoxifen-resistant breast cells in vitro and in vivo. In conclusion, our data reveal that targeting estrogen-regulated SLC7A11 and SLC3A2 enhances ferroptosis in ER+ breast cancer, offering a novel therapeutic option for patients with ER+ breast cancer, particularly those with endocrine resistance.

Compact and cGMP-compliant automated synthesis of [18F]FSPG on the Trasis AllinOne™

Background(S)-4-(3-18F-Fluoropropyl)-ʟ-glutamic acid ([18F]FSPG) is a positron emission tomography radiotracer used to image system xc−, an antiporter that is upregulated in several cancers. Not only does imaging system xc− with [18F]FSPG identify tumours, but it can also provide an early readout of response and resistance to therapy. Unfortunately, the clinical production of [18F]FSPG has been hampered by a lack of robust, cGMP-compliant methods. Here, we report the automated synthesis of [18F]FSPG on the Trasis AllinOne™, overcoming previous limitations to provide a user-friendly method ready for clinical adoption.ResultsThe optimised method provided [18F]FSPG in 33.5 ± 4.9% radiochemical yield in just 35 min when starting with 18–25 GBq. Importantly, this method could be scaled up to > 100 GBq starting activity with only a modest reduction in radiochemical yield, providing [18F]FSPG with a molar activity of 372 ± 65 GBq/µmol and excellent radiochemical purity (96.8 ± 1.1%). The formulated product was stable when produced with these high starting activities.ConclusionsWe have developed the first automated synthesis of [18F]FSPG on the Trasis AllinOne™. The method produces [18F]FSPG with excellent radiochemical purity and in high amounts suitable for large clinical trials and off-site distribution. The method expands the number of synthesis modules capable of producing [18F]FSPG and has been carefully designed for cGMP compliance to simplify regulatory approval for clinical production. The methods developed for the purification of high-activity [18F]FSPG are transferrable and should aid the development of clinical [18F]FSPG productions on other synthesis modules.

Nrf2 Regulates Basal Glutathione Production in Astrocytes.

Astrocytes produce and export glutathione (GSH), an important thiol antioxidant essential for protecting neural cells from oxidative stress and maintaining optimal brain health. While it has been established that oxidative stress increases GSH production in astrocytes, with Nrf2 acting as a critical transcription factor regulating key components of the GSH synthetic pathway, the role of Nrf2 in controlling constitutive GSH synthetic and release mechanisms remains incompletely investigated. Our data show that naïve primary mouse astrocytes cultured from the cerebral cortices of Nrf2 knockout (Nrf2-/-) pups have significantly less intracellular and extracellular GSH levels when compared to astrocytes cultured from Nrf2 wild-type (Nrf2+/+) pups. Key components of the GSH synthetic pathway, including xCT (the substrate-specific light chain of the substrate-importing transporter, system xc-), glutamate-cysteine ligase [catalytic (GCLc) and modifying (GCLm) subunits], were affected. To wit: qRT-PCR analysis demonstrates that naïve Nrf2-/- astrocytes have significantly lower basal mRNA levels of xCT and both GCL subunits compared to naïve Nrf2+/+ astrocytes. No change in mRNA levels of glutathione synthetase (GS) or the GSH exporting transporter, Mrp1, was found. Western blot analysis reveals reduced protein levels of both subunits of GCL, while (seleno)cystine uptake into Nrf2-/- astrocytes was reduced compared to Nrf2+/+ astrocytes, confirming decreased system xc- activity. These findings suggest that Nrf2 regulates the basal production of GSH in astrocytes through constitutive transcriptional regulation of GCL and xCT.

Understanding the Spectrum of Mild Clinical Outcomes and Novel Findings in Arterial Tortuosity Syndrome Among Qatari Patients: Implications of SLC2A10 Mutation.

Background/Objectives: Arterial Tortuosity Syndrome (ATS) is a rare, autosomal recessive connective tissue disorder characterized by arterial twists, abnormal bulges, constriction, and tears. Patients have distinctive features and disease manifestations. The syndrome's full clinical spectrum and course remain incompletely understood. Methods: We sought to review the medical records of Qatari patients who had ATS. The cohort study included 21 patients who were genetically confirmed by mutations in the SLC2A10 gene. Results: The study revealed that the NM_030777.4(SLC2A10):c.243C>G (p.Ser81Arg) mutation in SLC2A10 leads to mild outcomes of no mortality and less morbidity. Novel features such as a flat philtrum, bulbous noses, bilateral nasolacrimal duct obstruction, allergic conjunctivitis, latent nystagmus, café au lait spots, eczema, dermatitis, allergic reactions, bilateral temporomandibular joint cysts, bilateral syndactyly (toes), parapelvic cysts, kidney malrotation, vesicoureteral reflux, and nephrolithiasis were identified in our cohort. Furthermore, rare features previously documented in a limited number of patients, including leg length discrepancy, epilepsy, and migraine headaches, were also observed in our cohort. Conclusions: Our data contributes new insights into the life course of ATS in Qatari patients. These findings underscore the importance of effective education strategies through repeated counseling aimed at preventing cousin marriage and the syndrome within the cohort.

Implication of system xc- in Complete Freund's Adjuvant-induced peripheral inflammation and associated nociceptive sensitization.

Persistent inflammation leading to neuronal sensitization in pain pathways, are key features of chronic inflammatory pain. Alike macrophages in the periphery, glial cells exacerbate hypersensitivity by releasing proalgesic mediators in the central nervous system. Expressed by peripheral and central immune cells, the cystine-glutamate antiporter system xc- plays a significant role in inflammatory responses, but its involvement in chronic inflammatory pain remains underexplored. We herein investigated the contribution of this exchanger in nociceptive hypersensitivity triggered by a peripheral inflammatory insult. Complete Freund's adjuvant (CFA) was injected into the left hind paw of wild-type C57Bl/6 female mice, of xCT-deficient mice (specific subunit of system xc-) and of mice receiving the system xc- inhibitor sulfasalazine. Paw edema was measured over three weeks and pain-associated behaviors were evaluated. Additionally, pro-inflammatory cytokine levels were assessed in blood samples. CFA injection led to a persistent increase in paw edema and hypersensitivity to mechanical and thermal stimuli, which were less pronounced in xCT-deficient mice. This reduced sensitivity was accompanied by lower systemic pro-inflammatory cytokine levels in xCT-deficient mice. Accordingly, pharmacological inhibition of system xc- with sulfasalazine, either before or after pain induction, efficiently reduced the algesic and inflammatory responses to CFA in wild-type mice. Our findings reveal a critical role for system xc- in the pathophysiology of inflammatory pain. xCT deficiency reduces pain behaviors and peripheral inflammation, positioning system xc- as a promising therapeutic target for alleviating chronic inflammatory pain.

Monitoring of cancer ferroptosis with [18F]hGTS13, a system xc- specific radiotracer.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults, characterized by resistance to conventional therapies and poor survival. Ferroptosis, a form of regulated cell death driven by lipid peroxidation, has recently emerged as a promising therapeutic target for GBM treatment. However, there are currently no non-invasive imaging techniques to monitor the engagement of pro-ferroptotic compounds with their respective targets, or to monitor the efficacy of ferroptosis-based therapies. System xc-, an important player in cellular redox homeostasis, plays a critical role in ferroptosis by mediating the exchange of cystine for glutamate, thus regulating the availability of cysteine, a crucial precursor for glutathione synthesis, and influencing the cellular antioxidant defense system. We have recently reported the development and validation of [18F]hGTS13, a radiopharmaceutical specific for system xc-. Methods: In the current work, we characterized the sensitivity of various cell lines to pro-ferroptotic compounds and evaluated the ability of [18F]hGTS13 to distinguish between sensitive and resistant cell lines and monitor changes in response to ferroptosis-inducing investigational compounds. We then associated changes in [18F]hGTS13 uptake with cellular glutathione content. Furthermore, we evaluated [18F]hGTS13 uptake in a rat model of glioma, both before and after treatment with imidazole ketone erastin (IKE), a pro-ferroptotic inhibitor of system xc- activity. Results: Treatment with erastin2, a system xc- inhibitor, significantly decreased [18F]hGTS13 uptake and cellular glutathione content in vitro. Dynamic PET/CT imaging of C6 glioma-bearing rats with [18F]hGTS13 revealed high and sustained uptake within the intracranial glioma and this uptake was decreased upon pre-treatment with IKE. Conclusion: In summary, [18F]hGTS13 represents a promising tool to distinguish cell types that demonstrate sensitivity or resistance to ferroptosis-inducing therapies that target system xc-, and monitor the engagement of these drugs.

Acute hyperglycemia impedes spinal cord injury recovery via triggering excessive ferroptosis of endothelial cells.

Spinal cord injury (SCI) is a serious central nervous system injury that often causes sensory and motor dysfunction in patients. Diabetes seriously destroys the blood spinal cord barrier (BSCB) and aggravates SCI. Ferroptosis is a new type of programmed cell death. The role of ferroptosis in diabetes-medicated BSCB destruction has not been clearly elucidated. Here, we built a type 1 diabetes (T1D) combined with SCI rat model and confirmed that hyperglycemia exacerbates SCI-mediated BSCB destruction. Pathological mechanism demonstrated that except for apoptosis, the excessive ferroptosis is another caused factor for endothelial cells (ECs) loss under hyperglycemic condition. More importantly, ferrostatin-1(a ferroptosis inhibitor) treatment significantly inhibited the ferroptosis of ECs, and promoted the BSCB repair in T1D combined with SCI rat. The mechanism study further revealed that hyperglycemia not only induces the elevated reactive oxygen species (ROS) via activating RAGE, but also suppresses the xCT expression in system Xc- in ECs, which decreases GPX4 expression and induces ferroptosis. Additionally, hyperglycemia also accelerated the transfer of iron ions from serum to spinal cord after SCI. In summary, our results suggest that the excessive ferroptosis of ECs is essential for the severe BSCB destruction in T1D combined with spinal cord injury rat.

Selenomethionine alleviates T-2 toxin-induced articular chondrocyte ferroptosis via the system Xc-/GSH/GPX4 axis.

T-2 toxin can induce bone and cartilage development disorder, and oxidative stress plays an important role in it. It is well known that selenomethionine (Se-Met) has antioxidative stress properties and promotes the repair of cartilage lesion, but it remains unclear whether Se-Met can relieve damaged cartilage exposure to T-2 toxin. Here, the oxidative stress and ferroptosis of chondrocytes exposure to T-2 toxin were observed. Mechanistically, T-2 toxin increased ROS, lipid ROS, MDA and Fe2+ contents in chondrocytes, decreased GSH and GPX4 activity, and inhibited the system Xc-/GSH/GPX4 antioxidant axis. In addition, the mitochondria of chondrocytes shrunk and the mitochondrial crest decreased or disappeared. However, Fer-1 (Ferrostatin-1) inhibited ferroptosis induced by T-2 toxin in chondrocytes. The Se-Met alleviated lipid peroxidation, oxidative stress, and damaged mitochondrial in T-2 toxin-infected chondrocytes, enhanced antioxidant enzyme activity, and activated the system Xc-/GSH/GPX4 axis, thereby antagonizing ferroptosis of chondrocytes and alleviating articular cartilage damage. In conclusion, our findings highlight the essentiality of ferroptosis in chondrocyte caused by T-2 toxin, elucidate how Se-Met offers protection against this injury and provide research evidence for the drug treatment target of Kashin-Beck disease.

Aprepitant ameliorates vancomycin-induced kidney injury: Role of GPX4/system Xc- and oxidative damage.

Vancomycin, a glycopeptide antibiotic, is used in cases of drug-resistant bacterial infections, but unfortunately is associated with acute kidney injury (AKI). We here explore the protective potential of aprepitant against vancomycin-induced AKI. Vancomycin (500mg/kg/i.p) was given to rats for seven days and aprepitant (20mg/kg/p.o) was administered one day before and for seven days concomitant with vancomycin. At the end of the experiment, kidney function, oxidative stress, autophagy and ferroptosis markers were assessed. We show that aprepitant reduced kidney/body weight ratio, serum creatinine and blood urea nitrogen levels. It improved renal structure and enhanced the antioxidant machinery as indicated by elevated catalase activity and GSH levels and reduced renal MDA. Aprepitant managed to inhibit ferroptosis by decreasing system Xc- and GPX4 renal levels. As a result, levels of autophagic markers ATG3, LC3A and LC3B were attenuated. These results were confirmed by electron microscopy examination of cellular structures. In addition, aprepitant increased p62 protein expression. Moreover, aprepitant decreased the apoptotic marker cleaved caspase-3 levels. Our results suggest a new repurposed role for aprepitant in protecting against AKI. This protective effect relies on its antioxidant effect and the influence of inhibiting ferroptosis which resulted in downregulation of autophagy and apoptosis.

The effectiveness of sanggenon c in alleviating SLC7A11-induced ferroptosis in lung cancer was evaluated using in vivo, in vitro, and computational approaches.

Sanggenon c, a component in Morus alba L, has been proved to possess various biological activities. The aim of this study is to investigate whether sanggenon c can target SLC7A11 and inhibit lung cancer by regulating the ferroptosis mechanism. The levels of antioxidant factor, Fe 2+, and SLC7A11 were measured in the lungs of cancerous mice and human A 549 lung cancer cells. The computer-aided techniques were employed to validate the molecular docking and molecular dynamics simulations of sanggenon c and SLC7A11. The sanggenon c significantly inhibits lung cancer cell metastasis in vivo and A 549 cell proliferation in vitro by targeting the over-expression of SLC7A11, which inhibits GPX 4 and induces the release of ROS and MDA, effectively triggering ferroptosis. The interaction between sanggenon c and SLC7A11 exhibits a strong binding affinity, leading to the significant inhibition of the key protein SLC7A11. This restriction of system xc- transport induces ferroptosis in lung cancer. It epitomizes a groundbreaking inhibitor specifically designed to target SLC7A11.

Efficacy of glutathione inhibitor eprenetapopt against the vulnerability of glutathione metabolism in SMARCA4-, SMARCB1- and PBRM1-deficient cancer cells

Mutation of genes related to the SWI/SNF chromatin remodeling complex is detected in 20% of all cancers. The SWI/SNF chromatin remodeling complex comprises about 15 subunits and is classified into three subcomplexes: cBAF, PBAF, and ncBAF. Previously, we showed that ovarian clear cell carcinoma cells deficient in ARID1A, a subunit of the cBAF complex, are synthetic lethal with several genes required for glutathione (GSH) synthesis and are therefore sensitive to the GSH inhibitor eprenetapopt (APR-246). However, we do not know whether cancer cells deficient in SWI/SNF components other than ARID1A are selectively sensitive to treatment with eprenetapopt. Here, we show that SMARCA4-, SMARCB1-, and PBRM1-deficient cells are more sensitive to eprenetapopt than SWI/SNF-proficient cells. We found that deficiency of SMARCA4, SMARCB1, or PBRM1 attenuates transcription of the SLC7A11 gene (which supplies cysteine as a raw metabolic material for GSH synthesis) by the failure of recruitment of cBAF and PBAF to the promotor and enhancer regions of the SLC7A11 locus, thereby reducing basal levels of GSH. In addition, eprenetapopt decreased the amount of intracellular GSH and increased the intracellular amount of reactive oxygen species (ROS), followed by induction of apoptosis. Taken together, eprenetapopt could be a promising selective agent for SWI/SNF-deficient cancer cells derived from SMARCA4-deficient lung cancers, SMARCB1-deficient rhabdoid tumors, and PBRM1-deficient kidney cancers.

Harnessing ROS Amplification and GSH Depletion Using a Carrier-Free Nanodrug to Enhance Ferroptosis-Based Cancer Therapy.

Ferroptosis, a non-apoptotic form of cell death characterized by the production of reactive oxygen species (ROS) and massive accumulation of lipid peroxidation (LPO), shows significant promise in cancer therapy. However, the overexpression of glutathione (GSH) at the tumor site and insufficient ROS often result in unsatisfactory therapeutic efficacy. A multistage, GSH-consuming, and ROS-providing carrier-free nanodrug capable of efficiently loading copper ions (Cu2+), sorafenib (SRF), and chlorogenic acid (CGA) (Cu2+-CGA-SRF, CCS-NDs) is developed to mediate enhanced ferroptosis therapy. Through a reductive intracellular environment, Cu2+ in the CCS-NDs reacted with intracellular GSH, alleviating the antioxidant capacity of tumor tissues and triggering the release of drugs. Meanwhile, the released SRF inhibited system xc-, thereby blocking cystine uptake and reducing GSH synthesis in tumor cells. By depleting stored GSH and inhibiting its synthesis, CCS-NDs achieved efficient GSH depletion and increased accumulation of toxic LPO. More importantly, the high concentration of CGA in the CCS-NDs induced ROS generation, further promoting ferroptosis. Both in vitro and in vivo results demonstrated that CCS-NDs effectively triggered ferroptosis in tumor cells by inactivating glutathione peroxidase 4 and inducing LPO. Overall, the carrier-free nanodrug CCS-NDs offer a promising strategy for regulating GSH and LPO levels in ferroptosis-based cancer therapy.

Eye-acupuncture ameliorates ferroptosis of cerebral neuronal cells in CIRI rats by up-regulating System xc(-)-GSH-GPX4 antioxidant functional axis activities

To observe the effect of eye-acupuncture on the antioxidant function axis：System xc(-)-glutathione-glutathione peroxidase 4 (System xc［-］-GSH-GPX4) in the cortical tissue of ischemic penumbra of acute cerebral ischemia-reperfusion injury (CIRI) rats, so as to explore its underlying mechanism in improvement of CIRI by ameliorating the ferroptosis of neurons via antioxidant function axis. Male SD rats were randomly divided into sham operation, model, eye-acupuncture and GPX4-inhibitor groups, with 15 rats in each group. The CIRI model was replicated by occlusion of the middle cerebral artery and reperfusion for 24 h. For rats of the eye acupuncture group and inhibitor group, manual acupuncture stimulation was applied to bilateral eye-acupuncture-points "Gan"(Liver), "Shangjiao"(Upper-energizer), "Shen"(Kidney) and "Xiajiao"(Lower-energizer) of both eyes, 30 min, 12 h and 24 h after modeling, 3 times altogether. For rats of the inhibitor group, intraperitoneal injection of GPX4 inhibitor RSL3 (an activator of ferroptosis) 10 mg/kg (dissolved in 5% DMSO) was conducted 30 min before every acupuncture stimulation. The neurological function was assessed by using Garcia JH scoring method. The brain infarct size was detected after triphenyl tetrazolium chloride (TTC) staining. The ischemic penumbral cortex tissue of the brain was taken for observing morphological changes after H.E. staining, and for observing ultrastructural changes of the mitochondria by using transmission electron microscope. The contents of malondialdehyde (MDA) and GSH were detected by using photocolorimetric method, the content of ferrous ions (Fe2+) detected using spectrophotometry, and the activity of reactive oxygen species (ROS) assayed by ELISA. The expression levels of solute carrier family 7 member 11 (SLC7A11), solute carrier family 3 member 2 (SLC3A2) and GPX4 proteins of the ischemic penumbral cortex were detected by Western blot, and those of SLC7A11 and GPX4 mRNAs were detected using real-time quantitative PCR. Compared with the sham operation group, the Garcia JH neurological deficit score, GSH content, and expression levels of SLC7A11, SLC3A2 and GPX4 proteins, and GPX4 and SLC7A11 mRNAs were significantly decreased (P<0.01), and the contents of Fe2+ and MDA, and ROS activity considerably increased (P<0.01) in the model group. In contrast to the model group, the decreased and increased levels of the above mentioned indexes were reversed in the eye-acupuncture group (P<0.01, P<0.05) but not in the inhibitor group. The therapeutic effects of eye-acupuncture were reduced by GPX4 inhibitor in increasing the levels of Garcia JH neurological deficit score, GSH content, and expression levels of SLC7A11, SLC3A2, and GPX4 proteins, and GPX4 and SLC7A11 mRNAs, as well as in lowering the contents of Fe2+ and MDA and ROS activity(P<0.05, P<0.01). Results of TTC staining displayed that the brain tissue on the right side showed obvious gray infarct loci in the model group, which was evidently smaller in the eye-acupuncture group, but not in the inhibitor group. H.E. staining displayed disordered arrangement of cells, with shriveled or broken nucleus, and interstitial edema and vacuolation, and a number of large area typical cerebral infarction, and net-like necrotic loci with blurred necrotic lesion boundaries in the model group, which was apparently milder in the eye-acupuncture group. In the inhibitor group, an increased number of cerebral infarction foci, and disordered arrangement and severe injury of cells were found. Eye-acupuncture can ameliorate ferroptosis in neurons of CIRI rats by modulating System xc(-)-GSH-GPX4 antioxidant function axis.

Absence of gut microbiota alleviates iron overload-induced colitis by modulating ferroptosis in mice.

Iron overload disrupts gut microbiota and induces ferroptosis, contributing to colitis. However, whether gut microbiota directly drives iron overload-induced colitis and its underlying mechanism remain unclear. The study aimed to explore whether gut microbiota can directly regulate iron overload-induced colitis and its underling mechanism. Male C57BL/6N mice were fed with ferrous sulfate to establish an iron overload model. Antibiotics and DSS were used to create germ-free and colitis models, respectively. Results showed that iron overload caused disruption of systemic iron homeostasis via activating pro-inflammation response, which caused induction of ferroptosis and eventually resulted in colitis in mice. Notably, iron overload inhibited System Xc- and activated the nuclear factor E2-related factor 2/heme oxygenase-1 pathway, driving ferroptosis and colitis progression. Similar results were observed in mouse colon epithelial cells, which were treated with high doses ferric ammonium citrate. Additionally, iron overload exacerbated DSS-induced colitis by activating the ferroptosis and increasing harmful bacteria (e.g., Mucispirillum) abundance. Interestingly, eliminating gut microbiota attenuated iron overload-induced colitis, without affecting systemic inflammation through inhibiting ferroptosis of mice. Depletion of the gut microbiota partially mitigated the exacerbating effect of iron overload on DSS-induced colitis through inhibiting ferroptosis of mice. Iron overload activates ferroptosis in colonic cells, increases the relative abundance of harmful bacteria, and exacerbates DSS-induced colitis in mice. Iron overload exacerbates DSS-induced ferroptosis and colitis in a microbiota-dependent manner. Targeting gut microbiota may offer new strategies for managing iron overload-induced colitis.

Imaging NRF2 activation in non-small cell lung cancer with positron emission tomography

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. Currently, there is no means to non-invasively identify NRF2 activation in living subjects. Here, we show that positron emission tomography imaging with the system xc− radiotracer, [18F]FSPG, provides a sensitive and specific marker of NRF2 activation in orthotopic, patient-derived, and genetically engineered mouse models of NSCLC. We found a NRF2-related gene expression signature in a large cohort of NSCLC patients, suggesting an opportunity to preselect patients prior to [18F]FSPG imaging. Furthermore, we reveal that system xc− is a metabolic vulnerability that can be therapeutically targeted with an antibody-drug conjugate for sustained tumour growth suppression. Overall, our results establish [18F]FSPG as a predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

High Carbonyl Graphene Oxide Suppresses Colorectal Cancer Cell Proliferation and Migration by Inducing Ferroptosis via the System Xc-/GSH/GPX4 Axis.

Colorectal cancer (CRC) is characterized by a high rate of both incidence and mortality, and its treatment outcomes are often affected by recurrence and drug resistance. Ferroptosis, an iron-dependent programmed cell death mechanism triggered by lipid peroxidation, has recently gained attention as a potential therapeutic target. Graphene oxide (GO), known for its oxygen-containing functional groups, biocompatibility, and potential for functionalization, holds promise in cancer treatment. However, its role in ferroptosis induction in CRC remains underexplored. The objective of this study was to investigate the effects of High Carbonyl Graphene Oxide (HC-GO) on ferroptosis in CRC and elucidate the underlying mechanisms. In vitro assays were conducted to evaluate the impact of HC-GO on CRC cell proliferation, mitochondrial function, iron accumulation, lipid peroxidation, and reactive oxygen species (ROS) production. The ferroptosis inhibitor Fer-1 was used to confirm the role of ferroptosis in HC-GO's anti-tumor effects. In vivo, the anti-tumor activity of HC-GO was assessed in a CRC xenograft model, with organ toxicity evaluated. HC-GO significantly inhibited CRC cell proliferation, induced mitochondrial damage, and enhanced iron accumulation, lipid peroxidation, and ROS production. It also downregulated the ferroptosis-inhibiting proteins GPX4 and SLC7A11, which were reversed by Fer-1, confirming the involvement of ferroptosis in HC-GO's anti-cancer effects. In vivo, HC-GO significantly suppressed tumor growth without noticeable toxicity to vital organs. HC-GO triggered ferroptosis in CRC cells by suppressing the System Xc-/GSH/GPX4 pathway, providing a novel therapeutic strategy for CRC treatment. These findings suggest HC-GO as a promising nanomedicine for clinical application, warranting further investigation to explore its potential in CRC therapy.

Association of rs3755319 and rs4148325 of the &lt;i&gt;UGT1A1&lt;/i&gt; gene, rs2328136 of the &lt;i&gt;NUP153-AS&lt;/i&gt; gene, and rs16928809 of the &lt;i&gt;SLC22A18&lt;/i&gt; gene with benign unconjugated hyperbilirubinemia

Background: Benign unconjugated hyperbilirubinemia, also known as Gilbert's syndrome, is a common in the population moderate increase in total and unconjugated bilirubin concentrations in individuals without liver disease or hemolysis. Aim: To identify associations of rs3755319, rs4148325 of the UGT1A1 gene, rs2328136 of the NUP153-AS gene, and rs16928809 of the SLC22A18 gene with benign unconjugated hyperbilirubinemia. Methods: This case-control study included a group of individuals with benign unconjugated hyperbilirubinemia (n = 414, mean age 36.7 ± 15.9 years, 49.8% men) and a control group (n = 381, mean age 39.1 ± 15.9 years, 52.5% men). The sample was randomly selected from the participants of the MONICA project, screening of young people aged 25–44 years and a cross-sectional study of schoolchildren in Novosibirsk. DNA was isolated from venous blood by phenol-chloroform extraction or an express assay (PROBA-RAPID-GENETICS, DNA-Technology, Russia). Genotyping of the groups by rs3755319, rs4148325 of the UGT1A1 gene, rs2328136 of the NUP153-AS gene, and rs16928809 of the SLC22A18 gene was performed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results: No significant differences were found between the individuals with benign unconjugated hyperbilirubinemia and the control group by the genotypes and alleles of the nucleotide sequence variants of rs16928809 of the SLC22A18 gene (p 0.05). The CC genotype and the C allele of rs3755319 of the UGT1A1 gene were more common in the individuals with benign unconjugated hyperbilirubinemia, than in the control group (CC vs AC + AA: odds ratio (OR) = 21.1, 95% confidence interval (CI) 14.7–30.4, p 0.001; C vs A: OR = 12.4, 95% CI 9.4–16.4, p 0.001). The concentrations of total and unconjugated bilirubin were higher in the carriers of the CC genotype of rs3755319, compared to the carriers of the other two genotypes (p 0.05). rs4148325 of the UGT1A1 gene was in the linkage disequilibrium with the rs3064744 UGT1A1 variant. The GG genotype and the G allele rs2328136 of the NUP153-AS gene were more common in the individuals with benign unconjugated hyperbilirubinemia than in the control group (GG vs AG + AA: OR = 1.361, 95% CI 1.002–1.848, p = 0.048; G vs A: OR = 1.33, 95% CI 1.02–1.73, p = 0.034). Conclusion: The CC genotype and the C allele of rs3755319 of the UGT1A1 gene, the GG genotype and the G allele of rs2328136 of the NUP153-AS gene are the genotypes and alleles of risk for benign unconjugated hyperbilirubinemia. The rs16928809 of the SLC22A18 gene is not associated with benign unconjugated hyperbilirubinemia.